foods
Article
Oxidative Modification, Structural Conformation, and Gel
Properties of Pork Paste Protein Mediated by Oxygen
Concentration in Modified Atmosphere Packaging
Rui Liu 1 , Wen Guan 1 , Wei Lv 1 , Zhuangli Kang 2 , Qingling Wang 1 , Duxin Jin 1 , Xinxin Zhao 1 , Qingfeng Ge 1 ,
Mangang Wu 1 and Hai Yu 1, *
Citation: Liu, R.; Guan, W.; Lv, W.;
Kang, Z.; Wang, Q.; Jin, D.; Zhao, X.;
Ge, Q.; Wu, M.; Yu, H. Oxidative
Modification, Structural
Conformation, and Gel Properties of
Pork Paste Protein Mediated by
Oxygen Concentration in Modified
Atmosphere Packaging. Foods 2024,
13, 391. https://doi.org/10.3390/
foods13030391
Academic Editor: Vladimir Tomović
Received: 15 December 2023
Revised: 14 January 2024
Accepted: 16 January 2024
Published: 25 January 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Foods 2024, 13, 391. https://doi.org/10.3390/foods13030391
1 College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, China;
ruiliu@yzu.edu.cn (R.L.); mz120211938@stu.yzu.edu.cn (W.G.); mz120211959@stu.yzu.edu.cn (W.L.);
wangql891228@163.com (Q.W.); jinduxin@yzu.edu.cn (D.J.); xxzhao@yzu.edu.cn (X.Z.);
qfge@yzu.edu.cn (Q.G.); mgwu@yzu.edu.cn (M.W.)
2 School of Tourism and Cuisine, Engineering Technology Research Center of Yangzhou Prepared Cuisine,
Yangzhou University, Yangzhou 225127, China; kzlnj1988@163.com
* Correspondence: yuhai@yzu.edu.cn
Abstract: The objective of this study was to investigate the effect of pork oxidation through modified
atmosphere packaging (MAP) on gel characteristics of myofibrillar proteins (MP) during the heat-
induced gelation process. The pork longissimus thoracis (LT) was treated by MAP at varying oxygen
concentrations (0, 20, 40, 60, and 80% O2 ) with a 5-day storage at 4 ◦ C for the detection of MP oxidation
and gel properties. The findings showed the rise of O2 concentration resulted in a significant increase
of carbonyl content, disulfide bond, and particle size, and a decrease of sulfhydryl content and
MP solubility (p < 0.05). The gel textural properties and water retention ability were significantly
improved in MAP treatments of 0–60% O2 (p < 0.05), but deteriorated at 80% O2 level. As the
concentration of O2 increased, there was a marked decrease in the α-helix content within the gel,
accompanied by a simultaneous increase in β-sheet content (p < 0.05). Additionally, a judicious
oxidation treatment (60% O2 in MAP) proved beneficial for crafting dense and uniform gel networks.
Our data suggest that the oxidation treatment of pork mediated by O2 concentration in MAP is capable
of reinforcing protein hydrophobic interaction and disulfide bond formation, thus contributing to the
construction of superior gel structures and properties.
Keywords: pork paste; modified atmosphere packaging; protein oxidation; textural properties; water
holding capacity
1. Introduction
Modified atmosphere packaging (MAP), also known as protective gas packaging, is a
technology that involves extracting air from a package and injecting a pre-set gas mixture
into the package [1,2]. Due to its ability to slow down natural product deterioration, MAP
finds application in various product types, including meat, fish, fruits, and vegetables, to
achieve preservation effects [3]. Usually, MAP employs a combination of two or three kinds
of gases, typically carbon dioxide (CO2 ), oxygen (O2 ), and nitrogen (N2 ) [4]. In respect
to MAP of raw meat, O2 is able to bind with surface myoglobin, forming oxygenated
myoglobin, which increases the meat redness, and an increase of O2 concentration generally
increases color stability [5]. However, MAP occupies ample space, limits the storage
temperature, and the high content of O2 may lead to lipid and protein oxidation, thereby
impacting meat quality [6].
Recently, the relationships between meat oxidation and the sensorial quality, physico-
chemical properties, and nutritional characteristics in MAP have garnered considerable
https://www.mdpi.com/journal/foods
Foods 2024, 13, 391 2 of 19

attention [7,8]. High-oxygen MAP often consists of 70–80% O2 and 20–30% CO2 for main-
taining a vibrant red color in meat, but it also diminishes the edible quality of meat and af-
fects its tenderness and juiciness [9–12]. Clausen et al. [13] demonstrated how high-oxygen
MAP aggravated the oxidation of lipids and proteins and decreased protein degradation
and meat tenderness of beef. Bao et al. [14] observed that as the O2 concentration increased
(20–80%), protein oxidation levels elevated, subsequently leading to cross-link formation in
structural proteins. Those cross-links, in turn, increased mechanical strength, ultimately
resulting in tougher meat. Lund et al. [15] suggested that oxidation-induced myosin heavy
chain cross-linking through disulfide bonding represents another mechanism contributing
to the O2 -induced toughening of meat. Many studies have thus focused on the influence of
protein oxidation induced by O2 concentration in MAP on fresh meat quality. However, the
effect on meat processing characteristics, especially the gel properties of the myofibrillar
protein (MP), which are crucial functional attributes in the comminuted product matrix,
has received little attention.
The mechanism of thermogelation of MP gel properties of meat has been extensively
studied [16]. The MP undergoes denaturation and unfolding during heating, and the pro-
tein structure unfolds, exposing the functional groups that are originally in the protein [17].
The intermolecular interaction force is strengthened, the proteins aggregate with each other
to form larger polymers, and finally, the proteins are rearranged into a structurally stable,
viscoelastic three-dimensional gel network structure [18,19]. Disulfide bonds, in particular,
play a significant role in protein cross-linking, contributing to enhanced gel hardness and
water retention. Moderate oxidation-induced formation of disulfide bonds has been shown
to be crucial for improving gel quality [20]. Wang et al. [21] investigated the impact of O2
concentration (0, 20, 40, 60, and 80%) in MAP on beef MP gel properties during heating. It
was revealed that the 20% O2 treatment group produced a firmer and more elastic MP gel.
However, the internal mechanism underlying the linkage of protein oxidation induced by
MAP with gel properties warrants further investigation.
Up to the present, research concerning the oxidative modification of protein by O2
concentration in MAP have mostly emphasized clarification to the changes in raw meat
quality. However, there are few studies on the effect of O2 concentration in MAP on the
important functional properties of meat protein during the heat-induced gelation process.
Consequently, this study aims to explore the impact of pork oxidation levels in situ through
MAP with varying O2 concentrations (0, 20, 40, 60, and 80%) on gel characteristics of pork
paste during the heat-induced gelation process. This work will enrich and develop the
intrinsic relationship between the oxidative regulation of meat and protein gelation, thereby
providing a theoretical basis for improving the quality of gel products.

2. Materials and Methods

2.1. Sample Preparation
2.1.1. Raw Materials and Packaging
The pork was obtained from the 6-month-old crossbred pigs (Duroc × Landrace ×
Yorkshire) weighing 100 ± 10 kg (Sushi Meat Co., Ltd., Huai’an, Jiangsu, China). The
slaughtering process was strictly implemented by the regulations of the Operating Proce-
dures of Livestock and Poultry Slaughtering-Pig (GB/T 17236–2019) [22]. A total of 6 pork
longissimus thoracis (LT) muscles from the right side of the pig carcasses were selected
within 24 h post-slaughter. The pH was detected in the range of 5.5 to 5.8.
Visible connective tissue and external fat were meticulously removed from the sur-
face of LT muscles. Subsequently, each muscle was sectioned into 13 chops, measuring
approximately 100 g per chop (8 cm × 5 cm × 3 cm). The pH, weight, and color attributes
of the 0 d sample were immediately measured. Three chops of each LT muscle were taken
as 0 d biochemical meat samples for grinding and quick freezing with liquid nitrogen.
Then, the other 10 chops from each LT sample were assigned to five MAP treatments with
varying O2 concentrations (0, 20, 40, 60 and 80%). Thus, there were a total of 12 chops
from 6 LT muscle (2 chop/LT) set in each MAP treatment. All packages were N2 -balanced
Foods 2024, 13, 391 3 of 19

and contained 20% CO2 to inhibit microbial growth. Packaging was performed on a
packaging machine X-500D (Shanghai Tan Xin Packaging Technology Co., Ltd., Shanghai,
China) with a composite packaging bag (Dong Guang County Zhi Long Plastic Co., Ltd.,
Cangzhou, China) composed of polyamide/polypropylene. The O2 transmission rate was
12.707 cm3 /m2 /h/MPa. After the packaging was completed, the gas atmosphere (% O2 , %
N2 , and % CO2 ) in package was verified by a multi-in-one gas detector (GT-903, Shenzhen
Korno Electronic Technology Co., Ltd., Shenzhen, China), with no significant changes
observed. All packages were placed in a refrigerator (Qingdao Haier Co., Ltd., Qingdao,
China) for 5 days and the temperature was set to 4 ◦ C, which was confirmed by a portable
thermometer (ZG-8010, Ningbo Zhaoji Electrical Appliance Co., Ltd., Ningbo, China). After
5 days, the meat samples were ground and quick frozen with liquid nitrogen. Then, the
samples were stored at −80 ◦ C to enable further preparation of pork paste.

2.1.2. Gel Preparation of Pork Paste

The pork paste was made according to the method of [23] with slight modification.
The LT muscles were minced and then washed twice with deionized water (4 ◦ C, 2 volumes,
w/v), followed by one wash with 0.5% NaCl (4 ◦ C, 2 volumes, w/v). The resulting paste
was then centrifuged at 4000× g for 8 min to remove the supernatant. Subsequently, the
paste was dissolved with 2.5% (w/w) NaCl. Protein concentration was measured by Biuret
method and further adjusted to 100 mg/mL using distilled water. The gel was prepared
by the process of heat-induced pork paste. Before heating, the paste was centrifuged at
500× g for 3 min to eliminate air bubbles. Afterward, the paste was put into a glass vial
(5 cm height × 2.2 cm diameter), covered with aluminum foil, and then heated in an 80 ◦ C
water bath for 30 min. Finally, the gel was promptly chilled in ice water for 30 min and
stored at 4 ◦ C overnight.

2.2. Determination of Gel Quality

2.2.1. Color
The color difference and whiteness of the gel was assessed following the approach
outlined by Jeong et al. and Niu et al. [24,25], respectively. A colorimeter (CR-400, Konica
Minolta, Japan) was used and calibrated with a calibration plate prior to each measurement.
Measurements were taken at three different positions on each gel sample under the con-
ditions of an illuminant D65, a 10◦ standard observer angle and a diameter of 8 mm. The
average values of brightness (L*), redness (a*), and yellowness (b*) were calculated. The
color difference (∆E) and whiteness were determined using Formula (1) and Formula (2),
respectively. r
 2  2
2
∆E = L1 * − L2 * + ( a1 * − a2 * ) + b1 * − b2 * (1)
r
 2 2
2
Whiteness = 100 − 100 −L* + a* + b* (2)

where L1 *, a1 * and b1 * are the values on day 0 sample and L2 *, a2 * and b2 * are the values on
MAP treatment samples.

2.2.2. Texture Profile Analysis

After the gel was recovered to room temperature, textural properties were determined
by a TMS-Pro texture analyzer (Food Technology Corporation, Sterling, VA, USA) [26].
The gel was shaped 20 mm in diameter and 23 mm in height with a mold of steel. The
P/6 cylinder (diameter 12 mm) was selected as the test probe, the compression ratio was
50%, and extrusion took place twice. The test speed was set to 1 mm/s, while the pre-test
and post-test speeds were both 5 mm/s. Based on the obtained force-time curve, the
TPA attributes including hardness, cohesiveness, springiness, gumminess, and chewiness
were recorded.
Foods 2024, 13, 391 4 of 19

2.2.3. Water Holding Capacity

The determination of water holding capacity is based on a previously reported
method [25,27]. The pork paste was weighed before heating and recorded as W0 . Af-
ter cooking, the gel was extracted from the glass tube and gently dried using filter paper
and was weighted as W1 . The gel was then subjected to centrifugation at 10,000× g for
10 min at 4 ◦ C. Afterward, the exudate was poured and wiped dry with absorbent paper
and weighed as W2 . Cooking loss and centrifugation loss were calculated according to
Formula (3) and Formula (4), respectively.

W0 − W1
Cooking loss (%) = × 100% (3)
W0

W1 − W2
Centrifugation loss(%) = × 100% (4)
W1

2.3. Determination of Protein Oxidation of Pork Paste

2.3.1. Protein Surface Hydrophobicity
The determination of surface hydrophobicity was conducted following the method
described by Chelh et al. [28] and slightly modified. A protein suspension with a concen-
tration of 2 mg/mL was prepared, and 200 µL of 1 mg/mL bromophenol blue (BPB) was
added to 1 mL of the protein suspension. After being fully mixed, the suspension was
placed on a shaker and slowly shaken for 10 min. Then, the mixture was centrifuged at
4000× g for 15 min. The absorbance of the supernatant was measured at 532 nm, recorded
as A1 . As for the control, the protein suspension was replaced with 20 mM PBS, and the
absorbance value was recorded as A0 . The calculation formula was as follows:

A0 − A1
BPB (µg) = × 200 µg (5)
A0

2.3.2. Protein Solubility

According to the method developed by Wang et al. [29], 1 mL of 2 mg/mL protein
suspension was placed at 4 ◦ C for 1 h, and then centrifuged at 8000× g for 15 min. The
resulting supernatant was collected to determine protein concentration using the Biuret
method. The protein concentration was denoted as Cb before centrifugation and as Ca after
the centrifugation process. The calculation formula was as follows:

Ca
Protein solubility = ×100% (6)
Cb

2.3.3. Sulfhydryl and Disulfide Bond (S-S) Content

The determination of sulfhydryl groups and S-S groups of protein in pork paste was
slightly modified based on the method outlined by Cui et al. [30]. Briefly, protein aliquots
in buffer A (Tris-Gly) and buffer B (Tris-Gly-8M Urea) were treated with 5,5′ -dithiobis-
(2-nitrobenzoic acid) (DTNB) at 40 ◦ C for 25 min. The absorbance of two samples were
detected and recorded as SA and SB . The free and total sulfhydryl groups were calculated
using Formula (7) and Formula (8), respectively. As for protein S-S content, another aliquot
in buffer C (Tris-Gly-10M Urea) with β-mercaptoethanol (β-ME) was created at 25 ◦ C for
1 h, followed by trichloroacetic acid (TCA) treatment. After centrifugation, the pellet was
re-dissolved in buffer B and DTNB was added. The absorbance was recorded as SC . The
protein disulfide bond was calculated using Formula (9).

nmol
Free sulfhydryl content ( ) =73.53SA × D/C (7)
mg

nmol
Total sulfhydryl content ( ) =73.53SB × D/C (8)
mg
Foods 2024, 13, 391 5 of 19

nmol
S-S content ( ) =73.53SC × D/C − total sulfhydryl content (9)
mg
where D is the dilution coefficient and C (mg/mL) is the protein concentration in the
tested sample.

2.3.4. Dityrosine Content

The content of dityrosine was assayed following the method developed by
Davies et al. [31] with slight adjustments. Protein suspension at 2 mg/mL was mixed
with 20 mM PBS buffer containing 0.6 M of sodium chloride, and the mixture was cen-
trifuged (5000× g, 10 min, 4 ◦ C). The intensity of the fluorescence of the supernatant was
detected with an excitation wavelength of 325 nm and an emission wavelength of 425 nm.
Dityrosine content in the protein was obtained by dividing the fluorescence intensity by
protein concentration.

2.3.5. Carbonyl Content

The protein carbonyl of pork paste was measured by the derivatization of
2,4-dinitrophenylhydrazones (DNPH), which was described by Xu et al. [32]. The ab-
sorbance was recorded at 370 nm and protein content was assessed with the Biuret assay.
The calculation formula is as follows:
 
nmol A
Carbonyl content = 106 × (10)
mg C ×ε × b

where A indicates the absorbance of the sample, b is the optical path (0.54 cm), C is the
protein concentration (mg/mL), and ε is the absorption coefficient 22,000 M−1 cm−1 .

2.3.6. Particle Size

Particle size was determined using a previously established method with minor
adjustments [33]. Proteins were standardized to a concentration of 2 mg/mL with 50 mM
PBS (pH 6.0, 0.6 M NaCl). Subsequently, the particle size distribution, D3,2 (meaning
diameter in surface) and D4,3 (meaning diameter in volume) of pork paste proteins were
assessed by using a Masterizer 3000 laser particle size analyzer (Malvern Instruments,
Malvern, UK). The relative refractive index was set to 1.414, and the absorption rate
was 0.001.

2.3.7. SDS-PAGE
In order to observe the cross-linking of paste protein from pork through MAP treat-
ment, reducing and non-reducing gel electrophoresis were performed by referring to the
method of Grossi et al. [34] with slight adjustments. Protein samples (2 mg/mL) were
combined with an equivalent volume of loading buffer (100 mM Tris, 4% SDS, 20% Glycerol,
1% BPB), with or without 5% β-ME, and subsequently boiled for 5 min at 95 ◦ C. SDS–PAGE
was conducted using a 10% separating gel and a 4% acrylamide stacking gel. Standard pro-
tein (5 µL) was added to each gel as a reference marker, and 10 µg of protein samples were
loaded onto each well. The operating conditions were 90 V for the first 30 min and 120 V
for the latter 60 min. After the electrophoresis was completed, the gel was stained with
0.025% Coomassie brilliant blue G250 for 60 min, followed by destaining with a solution
composed of 10% methanol and 10% acetic acid. Protein profiles were scanned with a Geno
Sens 2100 Gel Imaging System (Qinxiang Scientific Instrument Co., Ltd., Shanghai, China).

2.4. Raman Spectra

The secondary structure of the gel protein was detected by using confocal laser Raman
spectroscopy. The gel was placed onto the sample cell. The detection parameters were set
as follows: the laser wavelength was 532 nm with a power of 20 mW and the number of
scans was 30, each with an exposure time of 15 s. Scanning was performed within the range
of 700 cm−1 to 2000 cm−1 at 25 ◦ C and the resolution was 1 cm−1 . Subsequent to data
Foods 2024, 13, 391 6 of 19

collection, the curve underwent smoothing, and the multi-point baseline was calibrated
using the spectral processing software Labspec version 5.0. The Alix calculation method
was used to analyze the secondary structure of the protein.

2.5. Chemical Forces

The gel underwent treatments with different specific chemicals for their capacity to
cleave distinct bonds. The following solutions were used: 0.6 M NaCl (S1), 0.6 M NaCl
+ 1.5 M urea (S2), 0.6 M NaCl + 8 M urea (S3), and 0.6 M NaCl + 8 M urea + 0.5 M β-ME
(S4). The procedures were conducted by following the description of Wu et al. [35]. S1,
S2, S3, and S4 correspond to ionic bond, hydrogen bond, hydrophobic interaction, and
disulfide bond, respectively, which were expressed as the percentage of soluble protein in
total protein.

2.6. Low-Field NMR

The water distribution of the gel was measured using a NiumagPQ001 low-field
nuclear magnetic resonance coimaging analyzer (Newmai Electronic Technology Co., Ltd.,
Shanghai, China) to determine the transverse relaxation time (T2 ). After the gel (5 g) stored
at 4 ◦ C was recovered to room temperature, it was transferred to the nuclear magnetic tube.
The relevant test parameters were set as follows: the resonance frequency was 40 MHz,
the interval pulses were 100 µs, and there were 15,000 scanning echoes, and 16 repeated
scans. The distribution index fitting analysis of T2 relaxation data was performed using the
Carr-Purcell-Meiboom-Gill (CPMG) sequence, and each sample was repeated six times.

2.7. Scanning Electron Microscopy (SEM)

The sample in the shape of 5 mm × 5 mm × 1 mm was cut from the intact gel and
then fixed with 2.5% glutaraldehyde for 12 h at 4 ◦ C. The fixed samples were rinsed 3 times
using 0.2 M PBS (pH 7.2), then dehydrated with a series of gradient ethanol at one time.
After freeze-drying, the gel was sprayed with gold. Finally, the gel was imaged by a
Gemini 300 SEM (Zeiss, Oberkochen, Germany) at an acceleration voltage of 5 kV and a
magnification of 2000 times [36].

2.8. Statistical Analysis

Analysis of variance (ANOVA) for the obtained data was carried out by using the SAS
statistical software (Version 9.1.3). The difference of means among treatments was evaluated
by Duncan’s multiple range test, and the results were presented as mean ± standard error.
The significance threshold was chosen at p < 0.05. Principal component analysis (PCA) and
Discriminant analysis (OPLS-DA) were performed using SIMCA software (Version 14.1,
Sartorius, Göttingen, Germany).

3. Results
3.1. Color, Texture and Water Holding Capacity
The properties of the gel from minced meat, including color, water holding capacity,
and texture are presented in Table 1. The color characteristics of meat products could affect
the satisfaction of consumers to a large extent, and the gel whiteness and ∆E values were
considered to be a useful indicator for the overall color evaluation [24,37]. As shown in
Table 1, with the increase of O2 concentration (0–80%) in MAP, ∆E, whiteness and L* values
of the gel slightly increased, but the difference was not significant (p > 0.05). The a* and b*
of gel decreased with O2 concentration from 0% to 20%, and then slightly increased in the
latter 40–80% O2 treatment. Lower a* values in MAP groups (0–80%) were observed when
compared to Day 0 samples. This may be due to the fact that the O2 concentration increases
meat oxidation, potentially leading to the unfolding of protein structure, which affects
the interaction between protein and water. The increased water absorption within the gel
network may contribute to a higher light reflection. That was evidenced by the detection
of water holding capacity, among which cooking loss and centrifugation loss of the gel
Foods 2024, 13, 391 7 of 19

significantly decreased within 0–60% of O2 concentration in MAP (p < 0.05). However,

both cooking loss and centrifugation loss in the 80% O2 of MAP group were higher than
that of the 60% O2 group (Table 1, p < 0.05), suggesting reduced water holding capacity
by high O2 MAP treatment. Similarly, Zhang et al. [26] showed that MP oxidation caused
by the increase of hydrogen peroxide (H2 O2 , 0–20 mM) concentration initially increased
gel water holding capacity and then decreased. Moreover, Zhou et al. [38] reported that
the water holding capacity of the gel was enhanced due to the moderate oxidation of the
protein, while the gel quality was reduced due to excessive oxidation. Mild oxidation
can promote cross-linking between proteins and make the gel network denser and more
uniform, thereby reducing the fluidity of water and fixing water in the gel network to
improve the water holding capacity of the gel. However, excessive oxidation leads to
increased pores in the gel and changes in the water states of the gel, leading to the weak
water holding capacity.

Table 1. Color, texture, and water holding capacity (cooking loss and centrifugation loss) of pork
paste gels from minced meat before storage (Day 0) and after being placed in refrigerated storage for
5 days in MAP with different O2 concentrations.

Parameter Day 0 0% 20% 40% 60% 80% p-Value

Brightness (L*) 86.83 ± 0.51 a 86.91 ± 0.41 a 86.69 ± 0.47 a 86.69 ± 0.52 a 88.09 ± 0.40 a 87.57 ± 0.52 a 0.274
Redness (a*) −0.77 ± 0.01 a −0.98 ± 0.01 bc −1.06 ± 0.04 c −0.87 ± 0.03 ab −0.98 ± 0.04 bc −0.86 ± 0.06 a 0.002
Yellowness (b*) 8.19 ± 0.06 a 8.01 ± 0.05 abc 7.68 ± 0.02 c 7.84 ± 0.08 bc 7.78 ± 0.11 bc 8.05 ± 0.18 ab 0.028
Color differences
- 0.60 ± 0.18 a 0.88 ± 0.14 a 0.77 ± 0.24 a 1.24 ± 0.20 a 1.04 ± 0.22 a 0.262
(∆E)
Whiteness 84.47 ± 0.45 a 84.62 ± 0.33 a 84.60 ± 0.42 a 84.52 ± 0.42 a 85.74 ± 0.28 a 85.15 ± 0.35 a 0.204
Hardness (N) 1.71 ± 0.09 e 2.32 ± 0.11 d 3.70 ± 0.10 c 4.50 ± 0.23 b 5.23 ± 0.12 a 4.13 ± 0.20 bc <0.001
Cohesiveness 0.23 ± 0.01 c 0.29 ± 0.01 b 0.35 ± 0.02 a 0.39 ± 0.02 a 0.39 ± 0.04 a 0.38 ± 0.01 a <0.001
Springiness (mm) 3.69 ± 0.16 b 4.92 ± 0.28 a 5.20 ± 0.18 a 5.47 ± 0.14 a 5.46 ± 0.10 a 5.21 ± 0.12 a <0.001
Gumminess 0.50 ± 0.02 d 0.67 ± 0.05 d 1.26 ± 0.04 c 1.77 ± 0.17 ab 2.01 ± 0.05 a 1.57 ± 0.10 b <0.001
Chewiness (mJ) 2.18 ± 0.17 d 3.30 ± 0.34 d 6.58 ± 0.33 c 9.73 ± 1.04 ab 11.02 ± 0.47 a 8.04 ± 0.54 bc <0.001
Cooking loss (%) 36.78 ± 0.11 a 35.89 ± 0.22 a 34.35 ± 0.35 b 34.61 ± 0.40 b 31.83 ± 0.41 c 33.84 ± 0.25 b <0.001
Centrifugation loss
33.76 ± 0.50 a 30.67 ± 0.36 b 29.20 ± 0.36 c 26.77 ± 0.15 d 24.84 ± 0.30 e 28.24 ± 0.25 c <0.001
(%)
Note: Mean values ± standard error. Different lowercase letters (a–e ) in the same line indicated significant
differences among treatment groups at p < 0.05.

Textural properties, including hardness, cohesiveness, springiness, gumminess, and

chewiness are essential for assessing the quality of comminuted meat products. The
increasing values in hardness, cohesiveness, springiness, gumminess, and chewiness of
gel were observed for pork under 0–60% O2 of MAP treatment, showing significantly
higher values than those of Day 0 pork (p < 0.05). However, the textural properties of gel
tended to decline when pork was exposed to 80% O2 of MAP. Similarly, the instron rupture
force of MP gel tended to increase when exposed to oxidizing conditions below 0.5 mM
H2 O2 in an iron-catalyzed oxidizing system, but decreased in oxidizing conditions over
1 mM H2 O2 and in metmyoglobin-oxidizing systems, as demonstrated in the study of
Xiong et al. [39]. Though proteins were oxidized during incubation in different oxidation
systems, intramolecular cross-linking formed to some extent to facilitate gel strength and
texture [40]. Otherwise, under strong oxidizing conditions, excessive protein cross-linking
produces larger protein aggregates, which may hinder ordered interactions of reactive
groups of proteins, while restraining the formation of a compact gel network [41,42].

3.2. Oxidation of the Pork Paste

3.2.1. Protein Surface Hydrophobicity and Solubility
The binding of BPB to protein molecules indicates the hydrophobicity of the protein
surface and the change in protein conformation [43]. As can be seen in Figure 1a, protein
surface hydrophobicity significantly increased over 0–80% O2 in MAP treatment groups,
which presented higher values than those of Day 0 samples. In addition, the solubility of
 3.2. Oxidation of the Pork Paste
3.2.1. Protein Surface Hydrophobicity and Solubility
The binding of BPB to protein molecules indicates the hydrophobicity of the protein
surface and the change in protein conformation [43]. As can be seen in Figure 1a, protein
Foods 2024, 13, 391 8 of 19
surface hydrophobicity significantly increased over 0–80% O2 in MAP treatment groups,
which presented higher values than those of Day 0 samples. In addition, the solubility of
pork protein in MAP gradually decreased with the increase of O2 concentrations (p < 0.05).
pork protein in
The change in MAP gradually decreased
hydrophobicity interactionwith
andthe increase conformation
structural of O2 concentrations (p < 0.05).
of proteins was
The change in hydrophobicity interaction and structural conformation of proteins
caused by the folding, unfolding, polymerization, and aggregation of proteins under the was
caused byexternal
action of the folding, unfolding,
factors polymerization,
[44]. Protein and been
oxidation has aggregation of proteins
demonstrated under the
to change the
action of external factors [44]. Protein oxidation has been demonstrated to change
structure of protein and influence protein solubility, which is in accordance with the cur- the
structure
rent studyof[45,46].
protein and influence protein solubility, which is in accordance with the current
study [45,46].

Figure 1. Surface hydrophobicity and protein solubility (a), active

active sulfhydryl
sulfhydryl and total
total sulfhydryl
sulfhydryl
groups (b), disulfide bonds (c), dityrosine contents (d), carbonyl contents (e), and particle
groups (b), disulfide bonds (c), dityrosine contents (d), carbonyl contents (e), and particle size size
distribution (f) of paste protein from pork before storage (Day 0) and after being placed in refrigerated
storage for 5 days in a modified atmosphere with different O2 concentrations. Note: the bar indicates
standard error of means. Different lowercase letters indicate significant differences among treatment
groups (p < 0.05).
Foods 2024, 13, 391 9 of 19

3.2.2. Sulfhydryl Content and Disulfide Bond

The reduction of the active sulfhydryl and total sulfhydryl groups of protein and the
formation of disulfide bonds were two important characteristics of protein oxidation. With
the increasing O2 concentration in MAP, the active sulfhydryl and total sulfhydryl groups
in pork paste proteins decreased significantly and the content of disulfide bonds gradually
increased (p < 0.05), as shown in Figure 1b and Figure 1c, respectively. This discovery
was consistent with the study of Delles et al. [47], which suggested that O2 induced
a pronounced depletion of active sulfhydryl groups and facilitated the development of
disulfide cross-links. This occurrence can be attributed to the generation of disulfide bridges
at both intra- and inter-molecular levels by the oxidation of protein cysteine residues.
Additionally, oxidation may also cause protein unfolding, leading to the exposure of
internal protein sulfhydryl groups, which can be counteracted by the concurrent formation
of disulfide bonds. Consequently, this dual process resulted in a reduction in the active
sulfhydryl content [48].

3.2.3. Dityrosine, Carbonyl Content, and Particle Size

The protein dityrosine and carbonyl content of pork paste are shown in Figure 1d
and Figure 1e, respectively. With the increase of O2 concentration in MAP, the dityrosine
and carbonyl content significantly increased (p < 0.05). When protein side chains were
exposed to reactive oxygen species (ROS), the generation of tyrosyl radicals occurs. Two
tyrosine radicals, whether situated on the same or different protein chains, readily combine
to produce dityrosine, which was another pattern of protein cross-linking [49]. Protein
carbonyl compounds were reported as the typical products of protein oxidation during
meat storage, which affected protein solubility and showed a negative relationship between
them [50].
The particle size distribution of protein in pork paste was detected and is presented
in Figure 1f. It was observed that the peak of smaller particle size generally increased
from the Day 0 sample to the 0–80% O2 in MAP samples. Notably, a new peak of higher
particles emerged from the 40% O2 in the MAP group, and the particle size became higher
and larger in the 60% and 80% O2 treatment groups. As is shown in Table S1, the D3,2 and
D4,3 were significantly increased with O2 concentration from 0 to 80% in MAP (p < 0.05,
Supplementary Table S1). It is implied that the average particle size of protein increased
with the rise of O2 in the MAP of pork. This result may be due to the formation of disulfide
bonds and macromolecular aggregates between the protein molecules, as evidenced by
Sun et al. [51], finding that protein oxidation is closely related to the changes in protein
particle size.

3.2.4. SDS-PAGE of the Paste Protein

The oxidation profiles of paste protein as affected by different O2 concentrations in
MAP were detected by using reduced gel and non-reduced gel electrophoresis. As seen
in Figure 2, with the increase of O2 concentration in MAP, the band intensity of protein
on the top of the stacking gel strengthened (band 1) and the intensity of protein bands 2,
3, and 4 were visibly weakened. Most changes in protein band intensity in non-reduced
gel (band 1–4, Figure 2a) among treatment groups seemed to disappear in reduced gels
(band 1′ –4′ , Figure 2b), suggesting the oxidation of proteins can be largely recovered by
the reducing agent. It also showed that the oxidation intensity of MP was exacerbated
with the increase of O2 concentration, resulting in the aggravation and cross-linking of MP.
Consistent with our finding (Figure 1), Li et al. [52] also found that protein intensities of
myosin heavy chain and actin were gradually decreased by the treatment of increasing
H2 O2 concentration, especially under the condition of high H2 O2 concentration. Moreover,
the large aggregates were mainly cross-linked by myosin and proteins in MP through
disulfide bonds.
 Foods 2024, 13, x FOR PEER REVIEW 10 of 19

concentration, especially under the condition of high H2O2 concentration. Moreover, the
Foods 2024, 13, 391 large aggregates were mainly cross-linked by myosin and proteins in MP through disul- 10 of 19
fide bonds.

Representative
Figure2.2.Representative
Figure SDS–PAGE
SDS–PAGE patterns
patterns of paste
of paste protein
protein in non-reduced
in non-reduced form ((a)form −β-ME)
((a)and
−β-ME)
and reduced
reduced form
form ((b) ((b) +β-ME)
+β-ME) from
from pork porkstorage
before before (Day
storage (Day
0) and 0) and
after after
being being
placed placed refrigerated
refrigerated stor-
age for 5 for
storage days in a modified
5 days atmosphere
in a modified with different
atmosphere O2 concentrations.
with different O2 concentrations.

3.3.Secondary
3.3. SecondaryStructure
Structure of of
thethe
GelGel
AsAsindicated
indicatedininthe the Raman
Raman spectra
spectra (Figure
(Figure 3a),3a), protein
protein secondary
secondary structures
structures of theofgel
the gel
were analyzed and the variations were found in groups of Day 0 samples and different O2 O2
were analyzed and the variations were found in groups of Day 0 samples and different
concentrationsin
concentrations inMAP.
MAP. Predominantly,
Predominantly, the the information
information on on protein
protein secondary
secondary structure
structure was
derived from the Amide I band (1600–1700 cm −1−1), primarily involving C-O stretching [53].
was derived from the Amide I band (1600–1700 cm ), primarily involving stretching
The The
bands from 1650–1658 cm −1−1 correspond to the α-helix, 1665–1680 cm −1 correspond
[53]. bands from 1650–1658 cm correspond to the α-helix, 1665–1680 cm−1 correspond
toβ-sheet, 1680–1690 −1 correspond to β-turn, and 1660–1665 cm−1 correspond
to β-sheet,1680–1690 cmcm−1 correspond to β-turn, and 1660–1665 cm−1 correspond to ran- to
random
dom coil coil [54,55].
[54,55]. WithWith the increase
the increase of O2ofconcentration
O2 concentration in MAP,
in MAP, the protein
the protein α-helix of gelof gel
α-helix
gradually
graduallydeclined
declined(p (p<<0.05),
0.05),while
whileβ-sheet
β-sheetincreased
increased(p(p<<0.05)
0.05)and
andminor
minorchanges
changesof ofβ-
β-turn
turn
and and random
random coil coil
were were observed
observed (Figure
(Figure 3b).3b). This
This suggests
suggests protein
protein structural
structural conver- of
conversions
sions of gel
gel from thefrom
α-helixthe to
α-helix
otherto other structures,
structures, which canwhich can possibly
possibly be attributed
be attributed to the Oto2 -induced
the
O 2-induced
protein proteinexpediting
oxidation oxidation expediting
the unfolding the unfolding of the
of the α-helix. α-helix.
The The enhancement
enhancement of the β-sheet
of the β-sheet
indicates the indicates
formation theofformation of inter-molecular
inter-molecular hydrogen bondinghydrogenbetween
bondingpeptide
betweenchains
pep- and
tide chains and an ordered rearrangement of protein molecules
an ordered rearrangement of protein molecules [56]. In addition, a high ratio of [56]. In addition, a high
β-sheet
Foods 2024, 13, x FOR PEER REVIEW 11 of 19
ratio
was shown to facilitate the assembly of a dense gel network, resulting in improvedinwater
of β-sheet was shown to facilitate the assembly of a dense gel network, resulting
improved
retention water
capacity retention capacity
[57], which [57], which
is proven is proven
by the presentby the present study.
study.

Figure 3.
3. Raman
Ramanspectra
spectra(a)
(a)and
andsecondary
secondarystructures (b)(b)
structures of pork paste
of pork gelsgels
paste fromfrom
minced meatmeat
minced before
before
storage (Day 0) and after being placed in refrigerated storage for 5 days in a modified atmosphere
storage (Day 0) and after being placed in refrigerated storage for 5 days in a modified atmosphere
with different O2 concentrations. Note: different lowercase letters indicate significant differences
with different O2 concentrations. Note: different lowercase letters indicate significant differences
among treatment groups (p < 0.05).
among treatment groups (p < 0.05).
3.4. Chemical Forces in the Gel
As shown in Figure 4, the intermolecular chemical forces of the gel were determined
among treatment groups. It was found that hydrophobic interaction and disulfide bonds
occupied a higher proportion in gel compared to ionic and hydrogen bonds, suggesting
that hydrophobic interaction and disulfide bonds contributed significantly to the overall
chemical forces in all gels, highlighting their dominant role in stabilizing the gel structure.
 Foods 2024, 13, 391 11 of 19

3.4. Chemical Forces in the Gel

As shown in Figure 4, the intermolecular chemical forces of the gel were determined
among treatment groups. It was found that hydrophobic interaction and disulfide bonds
occupied a higher proportion in gel compared to ionic and hydrogen bonds, suggesting
that hydrophobic interaction and disulfide bonds contributed significantly to the overall
chemical forces in all gels, highlighting their dominant role in stabilizing the gel structure.
These findings are aligned with previous reports [35,58]. In addition, O2 in MAP had
minimal impact on ionic and hydrogen bonds during heat-induced gelation while the
increasing O2 in MAP treatments notably intensified hydrophobic interactions within the
gels. The increase of hydrophobic interactions seemingly resulted from the unfolding
of protein conformation and the breakdown of protein aggregates by the attack of O2
in MAP. Most importantly, the disulfide bond in the gel exhibited a significant rise over
0–60% O2 treatment groups but displayed a declining trend in the 80% O2 treatment
group. Concurrently, the fluctuation in disulfide bonds was similar to the changes in the
gel texture (Table 1). This suggested a close connection between the elevated disulfide
bonds and the observed alterations in gel strength over 0–80% O2 of MAP treatments.
Nevertheless, the trend of disulfide bonds in pork paste protein was inconsistent with
that in gels, in particular in the 80% O2 treatment group, which exhibited higher disulfide
bonds in pork paste but a lower value in gel in comparison to that of the 60% O2 treatment
group. It is known that heating is capable of causing the cleavage of former disulfide bonds,
then protein unfolding and the activation of buried sulfhydryl groups into forming new
Foods 2024, 13, x FOR PEER REVIEW intermolecular disulfide bonds of MPs [59,60]. However, excessive oxidation may12lead of 19to
a decrease in disulfide bonds and cross-linking between proteins during the process of
heat-induced gelation, affecting the structure and texture of the final gel.

Chemicalforces
Figure4.4.Chemical
Figure forcesofofpork
porkpaste
pastegels
gelsfrom
frommeat
meatbefore
beforestorage
storage(Day(Day0)0)and
andafter
afterbeing
beingplaced
placed
ininrefrigerated
refrigeratedstorage
storageforfor5 5days
daysininaamodified
modifiedatmosphere
atmospherewithwithdifferent
differentOO2 2concentrations.
concentrations.Note:
Note:
the
thebar
barindicates
indicatesstandard
standarderror
errorofofmeans.
means.Different
Differentlowercase
lowercaseletters
lettersindicate
indicatesignificant
significantdifferences
differences
among
amongtreatment
treatmentgroups
groups(p(p<<0.05).
0.05).

3.5.Water
3.5. WaterDistribution
Distributionand
andMobility
Mobility
AsAsshown
shownininFigure
Figure5,5,the
theNMR
NMRcurves
curvesforforall
allgels
gelsexhibited
exhibitedthree
threedistinct peaks,TT2121
distinctpeaks,
(0–5ms),
(0–5 T22
ms),T22 (30–200
(30–200 ms),
ms), T23 T
andand 23 (700–2000
(700–2000 ms),ms),
which which signified
signified tightly
tightly bound bound
water,water,
im-
immobilized water, and free water, respectively [61]. In particular, a shorter
mobilized water, and free water, respectively [61]. In particular, a shorter relaxation time relaxation time
(T ) implies reduced water mobility and intensified interaction between
(T2) implies reduced water mobility and intensified interaction between water molecules
2 water molecules
andproteins
and proteinswithin
within the
the gel
gel structure
structure [62].
[62]. As
As presented
presentedininTable
Table2,2,with
withthe O2Oconcentration
the 2 concentra-
from 0% to 60%, T and
tion from 0% to 60%,21T21 and 22 T of gel decreased, from 1.18 ms to 0.79
T22 of gel decreased, from 1.18 ms to 0.79 ms, fromms, from79.25
79.25msms
to 73.07 ms, respectively. Nevertheless, when O concentration reached
to 73.07 ms, respectively. Nevertheless, when O2 2concentration reached 80%, an increase 80%, an increase
T21
ininT21 and
and T22Twas
22 was observed,
observed, suggesting
suggesting the water
the water fluidity
fluidity was raised
was raised and flowed
and flowed out
out more
easily during processing. Moreover, the proportions of peak areas corresponding to the
three states of water were shown as P21, P22, and P23. The immobilized water (P22) occupied
the major constituent in the gel (91–95%), which was followed by free water and bound
water. The P22 significantly increased from 92.50% to 94.82%, with an increase in O2 con-
centration (0–60%). Concurrently, P23 experienced a notable drop from 6.10% to 3.44%.
 Foods 2024, 13, 391 12 of 19

Foods 2024, 13, x FOR PEER REVIEW
more easily during processing. Moreover, the proportions of peak areas corresponding
to the three states of water were shown as P21 , P22 , and P23 . The immobilized water (P22 )
occupied the major constituent in the gel (91–95%), which was followed by free water and
bound water. The P22 significantly increased from 92.50% to 94.82%, with an increase in
O2 concentration (0–60%). Concurrently, P23 experienced a notable drop from 6.10% to
3.44%. This observation suggests that protein oxidation not only hinders water fluidity in
the gel but also promotes the transformation of free water into immobile water, indicating
increased water entrapment within the protein structure, leading to immobilization. These
13 of 19
were also in accordance with the detection of water holding capacity. According to chemical
forces in the gel, changes in inter- and intramolecular bonds within proteins, induced by
oxidation, influenced proton accessibility, thereby causing alterations in the relaxation
velocity of water in close proximity to protein molecules and its retention ability.

Figure 5. Water distribution of pork paste gels from minced meat before storage (Day 0) and
Figureafter bring distribution
5. Water placed in refrigerated storage
of pork paste for from
gels 5 days in a modified
minced atmosphere
meat before with
storage different
(Day 0) andOafter
2
bring concentrations.
placed in refrigerated storage for 5 days in a modified atmosphere with different O2 concen-
trations.
Table 2. Corresponding relaxation time (T2 ) and peak area proportion (P) of pork paste gels from
Tableminced meat before relaxation
2. Corresponding storage (Day 0) and
time (T2)after
andbeing
peak placed in refrigerated
area proportion (P) storage
of porkfor 5 days
paste gelsinfrom
a
modified
minced atmosphere
meat before with
storage different
(Day O2 after
0) and concentrations.
being placed in refrigerated storage for 5 days in a
modified atmosphere with different O2 concentrations.
Parameter Day 0 0% 20% 40% 60% 80% p Value
ParameterT21 (ms)Day 01.18 ± 0.03 b 0%1.30 ± 0.01 a 20% 1.07 ± 0.02 c 40%
0.88 ± 0.01 d 60%
0.79 ± 0.01 e 80%
1.19 ± 0.00 b p<0.001
Value
b b ± c ± c d ± a
T (ms) 79.25 ± 0.40
T21 (ms) 22 1.18 ± 0.03 b 78.23 ± 0.14
1.30 ± 0.01 a 75.76
1.07 ± 0.02 c 0.32 74.61
0.88 ± 0.01 0.75
d 0.7973.07 ± 0.67 1.19 ± 0.00
± 0.01 e 81.38 0.40
b <0.001
<0.001
a a
T23 (ms) 1256b ± 30.5 b 1275 ± 23.2 b 1312 ± c41.4 b 1438 ± 30.9 1420 ± 34.5 1217 ± 24.1a b 0.001
T22 (ms) 79.25 ± 0.40 78.23 ± 0.14 b 75.76 ± 0.32 74.61 ± 0.75 a73.07 ± 0.67 a 81.38 ± 0.40 b
c d <0.001
P21 (%) 1.52 ± 0.02 b 1.29 ± 0.06 c 1.55 ± 0.05 b 1.76 ± 0.04 1.88 ± 0.05 1.43 ± 0.02 <0.001
T23 (ms) P221256
(%) ± 30.5
b 1275
91.30 ± 0.25 d ± 23.2 b 1312
92.50 ± 0.45 c ±93.57
41.4±b0.191438
b ± 30.9
94.15 ± 0.08 ab1420
a ± 34.5
94.82 ± 0.15 a 1217
a ± 24.1
93.50
b
± 0.46 b 0.001
<0.001
a ab c cd d b
P21 (%) P23 1.52
(%) ± 0.02 b 1.29 ± 0.06
6.84 ± 0.33 6.10 ± 0.391.55 ±4.55
c 0.05± 0.24 1.764.29
b ± 0.04
± 0.15 1.883.44
a ± 0.05
± 0.23 1.43
a ±±
5.58 0.02
0.50b <0.001
<0.001
P22 (%) 91.30 ± 0.25 d 92.50 Note:
± 0.45Mean 93.57
c values ±± 0.19
standard 94.15
b ± 0.08 lowercase
error. Different ab 94.82 letters
± 0.15( ) in93.50
a
a–e
± 0.46
the same line indicate <0.001
b significant
differences between treatmentc groups at p < 0.05. cd
P23 (%) 6.84 ± 0.33 a 6.10 ± 0.39 ab 4.55 ± 0.24 4.29 ± 0.15 3.44 ± 0.23 d 5.58 ± 0.50 b <0.001
Note: 3.6.
Mean values ± standard
Ultrastructure of Gel error. Different lowercase letters ( ) in the same line indicate signif-
a–e

icant differences between treatment groups at p < 0.05.

Microstructural analysis was conducted to elucidate variations in the network struc-
ture of the gel subjected to MAP treatment at different O2 concentrations. As presented
3.6. Ultrastructure of Gel
in Figure 6, the control gel (Day 0) appeared to be a coarse three-dimensional gel net-
Microstructural analysisofwas
work. With the increase conducted to elucidate
O2 concentration, a compactvariations in the network
and homogenous struc-
gel network
was
ture of thegenerated, notably
gel subjected in thetreatment
to MAP group of 60% O2 , exhibiting
at different gel characteristics
O2 concentrations. of small, in
As presented
Figure 6, the control gel (Day 0) appeared to be a coarse three-dimensional gel network.
With the increase of O2 concentration, a compact and homogenous gel network was gen-
erated, notably in the group of 60% O2, exhibiting gel characteristics of small, uniform
pores and filamentous structure. However, in the 80% O2 treatment group, the gel struc-
Foods 2024, 13, 391 13 of 19

uniform pores and filamentous structure. However, in the 80% O2 treatment group, the
gel structure was tight and irregular, and large aggregates and pore sizes appeared in the
microstructure. Nyaisaba et al. [63] stated that the microstructure of heat-induced protein
gel was closely associated with the relative velocity of protein unfolding and aggregation.
The oxidation of protein, induced by different O2 concentrations in MAP can affect its gel
Foods 2024, 13, x FOR PEER REVIEW 14 of 19
properties via structural and conformational changes to the ultrastructural formation of gel
during heating.

Figure6.6.Microstructure
Figure Microstructureof of pork
pork paste
paste gelsgels
fromfrom minced
minced meatmeat before
before storage
storage (Day(Day
0) and0)after
and being
after being
placed in refrigerated storage for 5 days in a modified atmosphere with different O2 concentrations.
placed in refrigerated storage for 5 days in a modified atmosphere with different O2 concentrations.

3.7.
3.7.SDS-PAGE
SDS-PAGEof of
GelGel
The
Thenon-participating
non-participating proteins
proteinsin the gelgel
in the formation
formationare presented in Figure
are presented 7. It was
in Figure 7. It was
observed that the thick bands were low molecular weight proteins (<36 kDa).
observed that the thick bands were low molecular weight proteins (<36 kDa). A previous A previous
study
studydemonstrated
demonstrated thatthat
tropomyosin
tropomyosin (a doublet in 34–36
(a doublet in kDa)
34–36 and troponin
kDa) (about 20 (about
and troponin kDa) 20
had a marginal role in gel formation [64]. Consistent with the report
kDa) had a marginal role in gel formation [64]. Consistent with the report of Gao of Gao et al. [65],et al.
myosin heavy chain (MHC) and actin nearly vanished in all gels with reduced form and
[65], myosin heavy chain (MHC) and actin nearly vanished in all gels with reduced form
non-reduced forms, providing additional confirmation of their predominant role in the
and non-reduced forms, providing additional confirmation of their predominant role in
gelation process. With the increase in O2 concentration, the intensity of the bands 2, 3, and
the gelation process. With the increase in O2 concentration, the intensity of the bands 2, 3,
3′ was gradually weakened, but the intensity of the bands 4 and 4′ was strengthened in the
and 3′ was
respective gradually weakened,
non-reduced butand
gel (Figure 7a) the reduced
intensitygel
of (Figure
the bands7b).4In
and 4′ was itstrengthened
addition, should
in the respective non-reduced gel (Figure 7a)′ and reduced
′ gel
be noted that in reduced gels, a tenuous band 1 and 2 vanished with the increase (Figure 7b). In addition,
in O2 it
should be noted
concentration. The that in reduced
findings suggestedgels, a tenuous
that band 1′could
MAP treatment and 2′ vanishedpromote
effectively with the increase
more
in O2 concentration.
proteins to become involvedThe findings suggestedofthat
in the construction gel MAP
network treatment
structure.could effectively pro-
mote more proteins to become involved in the construction of gel network structure.
 the gelation process. With the increase in O2 concentration, the intensity of the bands
and 3′ was gradually weakened, but the intensity of the bands 4 and 4′ was strength
in the respective non-reduced gel (Figure 7a) and reduced gel (Figure 7b). In additio
should be noted that in reduced gels, a tenuous band 1′ and 2′ vanished with the incr
Foods 2024, 13, 391 in O2 concentration. The findings suggested that MAP treatment could 14 of effectively
19
mote more proteins to become involved in the construction of gel network structure

Figure 7. Representative
Figure 7.SDS–PAGE patterns
Representative of non-participating
SDS–PAGE proteins of porkproteins
patterns of non-participating paste gels frompaste gels
of pork
minced meat before storage
minced meat(Day 0)storage
before and after being
(Day placed
0) and afterin refrigerated
being placed instorage for 5 storage
refrigerated days in for 5 day
a modified atmosphere with different O2 concentrations. Samples were prepared in the absence
((a) −β-ME) or presence ((b) +β-ME) of 5% β-ME.

3.8. Dependencies between Protein Oxidation and Gel Properties

The linkage of protein oxidation and gel properties of minced pork in the Day 0 sample
and MAP samples with different O2 concentrations were holistically investigated using
principal component analysis (PCA) and discriminant analysis (OPLS-DA) (Figure 8). PCA
analysis using an unsupervised technique was used to explore the similarities and hidden
patterns between different groups [66]. It could be seen from Figure 8a that the data of
various indicators in different processing groups had good repeatability and could be
clearly distinguished. The data results of different treatment groups were clearly classified
according to the first two principal components (PC1 was 52.8%, PC2 was 16.8%), covering
69.6% of the total data variance. The separable points on the PCA score plot indicate that
there were significant differences in the degree of protein oxidation and gel properties
of the samples after MAP at different O2 concentrations (Figure 8b). In order to better
classify and interpret the protein oxidation degree and gel properties of different O2
concentration MAP treatments, as presented in Figure 8c, OPLS-DA analysis successfully
isolated the significant differences between different O2 concentrations in MAP treatment
groups. The variable importance in projection (VIP) score in Figure 8d assessed the most
important indicators of protein oxidation degree and gel properties affecting different
treatment groups, and identified a total of 11 indicators with significant contributions of
variables (protein solubility, particle size, α-Helix, β-Sheet, dityrosine, active sulfhydryls,
T22 , disulfide bond, chewiness, centrifugation loss, and β-Turn). Therefore, it can be seen
from Figure 8 that oxidation affected the properties of pork paste proteins and gels. There
is a certain internal relationship between protein oxidation and gel properties.
In summary, the mechanism of oxidation-induced changes of gel properties by O2
in MAP is illustrated in Figure 9. It is indicated that as the O2 concentration increases,
the oxidation degree of MP intensifies, leading to a more intricate protein cross-linking.
During the protein gel formation induced by heat treatment, the transition of protein
molecules from their native state to a denatured state encompasses alterations in secondary
and tertiary structure conformations. This process involves various chemical forces such
as ionic bonds, hydrogen bonds, hydrophobic interactions, and disulfide bonds. These
modifications ultimately govern the texture and structure of protein gels. Oxidation modi-
fication of the protein indirectly influences the gel quality. As the O2 concentration rises,
the three-dimensional network structure of the gel becomes more uniform, enhancing both
texture and water retention. However, when the O2 concentration reaches 80%, the protein
 dicate that there were significant differences in the degree of protein oxidation and gel
properties of the samples after MAP at different O2 concentrations (Figure 8b). In order to
better classify and interpret the protein oxidation degree and gel properties of different
O2 concentration MAP treatments, as presented in Figure 8c, OPLS-DA analysis success-
Foods 2024, 13, 391 fully isolated the significant differences between different O2 concentrations in MAP treat- 15 of 19
ment groups. The variable importance in projection (VIP) score in Figure 8d assessed the
most important indicators of protein oxidation degree and gel properties affecting differ-
ent treatment
aggregates groups,
into largerand identified
structures, a total diminishing
thereby of 11 indicators
thewith significant
distinctive contributions
characteristics and
of variables (protein solubility, particle size, α-Helix, β-Sheet, dityrosine, active
microstructure of the gel. Thus, our results show that the mechanism of oxidation-induced sulfhy-
dryls, T22, disulfide
enhancement of gel bond, chewiness,
properties centrifugation
is through loss, and β-Turn).
oxidative modification Therefore,
of protein it canwhich
structure, be
seen from Figure 8 that oxidation affected the properties of pork paste proteins and
aggravates protein cross-linking, but excessive oxidative modification will lead to deterio- gels.
There is
ration ofathe
certain internal relationship
gel, excessive between
denaturation, protein oxidation
and irregular aggregation andofgel properties.
internal molecules.

Foods 2024, 13, x FOR PEER REVIEW 16 of 19

before storage (Day 0) and after being placed in refrigerated storage for 5 days in a modified atmos-
phere with different O2 concentrations.

In summary, the mechanism of oxidation-induced changes of gel properties by O2 in

MAP is illustrated in Figure 9. It is indicated that as the O2 concentration increases, the
oxidation degree of MP intensifies, leading to a more intricate protein cross-linking. Dur-
ing the protein gel formation induced by heat treatment, the transition of protein mole-
cules from their native state to a denatured state encompasses alterations in secondary
and tertiary structure conformations. This process involves various chemical forces such
as ionic bonds, hydrogen bonds, hydrophobic interactions, and disulfide bonds. These
modifications ultimately govern the texture and structure of protein gels. Oxidation mod-
ification of the protein indirectly influences the gel quality. As the O2 concentration rises,
the three-dimensional network structure of the gel becomes more uniform, enhancing
both texture and water retention. However, when the O2 concentration reaches 80%, the
protein aggregates into larger structures, thereby diminishing the distinctive characteris-
tics and microstructure of the gel. Thus, our results show that the mechanism of oxidation-
induced enhancement of gel properties is through oxidative modification of protein struc-
Figure
Figure 8.
8.The
ture, which ThePCA analysis
aggravates
PCA (score
protein
analysis scatter
(score plot (a)
cross-linking,
scatter and(a)
plot loading
but scatter plot
andexcessive
loading (b)) and
plotOPLS-DA
oxidative
scatter and analysis
modification
(b)) will
OPLS-DA
(loading scatter
lead to (loading
analysis plot
deterioration (c) and
scatterof VIP values
the(c)gel,
plot (d))
andexcessive of various
VIP valuesdenaturation,indexes of
(d)) of various andpaste and
irregular
indexes gel from minced
of pasteaggregation pork
and gel fromof inter-
minced
nal molecules.
pork before storage (Day 0) and after being placed in refrigerated storage for 5 days in a modified
atmosphere with different O2 concentrations.

Figure 9. The mechanism diagram of pork paste gels from minced meat before storage (Day 0) and
Figure 9. The mechanism diagram of pork paste gels from minced meat before storage (Day 0) and
after being placed in refrigerated storage for 5 days in a modified atmosphere with different O2
after being placed in refrigerated storage for 5 days in a modified atmosphere with different O2
concentrations.
concentrations.

4. Conclusions
The primary outcome of this study was that moderate oxidation in MAP (~60% O2)
markedly improved the gelling performance of paste protein during thermally-induced
gelation, as corroborated by the enhanced gel texture and water retention capacity. More-
Foods 2024, 13, 391 16 of 19

4. Conclusions
The primary outcome of this study was that moderate oxidation in MAP (~60% O2 )
markedly improved the gelling performance of paste protein during thermally-induced
gelation, as corroborated by the enhanced gel texture and water retention capacity. More-
over, T2 relaxation analysis demonstrated that moderate oxidation can decrease the water
fluidity during the gelation of paste protein and prompted the transformation of free water
into immobilized water. The oxidation enhanced protein-protein interactions and triggered
a more compact and homogenous gel network. Gel properties, chemical forces, and the
ultrastructure of gels were shown to differ significantly between packaging with different
O2 concentrations. In the case of 60% O2 in MAP, the gel had greater textural properties,
stronger elasticity, and a denser structure. Collectively, the present study would be benefi-
cial for the application of MAP as a potential tool to regulate meat proteins with improved
gel performance via oxidative modification.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/foods13030391/s1, Table S1: The average particle size of pork
paste gels from minced meat before storage (Day 0) and after being place in refrigerated storage for
5 days in a modified atmosphere with different O2 concentrations.
Author Contributions: Conceptualization, R.L.; methodology, R.L., W.G. and W.L.; resources, R.L.,
Z.K., Q.W., D.J., X.Z., Q.G., M.W. and H.Y.; data curation, W.G.; writing—original draft preparation,
W.G.; writing—review and editing, R.L. and W.G.; visualization, W.G. and W.L.; funding acquisition,
R.L. and H.Y. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Outstanding Youth Science Fund Project of Natural Science
Foundation of Jiangsu Province (BK20230070), China; Qing Lan Project of Yangzhou University,
China; High-Level Talent Support Program of Yangzhou University, China. National Natural Science
Foundation of China (No. 32172276; No. 32101859), China; Jiangsu Provincial Key Research and
Development Program (BE2022333), China.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Acknowledgments: The technical was supported by Bingbing Shen from Guoke Testing Technology
Co., Ltd., Hefei, China.
Conflicts of Interest: The authors declare no conflict of interest.

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