LWT - Food Science and Technology 146 (2021) 111481

Contents lists available at ScienceDirect

LWT
journal homepage: www.elsevier.com/locate/lwt

Impact of frozen storage duration of raw pork on the formation of advanced

glycation end-products in meatballs
Ligang Yu *, Qian Li , Yong Li , Yukun Yang , Caixia Guo , Meiping Li
School of Life Science, Shanxi University, Taiyuan, 030006, China

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

Keywords: Advanced glycation end-products (AGEs) are harmful compounds produced during food processing. This study
Raw pork investigated the effects of frozen storage duration (− 18 ◦ C) of raw pork on AGEs generation in meatballs.
Frozen storage Meatballs were prepared using frozen raw pork (at 0, 30, 60, 90, and 120 days), and Nε-(carboxymethyl)lysine
Nε-(carboxymethyl)lysine
(CML)/Nε-(carboxyethyl)lysine (CEL) (typical AGEs) levels were determined. With increasing frozen storage
Nε-(carboxyethyl)lysine
Myofibrillar protein
times of raw pork, CML levels in meatballs increased from 13.26 (0 day) to 43.30 mg/kg protein (120 days), and
CEL levels increased from 12.37 (0 day) to 40.50 mg/kg protein (120 days). Furthermore, oxidative damage to
raw pork (carbonyls, total sulfhydryl groups, free amines, surface hydrophobicity, thiobarbituric acid-reactive
substances of residual lipids, and intrinsic tryptophan fluorescence intensity) was assessed during storage pe­
riods. A significant correlation between myofibrillar protein oxidation and CML/CEL levels was observed, sug­
gesting that oxidative damage to raw pork during frozen storage could promote the generation of CML and CEL
in meatballs. Therefore, controlling myofibrillar protein oxidation in raw pork may facilitate reduced AGEs levels
in meatballs.

1. Introduction formation during pork processing, indicating that CML content

Advanced glycation end-products (AGEs), which are reaction prod­
ucts of amino groups with carbonyl compounds, are predominantly
formed at advanced stages of the Maillard reaction (Zhu et al., 2020).
Studies have shown that AGEs are linked closely with some chronic and
neurodegenerative diseases, including diabetes, kidney disease, cardio­
vascular disease, Alzheimer’s disease, Parkinson’s disease, and human
aging (Goldin, Beckman, Schmidt, & Creager, 2006; Grillo & Colom­
batto, 2008). Therefore, the study of AGEs in foods is of current interest.
There are two main sources of AGEs: endogenous and exogenous
AGEs. Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine
(CEL) are often selected as representative AGEs because of their unique
stability (Srey et al., 2010). As a valuable nutrient source, meat is often
selected to study AGEs in meat/meat-related products (Xie et al., 2020).
Several studies have investigated AGEs formation in meat products.
Poojary et al. (2020) reported a method for identification and quanti­
fication of AGEs using liquid chromatography quadrupole-Orbitrap
mass spectrometry, and the result showed that AGEs was successfully
assessed in meat products. Mitra, Lametsch, Greco, and Ruizcarrascal
(2018) compared the effects of different heat treatments on AGEs
increased with increasing cooking temperature. Niu et al. (2017a) sug­
gested that the heat-induced formation of protein-bound CML and CEL
increased with increasing storage times of fish muscle (0 ◦ C). AGEs
levels in cooked meat depend on the type of meat, cooking method, and
final internal temperature (Chen & Smith, 2015). Srey et al. (2010) re­
ported that thiamin exhibited good inhibitory effects toward CML and
CEL generation. These studies focused on quantitative methods, influ­
encing factors and inhibition strategies of AGEs. However, the effects of
frozen storage times of raw pork on AGEs formation remain unclear.
Myofibrillar protein (MP) accounts for approximately 55–60% of
muscle proteins, and plays important roles in raw pork (Goll, Neti,
Mares, & Thompson, 2008). Usually, most of meat products are made
from frozen stored meat. Oxidative damage to lipid and protein will
inevitably occur during frozen storage of meat (Utrera, Morcuende, &
Estevez, 2014), which may affect the formation of AGEs in meat prod­
ucts. Therefore, the objectives of this study were to investigate the
changes in physical and chemical properties of MP during frozen storage
of raw pork and AGEs content in meatballs, and to estimate the corre­
lation between MP oxidation and AGEs levels, thereby providing some
valuable references and guidelines to reduce dietary AGEs intake.

* Corresponding author.
E-mail address: yuligang@sxu.edu.cn (L. Yu).

https://doi.org/10.1016/j.lwt.2021.111481
Received 7 January 2021; Received in revised form 8 April 2021; Accepted 9 April 2021
Available online 12 April 2021
0023-6438/© 2021 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
L. Yu et al.
2. Materials and methods
2.1. Materials and reagents
Raw pork was purchased from Meet All supermarket (Taiyuan,
Shanxi Province, China), and buried in ice transported to the lab.
Longissimus dorsi muscle was excised from raw pork, and individual
muscle samples (approximately 150 g) were vacuum-packaged and
stored at − 18 ◦ C for 0, 30, 60, 90, and 120 days. The proximate
composition of raw pork was 21.08% protein, 7.26% fat, and 70.94%
moisture.
Bovine serum albumin, sodium dodecylsulfate (SDS), guanidine hy­
drochloride, L-leucine, and 1,4-piperazinediethanesulfonic acid (PIPES)
were purchased from Solarbio (Beijing, China). Ethylenebis(oxy­
ethylenenitrilo)tetraacetic acid and 5,5′ -dithiobis-(2-nitrobenzoic acid)
(DTNB) were supplied by Rhawn (Shanghai, China). 2,4,6-Trinitro-ben­
zenesulfonic acid was obtained from Bioteke Corporation (Beijing,
China). Sodium 8-anilino-1-naphthalenesulfonate (ANS) was purchased
from Xianding Biotechnology Co., Ltd. (Shanghai, China). 2,4-Dinitro­
phenylhydrazine (DNPH) and trichloroacetic acid (TCA) were ob­
tained from Guangfu Fine Chemical Institute (Tianjin, China). 2-
Thiobarbituric acid (TBA) was supplied by Macklin Biochemical Co.,
Ltd. (Shanghai, China). CML, carboxy[2H2]methyl-lysine (D4-CML),
CEL, and carboxy[2H2]ethyl-lysine (D4-CEL) were purchased from Santa
Cruz Biotechnology, Inc. (Heidelberg, Germany). Nonafluoropentanoic
acid (NFPA) was supplied by J & K Chemical Ltd. (Shanghai, China).
Methanol and acetonitrile (both HPLC grade) were obtained from
Thermo Fisher Scientific (Fair Lawn, NJ, USA).
2.2. MP preparation
MP was prepared according to Park, Xiong, and Alderton (2007),
with some modifications. Approximately 30 g of raw pork (previously at
− 18 ◦ C) was thawed (4 ◦ C for 3 h) and sliced. The meat was homoge­
nized in a Midea blender for 1 min in 4 vol (w/v) of phosphate buffer
(10 mmol/L sodium phosphate, 0.1 mol/L NaCl, 2 mmol/L MgCl2, and 1
mmol/L EGTA, pH 7.0). The homogenate was centrifuged at 2000×g for
15 min at 4 ◦ C and the supernatant was removed. The precipitate was
washed twice under the same conditions described above, and then
added 4 vol (w/v) of NaCl (0.1 mol/L). The sample (pH 6.25) was mixed
thoroughly, filtered through gauze, and recentrifuged as described. The
supernatant was then removed. The MP prepared was stored at 4 ◦ C and
used within 48 h. Protein concentrations were determined by the Biuret
method (Gornall, Bardawill, & David, 1949).
2.3. Protein carbonyls
MP carbonyl levels were measured according to Liu, Xiong, and
Butterfield (2000), with minor modifications. The MP prepared was
diluted to 20–40 mg/mL in 0.6 mol/L NaCl. From this, 800 μL was added
to 8 mL of DNPH (10 mmol/L) reagent in the dark at 4 ◦ C for 1 h, and
homogenized every 10 min. Then, 8 mL of TCA (20%) was added to
precipitate proteins. Samples were centrifuged at 5000×g for 10 min at
4 ◦ C and the supernatant was removed. 16 mL of mixed solution of
ethanol and ethyl acetate (1:1, v/v) was added, and a glass rod was used
to pound the precipitate. The supernatant was discarded after standing
for 10 min. Then, under the conditions described above, the precipitate
was washed three times and dried. Dried sediments were incubated with
6 mL of guanidine hydrochloride (6 mol/L) in a water bath at 50 ◦ C for
30 min. Absorbance at 370 nm was recorded against a HCl (2 mol/L)
blank. The 22,400 L/mol⋅cm absorption coefficient was used to calculate
carbonyl levels, with results expressed as nmol carbonyl/mg protein.
2.4. Protein sulfhydryl groups
Levels of total sulfhydryl groups in MP were measured according to
2
LWT 146 (2021) 111481
Beveridge, Toma, and Nakai (1974) and Cao, Ai, True, and Xiong
(2018), with slight modifications. The MP prepared was diluted to 2
mg/mL 1 mL of diluted protein, 4 mL of phosphate buffer (8 mol/L urea
and 3% SDS, pH 7.4), and 1 mL of DTNB (10 mmol/L) were added to a
test tube, and mixed thoroughly. Samples were reacted in the dark for
15 min at room temperature. To measure total sulfhydryl groups in MP,
levels were calculated from the peak absorbance (at 412 nm). A phos­
phate buffer (0.1 mol/L) was used as a blank. Results were expressed as
nmol total sulfhydryl group/mg protein.
2.5. Determination of free amine levels
Free amines in MP were measured according to Lertittikul, Benjakul,
and Tanaka (2007), with some modifications. The MP prepared was
diluted to 4 mg/mL in 0.6 mol/L NaCl. 0.2 mL of diluted MP, 2 mL of
SDS (1%), and 1 mL of TNBS (0.01%) were transferred to a fresh tube.
Samples were placed in a water bath (50 ◦ C) to react in dark for 30 min,
after thorough mixing. Then, 2 mL of Na2SO3 (0.1 mol/L) was added.
The absorbance at 420 nm was recorded after the samples cooled to
room temperature, and distilled water was used as a blank. Free amines
were determined using an L-leucine standard curve and results expressed
as nmol free amine/mg protein.
2.6. Determination of surface hydrophobicity (So)
The So of MP was measured according to Cao et al. (2018), with
slight modifications. MP was adjusted to 0.04, 0.08, 0.15, 0.3, and 0.6
mg/mL using NaCl (0.6 mol/L) in phosphate buffer (0.1 mol/L, pH
6.25). MP (4 mL) was incubated with 20 μL of ANS (8 mmol/L) in
phosphate buffer (0.1 mol/L, pH 7.0) for 10 min in the dark at room
temperature. A fluorescence spectrometer (PerkinElmer LS-55) was used
to measure the samples fluorescence intensity (FI). NaCl (0.6 mol/L) in
phosphate buffer (0.1 mol/L, pH 6.25) was used as a blank. Excitation,
emission, and slit width wavelengths were set at 392, 492, and 5 nm,
respectively. The initial slope of the relative FI versus the MP concen­
tration was calculated using linear regression analysis, and designated as
the S0 of MP.
2.7. Determination of residual lipid oxidation in MP
The oxidation of residual lipids was measured based on the thio­
barbituric acid–reactive substances (TBARS) method described by Cao,
True, Chen, and Xiong (2016), with slight modifications. Approximately
2 g of MP was incubated with 3 mL of TBA (1%) and 17 mL of TCA
(2.5%) for 30 min at 95 ◦ C, and cooled to room temperature. Then, 10
mL reaction supernatant and 10 mL of chloroform were transferred to a
centrifuge tube, mixed, and centrifuged at 3622×g for 15 min 6 mL of
the supernatant and 3 mL of petroleum ether were transferred to and
mixed in a new centrifuge tube. Then, the samples were recentrifuged as
described. Samples were measured against a blank containing 2 mL of
PIPES (15 mmol/L), and the absorbance was 532 nm. Results were
expressed as mg malonaldehyde equivalent/kg protein.
2.8. Measurement of intrinsic tryptophan fluorescence
Intrinsic tryptophan fluorescence of MP was measured according to
Cao et al. (2018), with minor modifications. MP was diluted to 0.4
mg/mL using NaCl (0.6 mol/L) in phosphate buffer (0.1 mmol/L, pH
6.25). Intrinsic tryptophan fluorescence of samples was processed on a
fluorescence spectrometer (PerkinElmer LS-55). The excitation wave­
length and slit width were 279 and 5 nm, respectively. Fluorescence
emission spectra were recorded between 305 and 450 nm. Using the
same instrument parameters, the fluorescence emission spectra of the
background were recorded.
L. Yu et al.
2.9. Meatballs preparation
Meatballs were prepared according to Yu, He, Zeng, Zheng, and Chen
(2016), with slight modifications. Briefly, approximately 40 g of ground
meat was shaped into a 3-cm-diameter meatball using specific molds.
Meatballs were transferred to a beaker and placed in a water bath
(90 ◦ C) for 20 min until the internal temperature reached 75 ◦ C. After
cooling to room temperature, meatballs were sealed in a vacuum bag,
placed in a water bath (70 ◦ C) for 30 min, and cooled to 25 ◦ C.
2.10. Sample preparation
Samples for AGEs determination were prepared according to He,
Zeng, Zheng, He, and Chen (2014), with some modifications. Briefly,
200 μL of diluted acid hydrolysate (approximately 200 μg of protein)
was transferred to a centrifuge tube and dried at 50 ◦ C under nitrogen
gas. Then, 2 mL of NFPA (5 mmol/L), 150 μL of D4-CML (0.3 μg/mL),
and 150 μL of D4-CEL (0.3 μg/mL) were added in the centrifuge tube,
and mixed evenly for solid-phase extraction (SPE). Firstly, 3 mL of
methanol was added to the Oasis MCX cartridge (60 mg, 3 cc, 60 μm) to
wash the cartridge. Then, 3 mL of HCl (0.1 mol/L) was added at 2
mL/min. The sample was then added at a flow rate of approximately 0.5
mL/min. After this, 3 mL of HCl (0.1 mol/L) and 3 mL of methanol were
used to wash the cartridge, and 3 mL of 5% ammonia/methanol (v/v)
was then added to elute the sample, at approximately 0.5 mL/min. The
eluent was dried at 45 ◦ C under nitrogen gas, dissolved in ultrapure
water, filtered through a 0.22 μm membrane, and stored at − 18 ◦ C
before LC–MS/MS quantification of CML/CEL.
2.11. LC–MS/MS analysis
CML and CEL levels were determined using a standard addition
method using D4-CML and D4-CEL (Yu, He, et al., 2016). The peak area
ratios of CML/D4-CML and CEL/D4-CEL standards were used to estab­
lish the standard curve, respectively. Recovery studies for CML and CEL
were performed, and each sample was spiked using a specific CML and
CEL standard before SPE cleanup. The sample was analyzed on a Waters
X-Bridge column (2.1 × 100 mm, 3.5 μm). The column oven was set to
35 ◦ C. The mobile phases A and B were acetonitrile (100%) and NFPA (5
mmol/L) in water, respectively. Samples were separated using a
gradient elution of B into A at a flow rate of 0.3 mL/min, as follows:
0–0.1 min, 95% B; 0.1–5 min, 40% B; 5–9 min, 0% B, 9–20 min, 95% B.
The mass spectrometer was operated at the electron spray ionization
positive ion mode and ions were detected using multiple reaction
monitoring. The LC–MS/MS was operated as follows: source tempera­
ture (110 ◦ C), desolvation gas temperature (400 ◦ C), capillary voltage
(3.55 kV), cone voltage (30 V), cone gas flow rate (50 L/h), desolvation
gas flow rate (800 L/h), and collision gas flow rate (0.15 mL/min). Data
acquisition was performed using MassLynx 4.1 software (Waters, Mil­
ford, MA, USA). Calibration curves constructed by the linear regression
of peak area ratios of CML against D4-CML and CEL against D4-CEL,
respectively, were used to calculate CML and CEL levels in samples.
The results were expressed as mg CML/kg protein and mg CEL/kg pro­
tein, respectively. The recoveries of CML and CEL were texted at three
levels (100 ng/mL, 300 ng/mL, and 1000 ng/mL). The recovery ex­
periments were repeated six times for each level. Limit of detection
(LOD) and limit of quantification (LOQ) were calculated by diluting the
standard solutions to a specified signal-to-noise ratio of 3 and 10,
respectively.
2.12. Molecular docking
Molecular docking software Auto Dock (version 4.2) was used to
explain AGEs generation in meatballs. A three-dimensional (3D) struc­
ture of myosin was obtained from the RCSB Protein Databank (ID:
2FXO), deposited by Blankenfeldt, Thomä, Wray, Gautel, and
3
LWT 146 (2021) 111481
Schlichting (2006). AutoDock data were analyzed and visualized using
PyMOL (version 1.7).
2.13. Statistical analysis
A randomized complete block design was adopted. The experiments
were replicated three times, and the data were reported as means ±
standard deviation. Least significant difference tests using one-way an­
alyses of variance were conducted using Statistix software 9.0 to identify
significant differences (P < 0.05). IBM Corporation SPSS Statistics 20
was used for data correlation analysis (IBM Corp., Armonk, NY, USA).
3. Results and discussion
3.1. The performance of analytical method
As shown in Fig. 1, the retention times were as follows: CML, 5.56
min; CEL, 5.84 min; D4-CML, 5.56 min; D4-CEL, 5.84 min. Two product
ions (m/z 130 and m/z 84) for both CML and CEL were detected, which
were consistent with previous studies on determination of CML and CEL
(Zhang, Huang, Xiao, & Mitchell, 2011; Niu et al., 2017b). The method
for determination of CML and CEL was assessed using calibration,
sensitivity, and recovery. The linear ranges were wide enough to
determine CML (16.48–2110 ng/mL) and CEL (16.80–2150 ng/mL) in
samples. The correlation coefficients (R2) of CML and CEL were 0.9999
and 0.9997, respectively. The LOD of CML and CEL was 4.0 ng/mL and
4.3 ng/mL, respectively. The LOQ of CML and CEL was 12.4 ng/mL and
14.3 ng/mL, respectively. The recoveries of CML and CEL were tested at
three levels, and the results were 93%–105% for CML and 87%–95% for
CEL (Table 1). These were in line with those previously reported for
measurement of CML and CEL in food samples (He et al., 2014; Sun
et al., 2021). Therefore, this analytical method is suitable for quantifi­
cation of CML and CEL in meatballs.
3.2. CML and CEL levels in meatballs
CML and CEL levels in meatballs were shown in Fig. 2. When
compared with meatballs using fresh pork (0 day, 13.26 mg/kg protein),
CML levels were significantly (P < 0.05) increased in meatballs made
from raw pork stored for 120 days at − 18 ◦ C (43.30 mg/kg protein). For
CEL, CEL contents in meatballs using stored raw pork (90 days) were
obviously higher than those in meatballs made with fresh raw pork (0
day). As frozen storage times increased (120 days), the highest CEL
levels in meatballs were observed (P < 0.05). This observation was
consistent with previous reports. Niu et al. (2017a) indicated that the
heat-induced (30 min) formation of protein-bound CML increased by
172% and CEL increased by 191% in catfish muscle stored at 0 ◦ C for 21
days, as compared with that in heat-treated freshly catfish muscle.
Compared to fresh raw pork, raw pork stored for 8 days could lead to
82.5% increase of CML and 32.6% increase of CEL in sterile products
(Niu et al., 2018). Yu, Gao, et al. (2016) suggested that stored raw pork
could promote CML and CEL formation in sterilized meat products.
Therefore, frozen-stored raw pork could contribute to CML and CEL
generation in meatballs. During frozen storage of raw pork, protein
denaturation, lipid oxidation, and iron ion catalysis inevitably occur
(Kwak & Lim, 2004; Lund, Heinonen, Baron, & Estévez, 2011; Yu, Chai,
Zeng, He, & Chen, 2018), which may have promoted CML and CEL
accumulation in meatballs.
3.3. Carbonyls, total sulfhydryl groups, free amines, and So
Carbonylation of proteins is recognized as one of the most important
chemical modifications of oxidative stress (Villaverde & Estevez, 2013).
As frozen storage times increased, protein carbonyls increased signifi­
cantly from 0.29 to 0.85 nmol/mg protein (Fig. 3A). This result sug­
gested that oxidation degree of MP increased with the increasing frozen
L. Yu et al. LWT 146 (2021) 111481

Fig. 1. LC–MS/MS chromatograms of meatball samples (fresh pork) spiked with D4-CML and D4-CEL.

storage of raw pork. A similar conclusion was reached in previous study

Table 1
(Soyer, Ozalp, Dalmis, & Bilgin, 2010). Soyer et al. (2010) investigated
Linear range, correlation coefficients, LOD, LOQ, and recovery in LC-MS/MS
effects of freezing temperature and duration on protein oxidation in
analysis.
chicken meat (− 18 ◦ C for 120 days), which indicated that carbonyl
AGEs Linear range R2 LOD LOQ Recovery/% content markedly increased as frozen storage times increased. There­
(ng/mL) (ng/ (ng/
mL) mL)
100 300 1000 fore, the oxidation degree of MP in raw pork increased as the frozen
(ng/ (ng/ (ng/ storage increased.
mL) mL) mL)
Total sulfhydryl groups are recognized as a marker to determine
CML 16.48–2110 0.9999 4.0 12.4 105 93 ± 94 ± protein oxidation (Soyer et al., 2010). As presented in Fig. 3A, total
± 10 17 20 sulfhydryl groups decreased from 63.90 to 55.08 nmol/mg protein. The
CEL 16.80–2150 0.9997 4.3 14.3 91 ± 87 ± 95 ±
5 2 22
storage times (0–60 days) exerted significant (P < 0.05) effects on total
sulfhydryl groups. Zhang, Huang, and Xie (2019) observed that total
sulfhydryl groups could react with reactive oxygen species (ROS) to
form sulfonic acid, disulfide bonds, and common and other oxidation
products in pork during frozen storage, thereby decreasing total sulf­
hydryl groups. A similar trend was reported by previous studies (Soyer
et al., 2010; Xia, Kong, Jiang, Li, & Chen, 2012; Zhang et al., 2019).
Thus, total sulfhydryl groups decreased with increased storage times.
Changes in lysine contents are reflected commonly by measuring free
amines (Li, Xiong, & Chen, 2012). As shown in Fig. 3B, there were no
significant differences in free amines before 30 days of frozen storage. As
storage times increased (30–60 days), free amines markedly decreased
from 99.31 to 72.58 nmol/mg protein (P < 0.05). After 120 days of
frozen storage, free amines decreased to 55.90 nmol/mg protein. Feng
et al. (2016) observed that free amines of amino acid residues could be
deaminated and replaced with a carbonyl moiety, resulting in the loss of
free amines in sausages (day 0). Furthermore, active carbonyl–NH2 in­
teractions could lead to decreased free amino contents (Feng et al.,
2016; Li et al., 2012). Thus, this loss could be explained by protein ag­
gregation and the cross-linking of amino acid residues (Feng et al.,
2016). MP aggregation partially shielded the effects of MP unfolding
during frozen storage (Li et al., 2012). Therefore, MP would be oxidized
Fig. 2. The amounts of CML (black bars) and CEL (gray bars) in meatballs made
during frozen storage of raw pork.
from raw pork stored at − 18 ◦ C for 0, 30, 60, 90 and 120 days. Different letters
So is recognized as a marker to determine conformational changes in
(ab or A-C) above bars indicated significant difference (P < 0.05).
protein structures (Benjakul & Sutthipan, 2009). The fluorescent probe
ANS was used to determine So in MP. As storage times increased (0–30
days), a significant decrease (P < 0.05) was observed in So from 282.01

4
L. Yu et al. LWT 146 (2021) 111481

Fig. 3. Carbonyls (black bars), total sulfhydryl groups (gray bars), free amines (white bars), and So (red bars) of MP in raw pork stored at − 18 ◦ C for 0, 30, 60, 90,
and 120 days. Different letters (a-d or A-D) above bars indicated significant difference (P < 0.05). (For interpretation of the references to colour in this figure legend,
the reader is referred to the Web version of this article.)

to 152.32 (Fig. 3B). Wang et al. (2014) reported that So of gliadin
aqueous solutions of hydrated gliadin decreased after frozen storage.
ANS can be designed to bind well-defined hydrophobic cavities gener­
ated by the grouping of nonpolar residues on protein surfaces, rather
than individual hydrophobic residues randomly exposed on surfaces.
This decrease in So probably originated from protein aggregation, which
resulted from disulfide bond formation and hydrophobic interactions
during frozen storage (Leelapongwattana, Benjakul, Visessanguan, &
Howell, 2005). With extended storage times (30–120 days), a significant
increase (P < 0.05) was observed in So (from 152.32 to 250.64). This
may have been due to the formation of well-defined hydrophobic cav­
ities, and the exposure of grouping of nonpolar residues on protein
surfaces during frozen storage (Benjakul et al., 2009; Leelapongwattana
et al., 2005; Wang et al., 2014). These results discussed here suggested
that the degree of MP oxidation increased with increased frozen storage
times of raw pork.
tryptophan in meat (Zainudin, Poojary, Jongberg, & Lund, 2019). As
frozen storage times increased, an obvious decrease was observed in
intrinsic tryptophan FI (Fig. 4B). This result indicated that MP oxidation
increased with increasing frozen storage times. The present result was
consistent with those of Zainudin et al. (2019), who reported that
intrinsic tryptophan FI decreased significantly in beef stored under a
high-oxygen atmosphere with the extension of times. Tryptophan is
susceptible to oxidizing reagents, such as ROS (singlet oxygen) and
transition metals (iron and copper). Tryptophan may also be involved in
reactions with carbonyl compounds, such as aldehydes or α-keto acids
(Utrera et al., 2014; Xu et al., 2013). Hence, the loss of intrinsic tryp­
tophan may be due to the oxidative degradation of intrinsic tryptophan
during frozen storage of raw pork (Xu et al., 2013).
3.5. Correlation analysis

To further understand the effects of frozen storage times on meat

3.4. Changes of residual lipids and tryptophan fluorescence spectra in MP
MP still contains 0.49% fat (dry basis) from residual phospholipids
(Xiong, Park, & Ooizumi, 2009). The oxidative degree of muscle foods
was measured by TBARS (Cao et al., 2016). As presented in Fig. 4A, the
average TBARS content rose sharply (P < 0.05) from 0.07 ± 0.02 to 0.56
± 0.01 mg/kg protein with extended storage times, indicating that re­
sidual lipids in MP underwent oxidation rapidly. This increasing TBARS
trend agreed with previous studies that lipid oxidation increased in
patties (Hansen, Lauridsen, Skibsted, Moawad, & Andersen, 2004) and
chicken meat (Soyer et al., 2010) during frozen storage.
Tryptophan intrinsic fluorescence is often used to detect the protein
structure (Utrera et al., 2014). Fluorescence spectroscopy is a greatly
sensitive technique used widely for the measuring of intrinsic
quality characteristics, correlation analyses were conducted. Correlation
coefficients and significance levels of indicators were presented in
Table 2. The results showed that frozen storage times of raw pork were a
very significant positively correlated with carbonyls, TBARS, CML, CEL,
and AGEs. However, frozen storage times of raw pork exhibited a very
obvious negatively correlation with total sulfhydryl groups and free
amines (P < 0.01). Protein carbonyls were a marked positively corre­
lated with TBARS, CML, CEL, and AGEs (P < 0.01), but negatively
correlated with total sulfhydryl groups (P < 0.05) and free amine (P <
0.01). Significant positive correlations existed between total sulfhydryl
group levels and free amine contents (P < 0.01). Total sulfhydryl groups
and free amines were obvious negatively correlated with TBARS, CML
and AGEs. It was reasonable that total sulfhydryl groups and free amines
decreased, whereas TBARS, CML and AGEs levels increased with

Fig. 4. TBARS value of residual lipids (A) and fluorescence spectra corresponding to intrinsic tryptophan (B) of MP during frozen storage of raw pork (fresh pork:
solid line; 30 days: dash; 60 days: dot; 90 days: dash dot; 120 days: dash dot). Means with different letters significantly differ (P < 0.05).

5
L. Yu et al. LWT 146 (2021) 111481

Table 2
Pearson’s correlation coefficients among various indexes.
Storage times Carbonyls Total sulfhydryl groups Free amines S0 TBARS CML CEL AGEs

Storage times 1.000

Carbonyls 0.904** 1.000
Total sulfhydryl groups − 0.750** − 0.625* 1.000
Free amines − 0.838** − 0.720** 0.700** 1.000
S0 0.480 0.730 0.133 − 0.125 1.000
TBARS 0.949** 0.816** − 0.651** − 0.764** 0.172 1.000
CML 0.768** 0.838** − 0.581* − 0.582* 0.248 0.724** 1.000
CEL 0.821** 0.910** − 0.484 − 0.796** 0.105 0.757** 0.669** 1.000
AGEs 0.866** 0.952** − 0.588* − 0.741** 0.201 0.808** 0.931** 0.893** 1.000

Superscript asterisks indicate that Pearson’s correlations are significant between the variables: * means P＜0.05, ** means P＜0.01.

increasing frozen storage times of raw pork. TBARS levels in residual
lipids in MP were a very significant positively correlated with CML, CEL,
and AGEs (P < 0.01). Residual lipid oxidation promoted MP oxidation,
which may led to AGEs formation. These results were consistent with
those of Zhu et al. (2020), who reported that protein oxidation exerted
significant effects on CEL formation in four parts of braised chicken.
Therefore, our data indicated that MP in raw pork was gradually
oxidized with increased frozen storage times, which might promote
AGEs generation in meatballs.
3.6. Effects of frozen storage times on carbonyls, total sulfhydryl groups
and free amines in MP, and AGEs in meatballs
Usually, starting from the origin of the biplot, the vectors are formed
by connecting with the measurement index points. The correlation be­
tween two vectors could be evaluated by the cosine of the angle between
them (Rakshit et al., 2012). As presented in Fig. 5, an acute vector angles
was observed between carbonyls and AGEs, which suggested that car­
bonyls was positively correlation with AGEs. There were obtuse angles
between AGEs and total sulfhydryl groups or free amines, which
indicated that total sulfhydryl groups and free amines were negatively
correlation with AGEs. This result was consistent with the correlation
analysis described in Table 2.
Based on the research findings and discussion, a hypothesis was
proposed on the generation of CML and CEL in meatballs. This hy­
pothesis focused on myosin due to its high content in MP (myosin ac­
counts for 55% of total MP) (Ramachandran, Mohan, & Sankar, 2007).
Myosin is a hexamer, comprising two heavy and four light chains
(Blankenfeldt et al., 2006) (Fig. 6A). During storage and processing of
pork, oxidation of myosin and lipid will inevitably occur, which may
induce the unfolding of myosin heavy chain and exposure of amino acid
residues (Li et al., 2012) (Fig. 6B). These exposed amino acid residues
(such as lysine, glycine, or isoleucine) would promote AGEs formation in
meatballs. Therefore, frozen stored pork (more than 90 days) might
cause an increase of AGEs levels in meat products.
4. Conclusions
In summary, the effect of frozen stored pork on AGEs generation in
meatballs was investigated in this work. Protein carbonyls and TBARS

Fig. 5. Vector view of biplot for relationships among indicators (carbonyls, total sulfhydryl groups, free amines of MP, and AGEs content in meatballs).

6
L. Yu et al. LWT 146 (2021) 111481

Fig. 6. Schematic representation of muscle myosin (A) and proposed explanation for AGEs generation in meatballs (B).

value increased, whereas total sulfhydryl groups, free amines, and of the S2 subfragment. Proceedings of the National Academy of Sciences of the United
States of America, 103(47), 17713–17717.
intrinsic tryptophan FI decreased with the increasing frozen storage of
Cao, Y., Ai, N., True, A. D., & Xiong, Y. L. (2018). Effects of (-)-epigallocatechin-3-gallate
raw pork. So decreased initially (0–30 days), but increased as frozen incorporation on the physicochemical and oxidative stability of myofibrillar protein-
storage times progressed. A significant correlation was observed be­ soybean oil emulsions. Food Chemistry, 439–445.
tween MP oxidation and AGEs contents, which indicated frozen stored Cao, Y., True, A. D., Chen, J., & Xiong, Y. L. (2016). The dual role (anti- and pro-oxidant)
of gallic acid in mediating myofibrillar protein gelation and gel in vitro digestion.
pork (more than 90 days) might cause an increase of AGEs levels in Journal of Agricultural and Food Chemistry, 64(15), 3054–3061.
meatballs. Therefore, we recommend to use the raw pork stored within Chen, G., & Smith, J. S. (2015). Determination of advanced glycation endproducts in
90 days (− 18 ◦ C). This study could provide some valuable references cooked meat products. Food Chemistry, 168, 190–195.
Feng, X., Li, C., Jia, X., Guo, Y., Lei, N., Hackman, R. M., et al. (2016). Influence of
and guidelines for consumer dietary habits. sodium nitrite on protein oxidation and nitrosation of sausages subjected to
processing and storage. Meat Science, 116, 260–267.
Goldin, A., Beckman, J. A., Schmidt, A. M., & Creager, M. A. (2006). Advanced glycation
CRediT authorship contribution statement
end products sparking the development of diabetic vascular injury. Circulation, 114
(6), 597–605.
Ligang Yu: Investigation, Writing – original draft, Funding acquisi­ Goll, D. E., Neti, G., Mares, S. W., & Thompson, V. F. (2008). Myofibrillar protein
tion. Qian Li: Investigation, Writing – original draft, Data curation. turnover: The proteasome and the calpains. Journal of Animal Science, 86, E19–E35.
Gornall, A. G., Bardawill, C. J., & David, M. M. (1949). Determination of serum proteins
Yong Li: Writing – original draft. Yukun Yang: Writing – review & by means of the biuret reaction. Journal of Biological Chemistry, 177(2), 751–766.
editing, Funding acquisition. Caixia Guo: Validation. Meiping Li: Grillo, M. A., & Colombatto, S. (2008). Advanced glycation end-products (AGEs):
Conceptualization, Methodology. Involvement in aging and in neurodegenerative diseases. Amino Acids, 35(1), 29–36.
Hansen, E., Lauridsen, L., Skibsted, L. H., Moawad, R. K., & Andersen, M. L. (2004).
Oxidative stability of frozen pork patties: Effect of fluctuating temperature on lipid
oxidation. Meat Science, 68(2), 185–191.
Declaration of competing interest He, J., Zeng, M., Zheng, Z., He, Z., & Chen, J. (2014). Simultaneous determination of Nε-
(carboxymethyl) lysine and Nε-(carboxyethyl) lysine in cereal foods by LC–MS/MS.
The authors declare no conflict of interest for the publication of this European Food Research and Technology, 238(3), 367–374.
Kwak, E. J., & Lim, S. I. (2004). The effect of sugar, amino acid, metal ion, and NaCl on
article. model Maillard reaction under pH control. Amino Acids, 27(1), 85–90.
Leelapongwattana, K., Benjakul, S., Visessanguan, W., & Howell, N. K. (2005).
Physicochemical and biochemical changes during frozen storage of minced flesh of
Acknowledgements lizardfish (Saurida micropectoralis). Food Chemistry, 90(1), 141–150.
Lertittikul, W., Benjakul, S., & Tanaka, M. (2007). Characteristics and antioxidative
This research was supported by the National Natural Science Foun­ activity of Maillard reaction products from a porcine plasma protein-glucose model
system as influenced by pH. Food Chemistry, 100(2), 669–677.
dation of China (Grant No. 31801670).
Liu, G., Xiong, Y. L., & Butterfield, D. A. (2000). Chemical, physical, and gel-forming
properties of oxidized myofibrils and whey- and soy-protein isolates. Journal of Food
Abbreviation Science, 65(5), 811–818.
Li, C., Xiong, Y. L., & Chen, J. (2012). Oxidation-induced unfolding facilitates myosin
cross-linking in myofibrillar protein by microbial transglutaminase. Journal of
ANS Sodium 8-Anilino-1-naphthalenesulfonate Agricultural and Food Chemistry, 60(32), 8020–8027.
AGEs Advanced glycation end-products Lund, M. N., Heinonen, M., Baron, C. P., & Estévez, M. (2011). Protein oxidation in
muscle foods: A review. Molecular Nutrition & Food Research, 55(1), 83–95.
CML Nε-(carboxymethyl)lysine Mitra, B., Lametsch, R., Greco, I., & Ruizcarrascal, J. (2018). Advanced glycation end
CEL Nε-(carboxyethyl)lysine products, protein crosslinks and post translational modifications in pork subjected to
DNPH 2,4-Dinitrophenylhydrazine different heat treatments. Meat Science, 415–424.
Niu, L., Sun, X., Tang, J., Wang, J., Rasco, B. A., Lai, K., et al. (2017a). Free and protein-
D4-CML Carboxy[2H2]methyl-lysine
bound Nε -carboxymethyllysine and Nε -carboxyethyllysine in fish muscle: Biological
D4-CEL Carboxy[2H2]ethyl-lysine variation and effects of heat treatment. Journal of Food Composition and Analysis, 57,
FI Fluorescence intensity 56–63.
MP Myofibrillar protein Niu, L., Sun, X., Tang, J., Wang, J., Rasco, B. A., Lai, K., et al. (2017b). Formation of
advanced glycation end-products in fish muscle during heating: Relationship with
NFPA Nonafluoropentanoic acid fish freshness. Journal of Food Composition and Analysis, 63, 133–138.
PIPES 1,4-Piperazinediethanesulfonic acid Niu, L., Sun, X., Tang, J., Wang, J., Wang, J., Rasco, B. A., et al. (2018). Combination
So Surface hydrophobicity effects of salts and cold storage on the formation of protein-bound Nϵ-
(carboxymethyl)lysine and Nϵ-(carboxyethyl)lysine in raw and subsequently
TBA 2-Thiobarbituric acid commercially sterilized ground pork. Food Chemistry, 264, 455–461.
TBARS Thiobarbituric acid-reactive substances Park, D., Xiong, Y. L., & Alderton, A. L. (2007). Concentration effects of hydroxyl radical
TCA Trichloroacetic acid oxidizing systems on biochemical properties of porcine muscle myofibrillar protein.
Food Chemistry, 101(3), 1239–1246.
Poojary, M. M., Zhang, W., Greco, I., De Gobba, C., Olsen, K., & Lund, M. N. (2020).
References Liquid chromatography quadrupole-Orbitrap mass spectrometry for the
simultaneous analysis of advanced glycation end products and protein-derived cross-
Benjakul, S., & Sutthipan, N. (2009). Muscle changes in hard and soft shell crabs during links in food and biological matrices. Journal of Chromatography A, 1615, 460767.
frozen storage. LWT - Food Science and Technology, 42(3), 723–729. Rakshit, S., Ganapathy, K. N., Gomashe, S. S., Rathore, A., Ghorade, R. B.,
Beveridge, T., Toma, S. J., & Nakai, S. (1974). Determination of SH- and SS- groups in Kumar, M. V. N., et al. (2012). GGE biplot analysis to evaluate genotype,
some food proteins using Ellman’s reagent. Journal of Food Science, 39(1), 49–51. environment and their interactions in sorghum multi-location data. Euphytica, 185
Blankenfeldt, W., Thomä, N. H., Wray, J., Gautel, M., & Schlichting, I. (2006). Crystal (3), 465–479.
structures of human cardiac β-myosin II S2-Δ provide insight into the functional role

7
L. Yu et al.
Ramachandran, D., Mohan, M., & Sankar, T. V. (2007). Physicochemical characteristics
of muscle proteins from barracuda (Sphyraena jello) of different weight groups. LWT
- Food Science and Technology, 40(8), 1418–1426.
Soyer, A., Ozalp, B., Dalmis, U., & Bilgin, V. (2010). Effects of freezing temperature and
duration of frozen storage on lipid and protein oxidation in chicken meat. Food
Chemistry, 120(4), 1025–1030.
Srey, C., Hull, G. L., Connolly, L., Elliott, C. T., Del Castillon, M. D., & Ames, J. M. (2010).
Effect of inhibitor compounds on nε-(carboxymethyl)lysine (CML) and nε-
(carboxyethyl)lysine (CEL) formation in model foods. Journal of Agricultural and Food
Chemistry, 58(22), 12036–12041.
Sun, X., Li, X., Tang, J., Lai, K., Rasco, B. A., & Huang, Y. (2021). Formation of protein-
bound Nε-carboxymethyllysine and Nε-carboxyethyllysine in ground pork during
commercial sterilization as affected by the type and concentration of sugars. Food
Chemistry, 336, 127706.
Utrera, M., Morcuende, D., & Estevez, M. (2014). Fat content has a significant impact on
protein oxidation occurred during frozen storage of beef patties. LWT - Food Science
and Technology, 56(1), 62–68.
Villaverde, A., & Estevez, M. (2013). Carbonylation of myofibrillar proteins through the
maillard pathway: Effect of reducing sugars and reaction temperature. Journal of
Agricultural and Food Chemistry, 61(12), 3140–3147.
Wang, P., Tao, H., Wu, F., Yang, N., Chen, F., Jin, Z., et al. (2014). Effect of frozen storage
on the foaming properties of wheat gliadin. Food Chemistry, 164, 44–49.
Xia, X. F., Kong, B. H., Jiang, L. Z., Li, P. J., & Chen, Q. (2012). Changes in structural
properties of porcine myofibrillar protein induced by frozen storage. Advanced
Materials Research, 554–556, 1395–1399.
Xie, D., Zhuo, L., Xie, P., Liu, Y., Feng, B., & Wu, P. (2020). Spatiotemporal variations and
developments of water footprints of pig feeding and pork production in China (2004-
2013). Agriculture, Ecosystems & Environment, 297, 106932.
LWT 146 (2021) 111481
Xiong, Y. L., Park, D., & Ooizumi, T. (2009). Variation in the cross-linking pattern of
porcine myofibrillar protein exposed to three oxidative environments. Journal of
Agricultural and Food Chemistry, 57(1), 153–159.
Xu, D., Wang, X., Jiang, J., Yuan, F., Decker, E. A., & Gao, Y. (2013). Influence of pH,
EDTA, α-tocopherol, and WPI oxidation on the degradation of β-carotene in WPI-
stabilized oil-in-water emulsions. LWT - Food Science and Technology, 54(1),
236–241.
Yu, L., Chai, M., Zeng, M., He, Z., & Chen, J. (2018). Effect of lipid oxidation on the
formation of Nε-carboxymethyl-lysine and Nε-carboxyethyl-lysine in Chinese-style
sausage during storage. Food Chemistry, 269, 466–472.
Yu, L., Gao, C., Zeng, M., He, Z., Wang, L., Zhang, S., et al. (2016a). Effects of raw meat
and process procedure on Nε-carboxymethyl-lysine and Nε-carboxyethyl-lysine
formation in meat products. Food Science and Biotechnology, 25(4), 1163–1168.
Yu, L., He, Z., Zeng, M., Zheng, Z., & Chen, J. (2016). Effect of irradiation on Nε-
carboxymethyl-lysine and Nε-carboxyethyl-lysine formation in cooked meat products
during storage. Radiation Physics and Chemistry, 120, 73–80.
Zainudin, M. A., Poojary, M. M., Jongberg, S., & Lund, M. N. (2019). Light exposure
accelerates oxidative protein polymerization in beef stored in high oxygen
atmosphere. Food Chemistry, 299(30), 125132.
Zhang, G., Huang, G., Xiao, L., & Mitchell, A. E. (2011). Determination of advanced
glycation endproducts by LC-MS/MS in raw and roasted almonds (Prunus dulcis).
Journal of Agricultural and Food Chemistry, 59(22), 12037–12046.
Zhang, X., Huang, W., & Xie, J. (2019). Effect of different packaging methods on protein
oxidation and degradation of grouper (Epinephelus coioides) during refrigerated
storage. Foods, 8, 325.
Zhu, Z., Fang, R., Cheng, Y., Khan, I. A., Huang, J., Li, B., et al. (2020). Content of free
and protein-binding Nε-carboxymethyllysine and Nε-carboxyethyllysine in different
parts of braised chicken. Food Sciences and Nutrition, 8(2), 767–776.