Food Chemistry 376 (2022) 131881

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Effect of frozen storage on the lipid oxidation, protein oxidation, and flavor
profile of marinated raw beef meat
Sam Al-Dalali a, b, c, Cong Li a, b, Baocai Xu a, b, *
a
School of Food and Biological Engineering, Hefei University of Technology, Hefei 230601, China
b
China Light Industry Key Laboratory of Meat Microbial Control and Utilization, Hefei University of Technology, Hefei 230009, China
c
Department of Food Science and Technology, Faculty of Agriculture and Food Science, Ibb University, Ibb 70270, Yemen

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

Keywords: This study aimed to evaluate the effects of long-term frozen storage on the lipid oxidation, protein oxidation, and
Frozen storage flavor profile of marinated raw beef meat. Twenty-eight volatiles were identified in all the samples during
Raw beef meat different times of frozen storage using HS-SPME-GC–MS. Frozen storage affected the contents of flavor com­
Marination
pounds, in which their concentrations fluctuated along with the frozen storage. Partial least squares-discriminant
Lipid oxidation
Flavor profile
analysis screened six flavors as markers, indicating the effect of frozen storage in all the beef samples. They
GC–MS included octanal, 2-ethyl-1-hexanol, benzeneacetaldehyde, 1-heptanol, isoeugenol, and hexanal. Most of the
E-nose screened markers belonged to aldehydes and alcohols, indicating that these components were derived from lipid
oxidation. Thiobarbituric acid reactive substances significantly increased in the first two months of frozen
storage and then decreased slightly. Carbonyl content was increased linearly in all the samples during frozen
storage.

1. Introduction Antequera, 2010). Meat components interact during frozen storage

Beef is a vital global product, and thus the beef industry must ensure
the safe delivery of high-quality beef to frequently distant end-users.
Chilled and frozen storage have been used for this purpose and have
shown to be effective, as seen by their widespread use and scientific
confirmation (Holman, Coombs, Morris, Kerr, & Hopkins, 2017).
Freezing is one of the most significant strategies for preserving food
quality during long-term storage (Culleré, Ferreira, Venturini, Marco, &
Blanco, 2013), and minimizes quality loss (Sebranek, 1982). Frozen
storage is used to slow down unwanted metabolic processes in meat,
although the formation of ice crystals causes cell rupture and muscle
fiber damage (Sebranek, 1982). Frozen storage may alter physical
properties, such as texture and drip loss; chemical processes, such as
protein aggregation, denaturation, oxidation, color changes, lipolysis,
and lipid oxidation; and sensory properties of meat, including flavor and
sensory characteristics (Pérez-Palacios, Ruiz, Martín, Grau, &
because of the high amounts of oxidation catalysts (such as iron and
myoglobin) and lipids in meats and are thus vulnerable to oxidative
processes (Asghar, Gray, Buckley, Pearson, & Booren, 1988). The main
cause of degradation in stored muscle meats is lipid oxidation. The
critical component involved in loss of meat quality are oxidative re­
actions, which affect flavor, nutritional value, color, and texture. Lipid
oxidation is caused by the production of malondialdehyde (MDA) and
cholesterol oxidation products because of the creation of oxy free and
lipid-free radicals (Soyer, Özalp, Dalmış, & Bilgin, 2010). Proteins,
which are abundant in muscle cells, can be affected by oxidative pro­
cesses. Protein oxidation causes various changes in proteins, including
amino acid, protein-polymer formation, loss of solubility, and increase
in carbonyl groups (Soyer et al., 2010). Protein free radicals are form
when the side chains of proteins react with free radicals, which then
react with oxygen to generate peroxy radicals, decompose, and generate
carbonyl derivatives (Soyer et al., 2010). Such breakdown results in

Abbreviations: DNPH, 2,4-dinitrophenyl hydrazine; DVB-CAR-PDMS, divinylbenzene-carboxen-polydimethylsiloxane; E-nose, Electronic nose; FAME, fatty acid
methyl esters; GC-O-MS, gas chromatography-olfactometry-mass spectrometry; HS-SPME, headspace-solid phase microextraction; MDA, malondialdehyde; MS, mass
spectra; MUFA, monounsaturated fatty acids; PCA, principal component analysis; PLS-DA, partial least squares discriminant analysis; PUFA, polyunsaturated fatty
acids; RI, retention indices; SFA, saturated fatty acids; TBARS, thiobarbituric acid reactive substances; TBA, 2-thiobarbituric acid; TCA, trichloroacetic acid; VIP,
variable importance in projection.
* Corresponding author at: School of Food and Biological Engineering, Hefei University of Technology, Hefei 230601, China.
E-mail address: baocaixu@163.com (B. Xu).

https://doi.org/10.1016/j.foodchem.2021.131881
Received 27 June 2021; Received in revised form 14 December 2021; Accepted 14 December 2021
Available online 17 December 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.
S. Al-Dalali et al.
discoloration, loss of water-binding ability, increased roughness, and
exudate loss (Coombs, Holman, Friend, & Hopkins, 2017).
The effects of frozen storage on the volatile content in various meat
products have been explored. Soyer et al. (2010) evaluated the effects of
frozen temperature and storage time on protein and lipid oxidation in
chicken leg and breast meat. Frozen storage time substantially influ­
enced lipid oxidation. Increase in carbonyl groups and reduction in total
sulfhydryl groups in leg meat was observed after protein oxidation.
Özer, & Seçen (2018) reported that the thiobarbituric acid reactive
substances (TBARS) and carbonyl values of raw and cooked beef burgers
steadily rose of up to 3 months of frozen storage. Iglesias et al. (2009)
identified some potential markers for distinguishing between fresh fish
and fish subjected to freezing and thawing, such as 1-octen-3-ol or 1-
penten-3-ol. Vercellotti et al. (1988) proposed hexanal as a biomarker
for lipid oxidation in meats. (E,E)-2,4-Nonadienal, 1-octen-3-one,
hexanal, and trans-4,5-epoxy-(E)-2-decenal were identified as warmed-
over flavor markers for boiled beef (Konopka, Guth, & Grosch, 1995).
Qi et al. (2021) evaluated the effects of different frozen storage periods
(0–8 weeks) on the flavor of raw chicken meat. They found that frozen
storage time of raw meat enhances its flavor profile upon stewing,
indicating that short-term frozen storage of raw meat improves its flavor
profile. Sun, Zhang, & Song (2020) evaluated changes in flavor com­
pounds in cooked beef meatballs during frozen storage (up to 90 days)
using solid-phase microextraction (SPME) and solvent-assisted flavor
evaporation (SAFE) combined with gas chromatography–olfactometry-
mass spectrometry (GC-O-MS). Their sensory evaluation results showed
that general acceptance decreases with frozen storage. In addition, eight
compounds were identified as aroma-active in cooked beef meatballs,
including 1-octene-3-ol, diallyl disulfide, linalool, eugenol, 2-ethyl hexyl
acetate, α-pinene, hexanal, and anisole. However, most previous studies
about frozen storage of meat and meat products used different meat
species, unmarinated raw beef or cooked beef. Furthermore, no previous
studies investigated the effects of frozen storage on the flavor profile of
raw beef meat marinated in salt and sugar combinations. Therefore, this
study aimed to evaluate the effects of long-term frozen storage on the
protein and lipid oxidation and flavor profile of marinated raw beef
meat.
2. Material and methods
2.1. Samples
The beef meat used in this investigation was a single round primal
(M. semimembranosus, approximately 12–15 kg), from male Chinese
crossbred Xia-Nan cattle (24–36 months old; 7 days post-mortem; 19.89
% protein and 3.37 % fat). The primal was obtained from Metro Mall
(Hefei, Anhui, China), packaged, and transferred to the laboratory in an
icebox, where it was kept at 4 ◦ C for further processing.
2.2. Chemicals and reagents
Chemicals used in this study were octanal (99%), nonanal (96%),
trans-2-octen-1-ol (95%), and 1-heptanol (99%) (Shanghai Macklin
Biochemical Co. Ltd., Shanghai, China); 1-octen-3-ol (98%), and fur­
aneol, (Shanghai Aladdin Bio-chem Technology Co., Ltd., Shanghai,
China); ethylenediamine tetraacetic acid disodium salt dehydrate
(EDTA, 99.5%; Sisco Research Laboratories Pvt. Ltd. India); benzalde­
hyde (99%), 1-hexanol (99%), 2-ethyl-1-hexanol (99.6%), 2(5H)-fur­
anone (98%), hexanoic acid (99%), 2-thiobarbituric acid (98%), n-
alkanes (C7-C30), 1-octanol (99%), absolute ethanol, ethyl acetate, tri­
chloroacetic acid (TCA, 99%), and 2,4-dinitrophenyl hydrazine (DNPH)
(Sigma-Aldrich Co. Ltd., Shanghai, China); phenylethyl alcohol (98%)
(TCI Co., Ltd., Shanghai, China); HCl (36.5%-38%), and guanidine hy­
drochloride (99%; Thermo Fisher Chemical™, Shanghai, China).
2
Food Chemistry 376 (2022) 131881
2.3. Preparation of samples
After removing the visible connective tissue and external fat, the beef
primal was cut into 108 steaks (8 cm × 4 cm × 2 cm; 70.00 ± 2.00 g).
The 108 steaks were comprised of 3 marinade solution treatments × 3
tumbling replicates × 4 frozen storage periods × 3 technical replicates.
The steaks were then marinated in three different marinade solutions,
which were: (1) 25% cold water as a control (BS1, n = 36), (2) water
with 2% salt (BS2, n = 36), and (3) water with 2% salt and 0.5% sugar
(BS3, n = 36).
2.4. Tumbling process and vacuum packing procedures
Raw beef steaks were blended with the individual marinade solution
inside tumbling bowl in triplicate for each treatment (n = 12). A vacuum
tumbler (VT50, Suhner Holding AG, Herisau, Switzerland) was used for
2 h at 4 ◦ C. The rotating circle was programmed to turn on for 20 min
and then turn off for 10 min. A DZ-400/2S-vacuum packer (Shandong
Xiaokang Co., Ltd, Shandong, China) was used under atmospheric
pressure to pack each marinated beef steak in a polyethylene bag. All the
procedures were carried out in a 4 ◦ C chilled environment.
2.5. Freezing and sampling procedure
After the packaging procedure, packed steaks were stored in a freezer
at − 18 ◦ C. Samples were collected at different frozen storage periods (3
replicates × 3 marinade treatments × 3 tumbling replicates) of 0, 2, 4,
and 6 months, for further analysis.
2.6. Physicochemical measurements
2.6.1. Color measurement
A CR-400 colorimeter (2-degree observer and illuminant D65; Kon­
ica Minolta, Tokyo, Japan) was used in assessing lightness (L*), redness
(a*), and yellowness (b*) at five separate points on the surface of each
beef steak. The colorimeter was calibrated before measurement, using
the standard white plate (Y117 = 86.3, x = 0.3165, and y = 0.3242). All
the CIE values were determined on five separate points on three sub-
samples sectioned from the 108 steaks (3 marinade solution treat­
ments × 3 tumbling replicates × 4 frozen storage periods × 3 technical
replicates), totalling 324 sub-samples and 1620 color evaluations.
2.6.2. Ultimate pH determination
The ultimate pH of the beef samples was determined immediately (0
months) or from samples frozen for 6 months using a pH meter (MP512-
03, Hangzhou Tuqi Instrument Co., Ltd, China) after preparing a mixture
of 5 g sample and 45 mL of distilled water (homogenized Ultra-Turrax
T25, IKA-Labortehnik, Staufen, Germany) for 30 s at 3000 rpm. The
pH meter was calibrated at pH 4.0 and pH 6.8 (at 20 ◦ C) before the
measurement of the samples according to the method described by
Holman et al. (2017). Ultimate pH values were determined using three 5
g sub-samples sectioned from 54 steaks (3 marinade solution treatments
× 3 tumbling replicates × 2 frozen storage periods × 3 technical repli­
cates), totalling 162 sub-samples.
2.6.3. Lipid oxidation
The lipid oxidation of marinated raw beef meat was determined at
different times of frozen storage by measuring TBARSs according to the
method described by Maraschiello, Sárraga, & García (1999) with some
modifications. Briefly, 5 g of beef meat was soaked in ultrapure water
(20 mL) and homogenized by Ultra-Turrax T25 (IKA-Labortechnik,
Staufen, Germany) for 10 s at 13500 rpm. The homogenate was then
added to a cold 7.5% trichloroacetic acid (TCA; 50 mL) containing 1 g of
EDTA, which was moderately stirred at 50 ◦ C for 30 min. Then, the
mixture was filtered, and the supernatant was collected. 5 mL of su­
pernatant was mixed with 5 mL of 2-thiobarbituric acid (TBA, 0.02 M).
S. Al-Dalali et al.
In a 90 ◦ C water bath, the screw-capped tube was incubated for 30 min.
The TBARSs were measured in triplicate using a micro-plate reader
(Shimadzu, Japan) at 532 nm against a blank. Calibration curves were
measured at the same wavelength as that for the samples. A series of
concentrations from 1,1,3,3-tetraethoxypropane were prepared and
taken through the TBA procedure explained before for the samples. The
results were reported in µg MDA/kg sample. All the TBARS values were
determined in triplicate using three 5 g sub-samples sectioned from the
108 steaks (3 marinade solution treatments × 3 tumbling replicates × 4
frozen storage periods × 3 technical replicates), totalling 324 sub-
samples and 972 TBARS evaluations.
2.6.4. Protein oxidation
Protein oxidation of marinated raw beef meat during different times
of frozen storage was determined by measuring carbonyl contents after
derivatization with DNPH according to the method described by Berardo
et al. (2016). In brief, the protein was precipitated by mixing 3 g of beef
meat with 30 mL of phosphate buffer (20 mM, pH 6.5 containing 0.6 M
NaCl), and then four aliquots of 0.2 mL were treated with 1 mL of TCA
(10%). The supernatant was removed after centrifugation, and two ali­
quots were mixed with 0.5 mL of DNPH (10 mM) dissolved in HCl (2.0
M), and the other two aliquots were treated with 0.5 mL of HCl (2.0 M)
as blank. Then, 0.5 mL of TCA (20%) was added after 1 h of reaction at
37 ◦ C in the dark. After the centrifugation of the samples, the superna­
tants were discarded. Excess DNPH was washed three times with 1 mL of
a solution containing absolute ethanol and ethyl acetate (1:1, v/v).
Finally, 1 mL of guanidine hydrochloride (6.0 M) in phosphate buffer
(20 mM) was added to dissolve the pellets. Carbonyl content (nmol/mg
protein) was calculated according to the absorbance (determined in
triplicate) with a micro-plate reader (Shimadzu, Japan) at 280 and 370
nm as follows:
A370
Carbonylcontent(nmol/mgprotein)= ×106
εhydrazone370 ×(A280 − A370 ×0.43)
where εhydrazone370 = 22,000 M− 1⋅cm− 1. Carbonyl content were
determined in triplicate using three 3 g sub-samples sectioned from the
108 steaks (3 marinade solution treatments × 3 tumbling replicates × 4
frozen storage periods × 3 technical replicates), totalling 324 sub-
samples and 972 carbonyl content evaluations.
2.6.5. Fatty acids composition analysis
The fatty acid composition of beef samples was assessed according to
a previously described method (Gonzales-Barron et al., 2021) with
minor changes. Lipid was extracted from 5 g of beef samples with a
chloroform/methanol mixture (2:1; v/v) and ultrasonic shaking for 20
min. After the extraction, the mixture was allowed to stand for 2 h and
then filtered with a Whatman filter paper (no. 1). At 4 ◦ C and 10000 g,
the supernatant was centrifuged for 15 min. A constant flow of nitrogen
was used to evaporate the solvent. Then, 0.1 g of extracted lipids was
saponified at 60 ◦ C for 30 min with 100 µL of KOH (2 M) in methanol,
and 3 mL of n-hexane was added. After the mixture was agitated for 1
min, 2 g of anhydrous sodium sulfate was added and centrifuged at 1008
g for 5 min. The resulting fatty acid methyl esters (FAMEs) were
collected, filtered with a 0.22 µm filter membrane, and analyzed using
gas chromatography (GC, 7890A, Agilent Technologies) equipped with a
flame ionization detector. A DB-Wax column (30 m × 0.25 mm × 0.25
μm, Agilent, USA) was used to separate the FAMEs. The oven tempera­
ture was set at 50 ◦ C for 1 min, raised to 200 ◦ C at 25 ◦ C/min, ramped to
230 ◦ C at 3 ◦ C/min, and maintained for 18 min. The injection volume
was 1 µL in split mode at a split ratio of 1:50 and a 1 mL/min carrier gas
(He). The FAMEs were identified by comparing their retention times
with those of authentic standards. The FAMEs were quantified according
to the percentage of fatty acid peak area. FAMEs were quantified using
three 5 g sub-samples sectioned from 54 steaks (3 marinade solution
treatments × 3 tumbling replicates × 2 frozen storage periods × 3
3
Food Chemistry 376 (2022) 131881
technical replicates), totalling 162 sub-samples.
2.7. Analysis of volatile flavor profile
2.7.1. Extraction of volatile flavor compounds
Volatiles were extracted from beef samples at different periods of
frozen storage using headspace-solid phase microextraction (HS-SPME)
according to the literatures (Sun et al., 2020; Al-Dalali, Li, & Xu, 2021).
In a 40 mL headspace vial, 5 g of sample was weighed, and 10 µL of 2-
methyl-3-heptanone (0.42 mg/mL) was added as an internal standard.
The bottle was carefully covered with a silicon septum. After equili­
bration in a water bath for 20 min at 60 ◦ C, volatiles were extracted for
40 min under the same temperature by divinylbenzene-carboxen-
polydimethylsiloxane (DVB-CAR-PDMS; Supelco, PA, USA). At the
splitless mode, volatiles were thermally desorbed for 5 min at 250 ◦ C in
the injection port of GC–MS. Volatiles were extracted from injections in
two columns using three 5 g sub-samples sectioned from the 108 steaks
(3 marinade solution treatments × 3 tumbling replicates × 4 frozen
storage periods × 3 technical replicates), totalling 324 sub-samples and
648 extractions.
2.7.2. Separation of volatile flavor compounds
Volatiles were individually separated using DB-Wax (30 m × 0.25
mm × 0.25 μm) and DB-5MS (60 m × 0.25 mm × 0.25 μm) columns
(Agilent Technology, USA). Shimadzu GC–MS (GC–MS-QP2010 Ultra)
was used. The carrier gas was helium at a flow rate of 2 mL/min, and the
oven temperature was set at 40 ◦ C for 3 min, then raised to 200 ◦ C at
5 ◦ C/min, then at 10 ◦ C/min elevated to 250 ◦ C in the DB-5MS column
and 230 ◦ C in the DB-Wax column and maintained for 3 min. The MS
detector conditions were as follows: the ion source and line transfer
temperatures were adjusted to 230 ◦ C and 250 ◦ C, respectively. The
mass scan range was adjusted to 50–400 m/z with a 70 eV electron
ionization voltage.
2.7.3. Identification and quantitation of volatile flavor compounds
Volatiles were identified after matching their retention indices (RIs)
and mass spectra (MSs) in the DB-Wax and DB-5MS columns, with those
reported in the National Institute of Standard and Technology Library
(NIST 14). Some of them were positively confirmed by injecting their
standard chemicals. Finally, the concentrations of volatiles were deter­
mined with the internal standard method, and the following equation
was used:
( )
volatile
Peakarearatio internal ×con.of internalstandard
standard
Con.(μg/kg)= ×1000
Sampleweight
2.8. Electronic nose (E-nose) analysis
The odor profile of the beef samples at 0 and 6 months of frozen
storage were differentiated using an Alpha-MOS Fox Electronic Nose (E-
nose Fox 4000, France), according to the method described by Zhao
et al. (2020). In a 10 mL headspace vial, an accurate weight of a minced
beef sample (3 g) was placed. The airflow rate was fixed to 150 mL/min,
and analysis was conducted for 160 s at 60 ◦ C. The intensities of 18 metal
oxide sensors were measured and reported. E-nose analyses were
determined using four 3 g sub-samples sectioned from 54 steaks (3
marinade solution treatments × 3 tumbling replicates × 2 frozen storage
periods × 3 technical replicates), totalling 216 sub-samples.
2.9. Statistical analysis
All the data were presented as mean ± S.D. Three sub-samples were
randomly obtained from one beef round primal cut for examination at
four different times of frozen storage for each of three marinade treat­
ments (physical and chemical measures were independently processed
S. Al-Dalali et al. Food Chemistry 376 (2022) 131881

in triplicate, except color measurements which were processed in 5 Table 1

replicates, and E-nose measurements which were processed in 4 repli­ pH values, color values, TBARS (µg/kg), and carbonyl content (nmol/mg pro­
cates). Additionally, E-nose and ultimate pH measurements were con­ tein) of marinated raw beef meet during different times of frozen storage.
ducted at 0 and 6 months frozen storage only. The concentrations of Samples* Frozen storage (months)
carbonyl, TBARS, pH, flavor compounds, and color values were statis­ pH values
tically analyzed using one-way analysis of variance (ANOVA), and fatty 0 6
acid profile was statistically analyzed using t-test at different times of BS1 6.17 ± 0.01Ba 6.60 ± 0.09Ab
frozen storage and among three marinade solutions. SAS and a BS2 5.89 ± 0.04Bc 6.53 ± 0.02Ab
BS3 6.02 ± 0.03Bb 6.77 ± 0.13Aa
completely randomized design were used. The data were analyzed using
L*
PROC ANOVA procedure, in which beef samples and frozen storage 0 2 4 6
period were used as fixed effects and replications as random effects. The BS1 39.38 ± 29.84 ± 32.67 ± 2.49Ba 32.11 ±
mean differences were determined using Duncan’s multiple range test 1.73Ab 2.12Ca 1.45Ba
(P ≤ 0.05). The effect of frozen storage on the flavor profile of raw beef BS2 44.76 ± 27.47 ± 25.81 ± 1.12Cc 24.22 ±
1.32Aa 0.76Bc 2.23Db
meat in the marinated samples was statistically analyzed using principal BS3 34.36 ± 28.81 ± 27.02 ± 0.89Cb 19.58 ±
component analysis (PCA), partial least squares discriminant analysis 1.97Ac 1.41Bb 1.15Dc
(PLS-DA), and variable importance in projection (VIP) scores, based on a*
the concentration of quantified compounds during different times of BS1 40.59 ± 45.50 ± 43.00 ± 1.76Ca 47.61 ±
2.91Da 2.71Ba 1.78Aa
frozen storage. The influence of marination on the flavor profile of raw
BS2 31.29 ± 38.03 ± 37.30 ± 1.88Ac 37.62 ±
beef meat during frozen storage was statistically analyzed using PCA. 0.77Bb 1.54Ac 1.27Ac
MetaboAnalyst 5.0 was used to analyze Heatmap, PCA, PLS-DA, and BS3 30.44 ± 40.57 ± 40.19 ± 1.84Bb 43.02 ±
VIPs. The significance of different times of frozen storage was assessed 1.67Cb 2.42Bb 2.64Ab
using VIP analysis, and compounds with VIP of ≥1.0 exhibited sub­ b*
BS1 48.75 ± 56.43 ± 45.93 ± 2.14Ca 37.86 ±
stantial distinctiveness. Correlations among flavor compound, carbonyl
1.35Ba 2.54Aa 2.24Da
contents, TBARS, and pH values were analyzed using Unscrambler X BS2 44.97 ± 51.86 ± 45.59 ± 2.51Ba 24.54 ±
10.4 software. 1.82Bb 1.34Ac 0.59Cb
BS3 40.61 ± 53.39 ± 41.18 ± 1.49Bb 22.17 ±
1.78Bc 2.66Ab 1.15Cc
3. Results and discussion
TBARS values (µg/kg)
BS1 4.32 ± 0.12Dc 39.11 ± 32.99 ± 1.29Ba 26.61 ±
3.1. Physicochemical properties of beef meats 2.29Aa 0.85Ca
BS2 16.30 ± 18.79 ± 14.10 ± 1.57Cb 12.84 ±
3.1.1. Color measurements 0.39Bb 2.07Ab 0.34Db
BS3 18.80 ± 16.08 ± 9.24 ± 0.57Cc 5.40 ± 0.43Dc
The L*, a*, b* values of marinated beef meat at different times of 2.16Aa 2.17Bc
frozen storage are listed in Table 1, representing lightness, redness, and Carbonyl content (nmol/mg protein)
yellowness, respectively. Freezing effects were detected as linear BS1 12.42 ± 21.68 ± 20.563 ± 28.50 ±
decreased to L* values between 0 and 6 months, although the L* values 0.21Ca 1.81Ba 1.23Ba 2.63Ab
BS2 11.90 ± 15.21 ± 16.52 ± 0.87Bb 19.20 ±
tended to decrease at the last period (i.e., 6 months) of frozen storage. Its
0.10Da 0.55Cb 1.08Ac
value decreased from 39.38 at 0 months to 32.11 at 6 months (BS1), BS3 11.53 ± 14.86 ± 17.24 ± 1.22Bb 30.04 ±
from 44.76 to 24.44 (BS2), and from 34.36 to 19.58 (BS3; Table 1). 0.31Da 1.94Cb 2.07Aa
These decreases can be attributed to water loss during frozen storage,
The same letters (uppercase letters) within the same row are not significantly
and subsequent decreases in surface light reflectance and L* value different at the 95% confidence level. The same letters (lowercase letters) within
(Hughes, Oiseth, Purslow, & Warner, 2014). Moreover, marinade solu­ the same column are not significantly different at the 95% confidence level. *
tions showed an apparent significant effect on the L* values. Zhou, BS1, marinated beef in water; BS2, marinated beef in water and salt; and BS3,
Wang, Chen, & Yuan (2021) found that the L* values of beef meat marinated beef in water, salt, and sugar. 0, 2, 4, and 6 represented frozen storage
marinated with 20% water, 2% salt, and 3% phosphate and then cooked periods in months.
with spices were 24.34 and 40.91 in the outer and inner surfaces,
respectively. The BS2 treatment showed a high L* value at 0 months of weeks and remained relatively constant for the remaining frozen storage
frozen storage, reflecting the lightness of this sample. Such an increase period. In general, variances were found among the literature in terms of
can be ascribed to high water content (Martinez, Cilla, Beltran, & Ron­ color changes during frozen storage, because of many reasons, including
cales, 2006). Meanwhile, the BS3 samples showed a lower L* value than globin denaturation, which renders myoglobin vulnerable to auto-
BS1 and BS2 (34.36); this result can be attributed to the synergistic ef­ oxidation. MetMb-reducing enzyme activity is reduced in frozen meat.
fect of sugar and salt to absorb water, leading to reduced beef surface Lipid breakdown products reduce the redox stability of oxymyoglobin’s
lightness. Holman et al. (2017) reported that the chilling-then-freezing and may inhibit the enzymatic conversion of MetMb into myoglobin.
of beef meat slightly increased the L* values. Color changes in meat during frozen storage can be caused by physical
By contrast, frozen effects were detected as linear increased to a* processes, such as water freezing, thaw drip loss, and drying, which alter
values (indicative of red color) between 0 and 6 months. The value the concentrations of chemicals in the surface layer (Daszkiewicz,
increased from 40.59 to 47.61 (BS1), 31.29 to 37.62 (BS2), and 30.44 to Kubiak, & Panfil, 2018).
43.02 (BS3; Table 1). The a* values tended to increase over time be­
tween 4 and 6 months in all the samples. Frozen storage improved and 3.1.2. pH values
retained the red color of the frozen beef samples because myoglobin The pH of meat has a significant impact on its shelf life, color, and
oxidation was inhibited during frozen storage. However, among treat­ meat quality throughout production (Fernandez-Lopez, Zhi, Aleson-
ments within different storage periods (columns), it can be observed that Carbonell, Perez-Alvarez, & Kuri, 2005). Frozen storage showed a sig­
the control (BS1) presented the highest values, whereas BS2 and BS3 nificant effect (P ≤ 0.05) on the pH values of marinated raw beef meat.
showed reduction in redness (a* values; Table 1). The b* values showed The values increased with frozen storage (Table 1). The ultimate pH
slight increase in 2 months, and then a linear decrease was found be­ increased from 6.17 to 6.60 in BS1, from 5.89 to 6.53 in BS2, and from
tween 2 and 6 months of frozen storage (Table 1). Holman et al. (2017) 6.02 to 6.77 in BS3, from 0 and 6 months of frozen storage (Table 1).
found that frozen-only storage effects were visible, and b* declined at 12

4
S. Al-Dalali et al.
Seong et al. (2017) reported that the pH values of frozen horse meat
increased with increased frozen storage period. Such increase can be
attributed to differences in biochemical changes (e.g., glycolytic rate/or
others) among the meat muscles before or during frozen storage. Farouk
& Swan (1998) stated that differences in pH might be due to variation in
glycolysis, that is, whether or not glycolysis was completed during
frozen storage. In addition, changes (increases and decreases) in the pH
of meat during frozen storage at − 18 ◦ C can be associated with the
animals’ breed and examined muscle (Ablikim et al., 2016). Ziauddin,
Mahendrakar, Rao, & Amla (1993) proposed that the status of proteins
during frozen storage affects pH variations caused by changes in the
muscle’s buffering capacity. Holman et al. (2017) reported that the pH
of beef loins increased with continued chilled and frozen storage pe­
riods. Marination solution had significant effect on pH value at 0 months
of frozen storage. BS2 had the lowest pH (5.89), followed by BS3 (6.02),
and BS1 (6.17), as listed in Table 1. The low value of BS2 might be due to
the chloride anion in salt, which affected ion balance. In addition, BS2
and BS3 had greater increases in pH following frozen storage than BS1.
3.1.3. Lipid oxidation
The TBARS value is one of the most often used indicators for
detecting lipid oxidation in meat. During meat processing, oxidative
processes occur, and the oxidation of lipids produces aldehydes (Berardo
et al., 2016). The TBARS value of the control marinated raw beef meat
(BS1) increased with the frozen storage time, and the highest value was
obtained at 2 months of frozen storage (39.11 µg MDA/kg). This value
was considerably higher than that obtained at 0 months (4.32 µg MDA/
kg). The TBARS value slightly decreased with the frozen storage period
(Table 1). Such trend can be attributed to protein-lipid interactions, in
which MDA and hexanal formed adducts with lysine residues in meat
proteins. The formation of the adducts through Schiff base (SB)
arrangement would explain the apparent loss of MDA after 2 months of
frozen storage. Utrera & Estévez (2013) reported that proteins and lipid
oxidation products interact strongly to produce SB through condensa­
tion. In reality, lipid oxidation products (hydroperoxides and aldehydes)
may establish several types of covalent bonds with proteins through
hydrophobic interaction and hydrogen bonds, produce SB, and promote
polymerization. MDA and 4-hydroxy-2-nonenal may cross-link with the
free amino groups of lysine residues, decreasing their contents after a
period of frozen storage (Viljanen, Kivikari, & Heinonen, 2004).
Zhang et al. (2019) summarized the threshold value of TBARS in beef
meat for consumer acceptance from previous studies, where its value
ranged between 1.0 and 3.11 mg MDA/kg meat. Although, Zhang et al.
(2019) discovered that the TBARS values differed between the two
methods used. In the first method, sodium dodecyl sulfate and TBARS/
buffer reagent were used in extraction, and incubation was conducted at
95 ◦ C for 1 h, whereas in method 2, 20% TCA and 2 M phosphoric acid
were used, and incubation was conducted at room temperature for 12 h
in the darkness). A skewed distribution was obtained with the second
method, and most of the results were lower than 1.0 mg MDA/kg and the
highest value was 10.72 mg MDA/kg. However, regardless of method
used, the TBARS value had no significant effect on sensory data, indi­
cating that untrained consumers cannot notice abnormal flavor devel­
opment caused by high levels of lipid oxidation and are hence unaffected
when tasting beef samples. In our study, the TBARS values were lower
than the threshold value and decreased after long-term frozen storage.
Hence, the consumer cannot detect any rancid flavors in the samples of
this study. Adding salt in the marination process (BS2) significantly
increased the TBARS value at 0 months to approximately 16.30 µg MDA/
kg. The value continuously increased until 2 months of frozen storage
and then slightly decreased. The chloride anion in salt acted as a pro-
oxidant, causing fat to oxidize and rancidify. Long-term contact of salt
with fat results in the formation of peroxides, which are responsible for
rancid flavors (Petit et al., 2019). Meat lipid oxidation occurs when the
salt level is between 0.7% and 2.5% because salt suppresses antioxidant
enzymes, such as glutathione peroxidase, superoxide dismutase, and
5
Food Chemistry 376 (2022) 131881
catalase (Petit et al., 2019).
Shimizu, Kiriake, Ohtubo, & Sakai (2009) reported that MDA con­
tents increased in salted and control frozen yellowtail meat samples after
up to 20 weeks of frozen storage. The TBARS value of the salted samples
was significantly greater than that of the control samples after 4–20
weeks of storage. Interestingly, the addition of sugar to the marinade
solution of BS3 contributed significantly to reduction of lipid oxidation
during frozen storage. The TBARS value decreased from 18.80 µg MDA/
kg at 0 month to 5.40 µg MDA/kg at 6 months of frozen storage
(Table 1). This reduction can be attributed to sugar, which altered vis­
cosity, changed water activity, scavenged free radicals through hydroxyl
groups, and donated hydrogen. These findings were in line with those
reported by Vieira (2016), who stated that low glucose concentrations
(<0.09 M) enhance lipid oxidation rates but high amounts (>0.09)
decrease them. Reducing sugars (glucose and fructose) inhibit lipid
oxidation at high concentrations (>0.09). Sucrose inhibits lipid oxida­
tion. The binding of sugar to water may be responsible for the preven­
tion of lipid oxidation. Reducing sugars may also impact their capacity
to operate as hydrogen donors, potentially inactivating free radicals,
because of the Maillard reaction products produced (Vieira, 2016). The
oxidative alteration of lipids has long been thought to be a harmful
process that causes major changes in the chemical characteristics of
molecules, function loss, and formation of cytotoxic and genotoxic
chemicals, mainly oxidized lipid-derived aldehydes and peroxides
(Kumar & Tanwar, 2011). Soyer et al. (2010) found that the TBARS
content of non-marinated chicken breast and leg meat increased
significantly during frozen storage, and the fastest and most significant
increase in chicken leg meat occurred within the first 2 months.
3.1.4. Protein oxidation
The amino acid content and structural arrangement of proteins
determine their functional characteristics, such as gelation, solubility,
and emulsification potential in various food items. The oxidative
degradation of amino acids, particularly arginine, lysine, histidine, and
proline, can produce carbonyl compounds that can impact the func­
tioning of meat proteins (Sohaib et al., 2017). The most researched
oxidative modification in meat products is the formation of carbonyl
compounds, which are generally measured using the DNPH technique
(Estévez, 2011; Berardo et al., 2016). Table 1 shows that freezing has a
significant effect on carbonyl content in all the marinated samples. Its
content increased from 12.42 nmol/mg protein to 28.50 nmol/mg
protein in BS1, from 11.90 nmol/mg protein to 19.20 nmol/mg protein
in BS2, and from 11.53 nmol/mg protein to 30.04 nmol/mg protein in
BS3 samples at 0 and 6 months of frozen storage, respectively.
Marination solutions showed no effect at 0 months. By contrast, at 2
and 4 months of frozen storage, significant differences between BS1 and
other samples were found. No significant differences between BS2 and
BS3 at the same frozen storage period were found (Table 1). Protein
carbonyls are generated through three primary mechanisms, namely,
non-enzymatic glycation, metal-catalyzed oxidation, and adduct for­
mation with non-protein carbonyl chemicals. In the first pathway,
reducing sugars can produce carbonyls through the glycation of lysine
residues (Estévez, 2011). In this pathway, the added sugar in BS3 might
have been involved. The carbonyl content of BS3 increased during
frozen storage of up to 6 months, and the highest value (30.04 nmol/mg
protein) was obtained (Table 1). In the second pathway, carbonyls are
produced in the side chains of lysine, arginine, threonine, and proline
through a metal-catalyzed oxidation pathway (Berardo et al., 2016).
Through the Fenton reaction, metal ions, such as Fe2+, induce the
production of oxygen radicals from H2O2 and oxygen, and metal ions are
oxidized (Berardo et al., 2016). In the third pathway, carbonyl com­
pounds can be generated because of lipid oxidation products, such as
MDA, which may form adducts with proteins (Estévez, 2011; Berardo
et al., 2016).
In the present study, the results suggested that lipid oxidation
products participate in the formation of protein adducts, which
S. Al-Dalali et al. Food Chemistry 376 (2022) 131881

increased in amount over the course of frozen storage (Table 1). Protein
oxidation has been linked to lipid oxidation in different meat products
(Mercier, Gatellier, & Renerre, 1995; Shimizu et al., 2009; Soyer et al.,
2010). Given that the products of primary (hydroperoxides) and sec­
ondary (aldehydes) lipid oxidation can serve as substrates for protein
oxidation, once lipid oxidation begins, protein oxidation occurs (Soyer
et al., 2010). Shimizu et al. (2009) reported that the carbonyl content of
the control and salted yellowtail meat samples increased significantly
during the 20 weeks of frozen storage. Soyer et al. (2010) evaluated the
effect of frozen storage period and temperature in the protein oxidation
of chicken meat. They reported that protein oxidation in breast and leg
meat significantly increased following frozen storage period. They
stated that the frozen temperature showed no difference during the first
three months. In contrast, a significant difference was found with
continued frozen storage over than 3 months, especially at a frozen
temperature of − 7 ◦ C compared to of − 12 ◦ C and − 18 ◦ C. They proposed
that lipids and proteins are oxidized after being frozen for a long time.
The rate of protein oxidation significantly increased in chicken meat
stored at − 7 ◦ C, indicating that meat frozen at moderate temperature is
more susceptible to protein oxidation probably because of lipid oxida­
tion products or oxidation catalysts. Similarly, several studies reported
increasing carbonyl content during cold storage (Eymard, Baron, &
Jacobsen, 2009) and frozen storage (Baron, Kjærsgård, Jessen, &
Jacobsen, 2007). Increases in total carbonyls suggest that oxidative al­
terations occur in meat during frozen storage because carbonyls are the
main autoxidation products. When reactive oxygen species attack pro­
teins, carbonyl compounds are formed due to the interactions (Soyer
et al., 2010).
3.1.5. Fatty acids composition
Table 2 shows the fatty acid composition (% of peak area) and
changes in it throughout frozen storage (0 and 6 months) in marinated
∑ ilies, including alcohols (10 compounds), aldehydes (8), ethers (2), fu­
raw beef meat. In general, saturated fatty acids ( SFAs), mono­
∑ rans (2), phenols (3), and others (3), as listed in Table S1. The oxidative
unsaturated fatty acids ( MUFAs), and polyunsaturated fatty acids
∑ breakdown of lipids, the Maillard reaction, thiamine degradation, and
( PUFAs) increased after frozen storage in all beef samples with high
∑ ∑
SFA content. The SFAs increased from 11.805%, 11.280%, and
10.430% at 0 months to 19.735%, 15.520%, and 17.992% at 6 months
for the BS1, BS2, and BS3 samples, respectively (Table 2). Our findings
corroborated the results of Nazemroaya, Sahari, & Rezaei (2009). They
found that saturated fatty acid content increased after being frozen. The
∑ ∑ ∑
USFA ( MUFA and PUFA) increased during frozen storage from
5.785% to 12.710% (BS1), 6.295% to 9.650% (BS2), and 7.640% to
10.837% (BS3; Table 2). After five freeze–thaw cycles (in one cycle, the
fresh-packed muscle samples were frozen at − 20.0 ± 0.5 ◦ C for 24 h and
∑
thawed at 4 ± 0.5 ◦ C for 12 h), SFA content increased from 21.6% to
∑
35.3 %, and MUFA content increased from 16.1% to 30.5 % (He et al.,
2021), consistent with the findings of the present study. Oleic acid
(C18:1, cis-9) and palmitic (C16:0) were the most abundant mono­
unsaturated and saturated fatty acids in the beef samples, followed by
stearic acid (C18:0), as shown in Table 2. The free fatty acid content in
beef tissue and complicated lipid hydrolysis during the freeze–thaw
process can contribute to fatty acids in meat samples. The existence of
radicals, which are oxidation indicators, can cause oxidative changes in
frozen beef lipids (Dragoev, Kiosev, Danchev, Ioncheva, & Genov,
1998). In addition, the enzyme oxidation or auto-oxidation of oleic acid
(C18:1, cis-9) and linoleic acid (C18:2, cis-9,12) is extensively explained
∑ ∑
(Fig. S1). Finally, BS3 had the highest USFA and MUFA content,
followed by BS2 and BS1. Their content increased with frozen storage
period. Fatty acid composition was influenced by the marination solu­
tions and frozen storage.
3.2. Identification of flavor profile and their potential sources in beef meat
samples
The flavor profiles of marinated raw beef meat products were iden­
tified using HS-SPME-GC–MS. A total of 28 volatiles were identified in
all the samples, 16 of which were positively confirmed by their authentic
chemicals. The remaining 12 volatiles were tentatively identified by
matching their MS and RIs in two different columns (i.e., DB-Wax and
DB-5MS) with those reported in the literature and the NIST library. The
identified flavor chemicals were obtained from various chemical fam­
metabolite interaction from these reactions create the majority of flavor
components in meat and meat products (Ramalingam, Song, & Hwang,
2019). The potential precursors and metabolic pathways of the main
flavor components were hypothesized on the basis of the literature and
with MetaCyc platforms and used in exploring the mechanism of flavor
formation in meat and meat products. Flavor compounds in marinated

Table 2
Fatty acid composition (% of peak area) in marinated beef samples during frozen storage periods.
Samples* BS1 BS2 BS3 Pr >|t|*

FAs 0 6 0 6 0 6

C10:0 0.200 ± 0.001 0.180 ± 0.014 0.190 ± 0.004 0.865 ± 0.035 0.200 ± 0.005 0.843 ± 0.040 <0.0001
C11:0 0.045 ± 0.007 0.040 ± 0.001 0.050 ± 0.001 0.050 ± 0.001 0.045 ± 0.007 0.044 ± 0.002 <0.0001
C12:0 0.595 ± 0.007 0.595 ± 0.016 0.600 ± 0.015 0.660 ± 0.084 0.525 ± 0.014 0.545 ± 0.003 <0.0001
C13:0 0.095 ± 0.003 0.115 ± 0.002 0.105 ± 0.003 0.115 ± 0.007 0.065 ± 0.007 0.105 ± 0.007 <0.0001
C14:0 0.255 ± 0.077 0.705 ± 0.038 0.175 ± 0.007 0.460 ± 0.014 0.180 ± 0.002 0.430 ± 0.004 <0.0001
C14:1, cis-9 0.115 ± 0.007 0.115 ± 0.007 0.045 ± 0.007 0.065 ± 0.021 0.060 ± 0.001 0.020 ± 0.001 <0.0001
C15:0 0.220 ± 0.084 0.095 ± 0.007 0.070 ± 0.001 0.095 ± 0.003 0.060 ± 0.001 0.035 ± 0.001 <0.0001
C15:1, cis-10 0.175 ± 0.007 0.170 ± 0.005 0.175 ± 0.006 0.150 ± 0.002 0.145 ± 0.004 0.080 ± 0.002 <0.0001
C16:0 5.43 ± 0.933 9.810 ± 0.356 5.655 ± 0.048 7.675 ± 0.135 5.385 ± 0.139 8.840 ± 0.011 <0.0001
C16:1, cis-9 0.145 ± 0.009 0.535 ± 0.004 0.215 ± 0.049 0.420 ± 0.024 0.285 ± 0.063 0.482 ± 0.063 <0.0001
C17:0 0.140 ± 0.007 0.255 ± 0.002 0.155 ± 0.007 0.370 ± 0.002 0.165 ± 0.002 0.400 ± 0.005 <0.0001
C17:1, cis-10 0.230 ± 0.008 0.175 ± 0.006 0.210 ± 0.006 0.090 ± 0.003 0.205 ± 0.006 0.035 ± 0.001 <0.0001
C18:0 4.745 ± 0.756 7.765 ± 0.266 4.190 ± 0.268 5.065 ± 0.035 3.705 ± 0.137 6.625 ± 0.075 <0.0001
C18:1, cis-9 4.500 ± 0.195 10.955 ± 0.784 5.230 ± 0.480 8.145 ± 0.297 6.445 ± 0.201 9.480 ± 0.059 <0.0001
C18:2, cis-9,12 0.480 ± 0.098 0.650 ± 0.056 0.250 ± 0.012 0.650 ± 0.005 0.340 ± 0.004 0.595 ± 0.001 <0.0001
C18:3, cis-6,9,12 0.140 ± 0.001 0.110 ± 0.002 0.170 ± 0.003 0.130 ± 0.001 0.160 ± 0.003 0.145 ± 0.001 <0.0001
C20:0 0.080 ± 0.004 0.175 ± 0.001 0.090 ± 0.004 0.165 ± 0.002 0.100 ± 0.003 0.125 ± 0.001 <0.0001
∑
SFA 11.805 ± 0.324 19.735 ± 1.104 11.280 ± 0.924 15.520 ± 0.604 10.430 ± 0.434 17.992 ± 1.224 <0.0001
∑
MUFA 5.165 ± 0.354 11.950 ± 0.874 5.875 ± 0.464 8.870 ± 0.764 7.140 ± 0.654 10.097 ± 1.204 <0.0001
∑
PUFA 0.620 ± 0.004 0.760 ± 0.006 0.420 ± 0.007 0.780 ± 0.003 0.500 ± 0.001 0.740 ± 0.002 <0.0001
∑
USFA 5.785 ± 0.423 12.710 ± 1.022 6.295 ± 0.867 9.650 ± 0.537 7.640 ± 0.528 10.837 ± 1.106 <0.0001

* BS1, marinated beef in water; BS2, marinated beef in water and salt; and BS3, marinated beef in water, salt, and sugar. 0, 2, 4, and 6 represented frozen storage
periods in months. * the p-values associated with the t values showed statistically significant in all samples and all variables (FAs).

6
S. Al-Dalali et al.
raw beef meat were produced mostly through amino and fatty acid
metabolism. For instance, octanal, hexanal, nonanal, 1-octen-3-ol, 1-
heptanol, and 1-octanol are common found in meat products because of
their low thresholds (Yin et al., 2021). They were mostly derived from
the enzyme-oxidation or auto-oxidation of unsaturated fatty acids, such
as linolenic, linoleic, and oleic acids (see Fig. S1), which were the most
abundant unsaturated fatty acids in beef meat, accounting for 0.14%,
0.48%, and 4.5% of the total percentage, respectively (Table 2).
Monounsaturated fatty acids, such as oleic acid, can undergo enzyme-
oxidation or auto-oxidation and produce various kinds of hydroperox­
ides, such as 8-, 9-, 10-, 11- or 13-ROOH, which are then decomposed
into aldehydes through homolysis by hydroperoxide lyase (Wu et al.,
2020). The aldehydes can be partially reduced to matching alcohols by
aldehyde reductase. For instance, octanal and nonanal may be produced
through the homolysis of 8- and 9-ROOH, respectively (Wu et al., 2020).
Flavor compounds are being generated from polyunsaturated fatty
acids, such as linoleic, linolenic, eicosapentaenoic, and arachidonic
acids, by generating various hydroperoxides after homolysis (Benet
et al., 2015). Hexanal is largely driven by the alkoxy radical-scission of
13-ROOH derived from linoleic acid. 1-Octen-3-ol is produced by the
catabolism of arachidonic and linoleic acids (Wu et al., 2020). Hexanal is
often regarded as an excellent predictor of fat oxidation level (Yin et al.,
2021). The only precursor for 3-methyl-1-butanal synthesis is L-leucine
through the decomposition into α-ketoisocaproic acid by transaminase,
which is further degraded by decarboxylase into 3-methyl-1-butanal and
then to 3-methyl-1-butanol (Fig. S1) (Wu et al., 2020). The Maillard
reaction produces aldehydes with branching or complex structures, such
as benzaldehyde, which can be identified by a Maillard reaction system
model containing phenyl glycine (Whitfield, & Mottram, 1992). 1-Hex­
anol is produced through the reduction of hexanal or through linoleic
acid oxidation (Merlo et al., 2021). Farnesyl pyrophosphate synthase is
an enzyme that catalyzes the conversion of prenyl diphosphate into
geranyl diphosphate. Then, geranyl diphosphate is converted to (3R)-
linalool by linalool synthase (Jia, Crock, Lu, Croteau, & Chen, 1999).
Benzene ring ethers (i.e., estragole and anethole) are essential to the
development of flavor and enhancement of a mild beef flavor (Zhou
et al., 2021). The precursors of these compounds are phenylpropanoids
derived from phenylpropanoid biosynthesis. Estragole (also called
methylchavicol) is produced through methyl group transformation
facilitated by s-adenosyl-L-methionine (SAMe) from chavicol to meth­
ylchavicol (i.e., estragole). While, anethole is produced through the
transformation of coumaryl acetate to trans-anol under the action of anol
synthase, which is further transformed to anethole by the aid of SAMe
(MetaCyc platforms). Furans are typically generated during carbohy­
drate breakdown induced by the lipid oxidation and decomposition of
amino acids. They may be produced by the rearrangement and thermal
disintegration of cellulose and hemicellulose (Yin et al., 2021). Furaneol
can be produced through the conversion of D-glucose into β-D-fructose
1,6-bisphosphate by glycolytic enzymes (i.e., glucose-6-phosphate
isomerase) and then transformed into furanones (Wein, Lewinsohn, &
Schwab, 2001). Furaneol can also be considered an intermediate for the
formation of other flavor chemicals, such as furanthiols, thiophenes, and
other sulfur compounds, and has been identified in sheep meat, fried
prawn meat, roasted goose’s leg meat, and beef broth (Gąsior et al.,
2021). Most phenols are produced during the pyrolysis of lignin or
through the decomposition activities of microorganisms (Gąsior et al.,
2021), and they are known as the standard flavor characteristics of
smoked food, which they are contributing to the woody and smoky notes
(Yin et al., 2021). Additionally, Gąsior et al. (2021) reported that the
presence of phenolic chemicals in meat could become from beef given
grass and/or grain diets.
3.3. Dynamic changes in flavor compounds during the frozen storage of
beef meat
Dynamic changes in volatile flavor compounds during the frozen
7
Food Chemistry 376 (2022) 131881
storage of beef meat samples were determined using HS-SPME-GC–MS.
The marinated beef meat products were analyzed after being frozen and
kept for 0, 2, 4, and 6 months, and a total of 28 volatiles were identified
and quantified (Table S1 and Fig. 1). The total concentrations of the
quantified chemical groups fluctuated between decrease, increase, and
then decrease. In general, their concentrations decreased with
increasing frozen storage period (Table S1). The total concentrations of
aldehydes increased with frozen storage period (from 13.36 µg/kg to
33.55 µg/kg) from 0 and 4 months in BS1. In BS2, the concentration
increased from 58.96 µg/kg at 0 months to 61.25 µg/kg at 2 months of
frozen storage and then decreased to 19.5 µg/kg at 6 months (Table S1
and Fig. 1A). The total concentrations of alcohols decreased with
increasing frozen storage period up to 6 months in all beef samples. The
total concentration decreased from 34.92 µg/kg to 16.55 µg/kg (BS1),
75.67 µg/kg to 16.40 µg/kg (BS2), and 79.51 µg/kg to 25.84 µg/kg (BS3)
at 6 months of frozen storage (Fig. 1A). Merlo et al. (2021) reported no
apparent linear decreasing or increasing patterns in the volatiles. Similar
variations in volatiles in meat products during refrigerated storage have
been reported (Zhang et al., 2020). Qi et al. (2021) stated that with
longer frozen storage times for raw meat, the overall amount of volatile
chemicals increased first, then decreased, and then increased, and the
ketones and aldehydes fell in line. Meanwhile, the levels of hydrocar­
bons, alcohols, esters, and furans increased, and then decreased.
Lipolysis, proteolysis, and oxidation are the primary biochemical
processes that occur during the post-mortem ripening of meat and affect
the juiciness, tenderness, and flavor of meat. Flavor precursors, such as
sugars, peptides, amino acids, organic acids, and adenine nucleotide
breakdown products, are the chemicals produced during post-mortem
and influence the formation of meat flavor and during frozen storage
(Kosowska, Majcher, & Fortuna, 2017). These precursors are degraded
during frozen storage or participate in the Maillard reaction during the
cooking process and lipid degradation during frozen storage, forming
flavor compounds. For instance, nonanal, pentanal, 1-octen-3-ol, octa­
nal, octanol, hexanal, hexanol, heptanal, heptanol, and hexanoic acids
are formed primarily as a result of lipid oxidation processes, and 3-
methyl-1-butanol forms as a result of the Strecker degradation of an
amino acid (i.e., leucine). The flavor of non-ripened meat is weak and
tasteless, but the flavor of matured beef is enhanced and intensified. The
fatty flavor, beef-like, broth-lik, sweet, and caramel flavors are enhanced
by ripening for up to 14 days during chilled storage (Kosowska et al.,
2017).
In addition, the marination process contributes to the number and
content of identified compounds. Fig. 2B, E, and H illustrate that BS2
and BS3 had high content of volatile flavor compounds compared with
BS1 at 0 month of frozen storage. Specifically, 1-hexanol, octanal,
estragole, hexanoic acid, trans-2-octen-1-ol, tetradecanal, pentadecanal,
1-heptanol, 3-allyl-2-methoxyphenol, and hexanal were detected only in
BS2 and BS3 (Table S1), in which marinade content (i.e., salt) acceler­
ated lipid oxidation and led to the formation of the compounds. Alco­
hols, aldehydes, and ethers were the chemical groups with the highest
concentrations in the beef samples.
Ten alcohols were identified and quantified, including 2-ethyl-1-hex­
anol, 1-hexanol, 1-octanol, 3-methyl-1-butanol, trans-2-octen-1-ol, 1-
octen-3-ol, 1-heptanol, 4-carvomenthenol, phenylethyl alcohol, and
linalool. Seven of these alcohols were detected in BS1, 10 were identified
in BS2, and nine were identified in BS3. Moreover, 1-hexanol, trans-2-
octen-1-ol, and 1-heptanol were detected because of lipid oxidation in
BS2, in which added salt accelerated lipid oxidation and promoted the
formation of the compounds. As shown in Table S1, some alcohols
decreased with increasing frozen storage period. By contrast, 1-heptanol
was generated at 4 months of frozen storage in BS2 with a concentration
of 2.49 µg/kg. Alcohols, which may be generated from fat oxidation, are
the major aroma components of cooked beef meatballs during storage,
especially 1-octen-3-ol, hexanol, and 1-octanol (Sun et al., 2020).
Eight aldehydes were identified and quantified at different times of
frozen storage, including benzaldehyde, benzeneacetaldehyde, octanal,
S. Al-Dalali et al.
Fig. 1. (A) Total concentrations (µg/kg) of different volatile flavor groups in marinated raw beef meat samples; and (B) Heatmap elucidates the different con­
centrations of the volatile flavor compounds in the marinated raw beef meat during different times of frozen storage.
nonanal, tetradecanal, pentadecanal, hexadecanal, and hexanal. Their
concentrations fluctuated during continuous frozen storage, where they
increased, then decreased, and then increased for some of them. Some
aldehydes disappeared after 2 months of frozen storage, such as ben­
zeneacetaldehyde, octanal, tetradecanal, and hexanal. Aldehydes have
green, fatty, grassy, and fresh characteristics, and these compounds are
produced from oleic acid. For example, octanal accounts for the green,
meaty, fresh, fruity, and grassy notes, whereas nonanal contributes
fruity and sweet aroma notes (Merlo et al., 2021). Sun et al. (2020)
found that these aldehydes are the major components in beef, particu­
larly hexanal, pentanal, benzaldehyde, heptanal, and (E)-2-heptenal.
Regarding phenols, 3,5-di-t-butylphenol, 3-allyl-2-methoxyphenol,
and isoeugenol formed during frozen storage. 3,5-Di-t-butylphenol
formed and had concentrations of 0.40, 0.30, and 0.49 µg/kg at 6
months of frozen storage in BS1, BS2, and BS3, respectively (Table S1).
3-Allyl-2-methoxyphenol was generated at 6 months of frozen storage in
BS2 and BS3, with content of 6.61 and 3.76 µg/kg beef, respectively.
Isoeugenol was generated at 2 months of frozen storage in all the sam­
ples, and its content increased with frozen storage time. Phenols are
produced during the pyrolysis of lignin or through the decomposition
activity of microorganisms (Gąsior et al., 2021).
Furans and acids disappeared during frozen storage and were only
detected at 0 months. Furans can come from a variety of sources. Furans
are mainly synthesized through the lipid oxidation of free fatty acids
(Merlo et al., 2021). Two acids were determined in all the samples at 0
months, including hexanoic acid and acetic acid. Merlo et al. (2021)
reported these two acids in smoked bacon. They stated that carbohy­
drate fermentation in microorganisms is the most likely source of
organic acids in fermented meat products. Our results were in line with
those reported by Merlo et al. (2021), who stated that samples frozen for
up to 30 days contained higher amounts of volatiles than the samples
stored for longer periods. Key metabolic changes, such as lipid oxida­
tion, which promotes the production of volatiles, may have occurred in
the first 2 months of storage and may be related to this behavior. Such
findings are supported by the results of TBARS, which showed the
highest values at 2 months of frozen storage and then decreased with
increasing frozen storage period (Table 1) (Merlo et al., 2021). Sun et al.
(2020) found that the odor-active components in beef meatballs dis­
appeared after storage, and the flavor dilution factor changed as well.
The conversion of aldehydes (i.e., hexanal) into alcohols (i.e., hexanol)
8
Food Chemistry 376 (2022) 131881
before they could accumulate was another aspect that may have
inhibited the formation of volatiles during frozen storage periods, as
shown in Table S1. Moreover, some volatile compounds disappeared
over time for several reasons, including oxidation, volatilization due to
low boiling points during the preparation and extraction of flavor
compounds, Strecker degradation, and the chemical reaction among
different compounds.
Fig. 2 illustrates the PCA analysis and VIP scores of marinated raw
beef meat at different times of frozen storage. Fig. 2C, F, and I show the
important flavors (VIP ≥ 1.0) screened by PLS-DA at different times of
frozen storage. The sums of PC-1 and PC-2 in Fig. 2A, D, and G were
93.9%, 89.1%, and 90.2%, respectively, which explained nearly 90.0%
of the total variance in the effect of frozen storage periods on flavor
profile of individual marinated raw beef meat.
Fig. 2B, E, and H clearly show the correlation and distribution in each
frozen storage period and corresponding chemicals in each marinated
sample. According to the sum of the first two components, frozen storage
affected the aroma profiles of tested samples. These results were further
explored through GC–MS. PCA analysis showed that 1-hexanol, 2-ethyl-
1-hexanol, 3-methyl-1-butanol, 1-octen-3-ol, phenylethyl alcohol, and
linalool were present in all the samples at 0 month of frozen storage
(Table S1). This finding was supported by the VIP results, as shown in
Fig. 2 and consistent with the findings of Merlo et al. (2021), who stated
that 1-octen-3-ol is the major alcohol in meat products.
3.4. Screening of potential flavor compound markers during frozen
storage
The samples yielded a total of 28 volatile flavor components. The
effect of frozen storage on each marinated sample was determined with
the VIP method, and potential marker flavor components were screened.
When the VIP of a flavor component was equal to 1.0 or higher, it was
used as a marker to distinguish the effect of frozen storage period
(Huang et al., 2019). A high VIP increased the likelihood of a compound
to be recognized. Fig. 2C, F, and I summarize the important flavor
compounds identified through PLS-DA, where 9 markers have distin­
guished the effect of freezing in the BS2 sample. Among them, 6 flavor
compounds were screened at the frozen storage periods of 2 and 4
months, including tetradecanal, octanal, 2-ethyl-1-hexanol, benzenea­
cetaldehyde, 1-heptanol, and isoeugenol (see Fig. 2F). Seven markers
S. Al-Dalali et al. Food Chemistry 376 (2022) 131881

Fig. 2. PCA analysis and VIP scores of marinated raw beef meat: (A, D, and G) the score plots of BS1, BS2, and BS3 during different times of frozen storage; (B, E, and
H) the loading plots of BS1, BS2, and BS3 based on the concentration of quantified compounds during different times of frozen storage; and (C, F, and I) the Important
flavors (VIP ≥ 1.0) screened by PLS-DA during different times of frozen storage. The colored boxes on the right of Figs. C, F, and I represented the relative con­
centrations of the flavor compounds during different times of frozen storage. * The full name of the compounds with a star in Figs. C, F, and I is 3-methyl-1-butanol, 4-
carvomenthenol, 2-ethyl-1-hexanol, 3,5-di-t-butylphenol, phenylethyl alcohol, 2,6,11-trimethyl-dodecane, benzeneacetaldehyde, and 3-allyl-2-methoxyphenol. BS1-
0, BS1-2, BS1-4, and BS1-6 represented marinated beef in water at 0, 2, 4, and 6 months of frozen storage periods. BS2-0, BS2-2, BS2-4, and BS2-6 represented
marinated beef in water and salt at 0, 2, 4, and 6 months of frozen storage periods. BS3-0, BS3-2, BS3-4, and BS3-6 represented marinated beef in water, salt, and
sugar at 0, 2, 4, and 6 months of frozen storage periods.

were used in discriminating among the effects of freezing in BS3,
including benzeneacetaldehyde, hexanal, and isoeugenol (Fig. 2I). As
expected, most of the screened markers for the frozen process belonged
to aldehydes and alcohols, indicating that these components were
derived from lipid oxidation during frozen storage.
3.5. Effect of marination on the flavor profiles of marinated beef meat
Of the 28 volatiles identified by HS-SPME-GC–MS, 18 were identified
in BS1, 28 were identified in BS2, and 27 were identified in BS3, as listed
in Table S1 and Fig. S2. The oxidative breakdown of lipids, the Maillard
reaction, thiamine degradation, and metabolite interaction from these
reactions produce the majority of flavor components in meat products
(Ramalingam et al., 2019). The marination of beef steaks in various
marinade solutions, particularly the marinade solutions employed in
BS2 and BS3 samples, contributed to the flavor profiles. These findings
agreed with the TBARS results, in which the values of BS2 and BS3 were
higher than the result of the control (Table 1). As shown in Figs. S2C, F,
and I, phenylethyl alcohol, 1-hexanol, and estragole were markers in
BS2 and contributed to sweet, green, and spicy notes, respectively,
whereas phenylethyl alcohol, 1-hexanol, 3-methyl-1-butanol (fruity),
and estragole were markers in BS3. Meanwhile, 3-methyl-1-butanol and

9
S. Al-Dalali et al.
4-carvomenthenol (spicy and woody) were recognized as markers in
BS1. Fig. S2 illustrates the PCA analysis results for marinated raw beef
samples with different marinade solutions. The sum of PC-1 and PC-2 in
Figs. S2A, C, E, and G were 100% for all of them, which explained all the
variances concerning the effect of the marination process on the flavor
profile of beef meat at different times of frozen storage. Figs. S2B, D, F,
and H clearly show the correlation and distribution in each marinade
solution and corresponding chemicals in each frozen storage. According
to the sum of the first two components, it can be concluded that the
marination process contributed to the aroma profiles of the tested
samples.
3.6. Electronic nose (E-nose) results
An E-nose is a rapid and effective tool for detecting a product’s
aroma that does not require specific sample preparation. The metal
oxide sensors in an E-nose imitate the actions of a biological olfactory
organism. Therefore, they can distinguish among the flavor profiles of
meat products by simulating the sense of smell of biological organisms
(Di Rosa, Leone, Cheli, & Chiofalo, 2017). Sensors P30/2, P40/2, P30/1,
PA/2, T70/2, P40/1, P10/2, P10/1, TA/2, and T30/1 clearly distin­
guished the differences among the different marinated samples at 0 and
6 months of frozen storage, especially the BS2 sample, which showed the
highest response intensities compared to the other samples (Fig. 3). The
response intensities of the metal oxides decreased with increasing frozen
storage period of 6 months. This finding was in line with the GC–MS
results; in which the concentrations of most quantified compounds
decreased with increasing frozen storage.
3.7. Correlation analysis between lipid and protein oxidation and flavor
changes through partial least squares discriminant analysis (PLS-DA)
After data normalization, PLS-DA was used in elucidating the cor­
relations among lipid oxidation, protein oxidation, and flavor changes in
the marinated samples at different times of frozen storage. The results
clearly showed the correlation, as demonstrated in Fig. 4A and B, in
terms of volatile flavor components, TBARS, carbonyl content, and pH
value. Factor-1 explained 74% of the total variance, and factor-2
LY2/LG
TA/2
0.50
T40/1 0.45
0.40
0.35
0.30
T40/2 0.25
0.20
0.15
0.10
0.05
P30/2 0.00
-0.05
-0.10
P40/2
P30/1
PA/2
T70/2
P40/1
Fig. 3. E-nose data for marinated raw beef meet during different times of frozen storage: data of sensor responses. BS1-0, BS2-0, and BS3-0 represented marinated
beef in water, water and salt, water and salt and sugar at 0 months of frozen storage period, respectively. BS1-6, BS2-6, and BS3-6 represented marinated beef in
water, water and salt, water and salt and sugar at 6 months of frozen storage period, respectively.
Food Chemistry 376 (2022) 131881
explained 42% of the total variance, indicating that the top two fac­
tors clearly determined the correlations among the variables. The sam­
ples varied in terms of the variables. In the first factor, all the samples
subjected to frozen storage durations for 4–6 months were close
together, indicating similarity, as shown in Fig. 4A. These samples
showed the characteristic flavor components belonging to alcohols, al­
dehydes, ethers, and phenols and high carbonyl content and pH values,
confirming that most of the components were generated because of lipid
and protein oxidation during frozen storage. All the samples at 2 months
of frozen storage were located in the second factor, which presented
high contents of lipid-derived components, such as hexanal, pentade­
canal, tetradecanal, benzeneacetaldehyde, octanal, benzaldehyde,
nonanal, and 2-ethyl-1-hexanol and exhibited high TBARS levels
(Fig. 4B). These components were the major compounds generated
through lipid degradation. All the samples at 0 months of frozen storage
were positioned at the top of the second factor, presenting high levels of
hexanoic acid, estragole, acetic acid, phenylethyl alcohol, and 2(5H)-
furanone. This result might indicate that lipid and protein oxidation and
the marinade solution contents were responsible for flavor formation
and changes of raw beef meat during different durations of frozen
storage.
4. Conclusions
A total of 28 volatile flavor compounds were identified in all the
samples. The concentration of each identified compound fluctuated with
frozen storage (increasing, decreasing, or increasing and then
decreasing). This finding indicates that frozen storage significantly
affected the flavor profiles of beef samples. Marinade solutions
contributed to the flavor profile given that 18 compounds were identi­
fied in BS1, 28 compounds were identified in BS2, and 27 compounds
were identified in BS3. Furthermore, lipid oxidation occurred during the
first 2 months of frozen storage. The TBARS values increased to
approximately 39.11 and 18.79 µg MDA/kg for BS1 and BS2, respec­
tively, and then slightly decreased with continued frozen storage dura­
tion to 6 months. Carbonyl content linearly increased with frozen
storage period, indicating that lipid degradation and marinade solutions
were the main factors that influenced the aroma profile of raw beef
LY2/G BS1-0
BS2-0
LY2/AA
BS3-0
BS1-6
LY2/GH BS2-6
BS3-6
LY2/gCTl
LY2/gCT
T30/1
P10/1
P10/2
10
S. Al-Dalali et al.
Fig. 4. Correlation analysis between flavor compound, carbonyl contents, TBARS, and pH values: (A) the score plot of PCA, and (B) the correlation loading plot.
meat. The methods explored in this study are effective in detecting
frozen and marinated beef markers. However, additional studies using
different number of animals are needed before definitive conclusions
can be formulated.
CRediT authorship contribution statement
Sam Al-Dalali: Conceptualization, Data curation, Formal analysis,
Investigation, Methodology, Software, Validation, Visualization,
Writing - original draft, Writing - review & editing. Cong Li: Method­
ology. Baocai Xu: Funding acquisition, Project administration, Re­
sources, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was funded by the Fundamental Research Funds for the
Central Universities of China (Grant No. JZ2021HGQB0277), National
Key R&D Program of China (Grant No. 2019YFC1606200) and by
project number 202003b06020023.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2021.131881.
11
Food Chemistry 376 (2022) 131881
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