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

because of the high amounts of oxidation catalysts (such as iron and
Beef is a vital global product, and thus the beef industry must ensure myoglobin) and lipids in meats and are thus vulnerable to oxidative
the safe delivery of high-quality beef to frequently distant end-users. processes (Asghar, Gray, Buckley, Pearson, & Booren, 1988). The main
Chilled and frozen storage have been used for this purpose and have cause of degradation in stored muscle meats is lipid oxidation. The
shown to be effective, as seen by their widespread use and scientific critical component involved in loss of meat quality are oxidative re­
confirmation (Holman, Coombs, Morris, Kerr, & Hopkins, 2017). actions, which affect flavor, nutritional value, color, and texture. Lipid
Freezing is one of the most significant strategies for preserving food oxidation is caused by the production of malondialdehyde (MDA) and
quality during long-term storage (Culleré, Ferreira, Venturini, Marco, & cholesterol oxidation products because of the creation of oxy free and
Blanco, 2013), and minimizes quality loss (Sebranek, 1982). Frozen lipid-free radicals (Soyer, Özalp, Dalmış, & Bilgin, 2010). Proteins,
storage is used to slow down unwanted metabolic processes in meat, which are abundant in muscle cells, can be affected by oxidative pro­
although the formation of ice crystals causes cell rupture and muscle cesses. Protein oxidation causes various changes in proteins, including
fiber damage (Sebranek, 1982). Frozen storage may alter physical amino acid, protein-polymer formation, loss of solubility, and increase
properties, such as texture and drip loss; chemical processes, such as in carbonyl groups (Soyer et al., 2010). Protein free radicals are form
protein aggregation, denaturation, oxidation, color changes, lipolysis, when the side chains of proteins react with free radicals, which then
and lipid oxidation; and sensory properties of meat, including flavor and react with oxygen to generate peroxy radicals, decompose, and generate
sensory characteristics (Pérez-Palacios, Ruiz, Martín, Grau, & 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: [email protected] (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. Food Chemistry 376 (2022) 131881

discoloration, loss of water-binding ability, increased roughness, and 2.3. Preparation of samples
exudate loss (Coombs, Holman, Friend, & Hopkins, 2017).
The effects of frozen storage on the volatile content in various meat After removing the visible connective tissue and external fat, the beef
products have been explored. Soyer et al. (2010) evaluated the effects of primal was cut into 108 steaks (8 cm × 4 cm × 2 cm; 70.00 ± 2.00 g).
frozen temperature and storage time on protein and lipid oxidation in The 108 steaks were comprised of 3 marinade solution treatments × 3
chicken leg and breast meat. Frozen storage time substantially influ­ tumbling replicates × 4 frozen storage periods × 3 technical replicates.
enced lipid oxidation. Increase in carbonyl groups and reduction in total The steaks were then marinated in three different marinade solutions,
sulfhydryl groups in leg meat was observed after protein oxidation. which were: (1) 25% cold water as a control (BS1, n = 36), (2) water
Özer, & Seçen (2018) reported that the thiobarbituric acid reactive with 2% salt (BS2, n = 36), and (3) water with 2% salt and 0.5% sugar
substances (TBARS) and carbonyl values of raw and cooked beef burgers (BS3, n = 36).
steadily rose of up to 3 months of frozen storage. Iglesias et al. (2009)
identified some potential markers for distinguishing between fresh fish 2.4. Tumbling process and vacuum packing procedures
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 Raw beef steaks were blended with the individual marinade solution
for lipid oxidation in meats. (E,E)-2,4-Nonadienal, 1-octen-3-one, inside tumbling bowl in triplicate for each treatment (n = 12). A vacuum
hexanal, and trans-4,5-epoxy-(E)-2-decenal were identified as warmed- tumbler (VT50, Suhner Holding AG, Herisau, Switzerland) was used for
over flavor markers for boiled beef (Konopka, Guth, & Grosch, 1995). 2 h at 4 ◦ C. The rotating circle was programmed to turn on for 20 min
Qi et al. (2021) evaluated the effects of different frozen storage periods and then turn off for 10 min. A DZ-400/2S-vacuum packer (Shandong
(0–8 weeks) on the flavor of raw chicken meat. They found that frozen Xiaokang Co., Ltd, Shandong, China) was used under atmospheric
storage time of raw meat enhances its flavor profile upon stewing, pressure to pack each marinated beef steak in a polyethylene bag. All the
indicating that short-term frozen storage of raw meat improves its flavor procedures were carried out in a 4 ◦ C chilled environment.
profile. Sun, Zhang, & Song (2020) evaluated changes in flavor com­
pounds in cooked beef meatballs during frozen storage (up to 90 days) 2.5. Freezing and sampling procedure
using solid-phase microextraction (SPME) and solvent-assisted flavor
evaporation (SAFE) combined with gas chromatography–olfactometry- After the packaging procedure, packed steaks were stored in a freezer
mass spectrometry (GC-O-MS). Their sensory evaluation results showed at − 18 ◦ C. Samples were collected at different frozen storage periods (3
that general acceptance decreases with frozen storage. In addition, eight replicates × 3 marinade treatments × 3 tumbling replicates) of 0, 2, 4,
compounds were identified as aroma-active in cooked beef meatballs, and 6 months, for further analysis.
including 1-octene-3-ol, diallyl disulfide, linalool, eugenol, 2-ethyl hexyl
acetate, α-pinene, hexanal, and anisole. However, most previous studies 2.6. Physicochemical measurements
about frozen storage of meat and meat products used different meat
species, unmarinated raw beef or cooked beef. Furthermore, no previous 2.6.1. Color measurement
studies investigated the effects of frozen storage on the flavor profile of A CR-400 colorimeter (2-degree observer and illuminant D65; Kon­
raw beef meat marinated in salt and sugar combinations. Therefore, this ica Minolta, Tokyo, Japan) was used in assessing lightness (L*), redness
study aimed to evaluate the effects of long-term frozen storage on the (a*), and yellowness (b*) at five separate points on the surface of each
protein and lipid oxidation and flavor profile of marinated raw beef beef steak. The colorimeter was calibrated before measurement, using
meat. 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-
2. Material and methods samples sectioned from the 108 steaks (3 marinade solution treat­
ments × 3 tumbling replicates × 4 frozen storage periods × 3 technical
2.1. Samples replicates), totalling 324 sub-samples and 1620 color evaluations.

The beef meat used in this investigation was a single round primal 2.6.2. Ultimate pH determination
(M. semimembranosus, approximately 12–15 kg), from male Chinese The ultimate pH of the beef samples was determined immediately (0
crossbred Xia-Nan cattle (24–36 months old; 7 days post-mortem; 19.89 months) or from samples frozen for 6 months using a pH meter (MP512-
% protein and 3.37 % fat). The primal was obtained from Metro Mall 03, Hangzhou Tuqi Instrument Co., Ltd, China) after preparing a mixture
(Hefei, Anhui, China), packaged, and transferred to the laboratory in an of 5 g sample and 45 mL of distilled water (homogenized Ultra-Turrax
icebox, where it was kept at 4 ◦ C for further processing. 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
2.2. Chemicals and reagents measurement of the samples according to the method described by
Holman et al. (2017). Ultimate pH values were determined using three 5
Chemicals used in this study were octanal (99%), nonanal (96%), g sub-samples sectioned from 54 steaks (3 marinade solution treatments
trans-2-octen-1-ol (95%), and 1-heptanol (99%) (Shanghai Macklin × 3 tumbling replicates × 2 frozen storage periods × 3 technical repli­
Biochemical Co. Ltd., Shanghai, China); 1-octen-3-ol (98%), and fur­ cates), totalling 162 sub-samples.
aneol, (Shanghai Aladdin Bio-chem Technology Co., Ltd., Shanghai,
China); ethylenediamine tetraacetic acid disodium salt dehydrate 2.6.3. Lipid oxidation
(EDTA, 99.5%; Sisco Research Laboratories Pvt. Ltd. India); benzalde­ The lipid oxidation of marinated raw beef meat was determined at
hyde (99%), 1-hexanol (99%), 2-ethyl-1-hexanol (99.6%), 2(5H)-fur­ different times of frozen storage by measuring TBARSs according to the
anone (98%), hexanoic acid (99%), 2-thiobarbituric acid (98%), n- method described by Maraschiello, Sárraga, & García (1999) with some
alkanes (C7-C30), 1-octanol (99%), absolute ethanol, ethyl acetate, tri­ modifications. Briefly, 5 g of beef meat was soaked in ultrapure water
chloroacetic acid (TCA, 99%), and 2,4-dinitrophenyl hydrazine (DNPH) (20 mL) and homogenized by Ultra-Turrax T25 (IKA-Labortechnik,
(Sigma-Aldrich Co. Ltd., Shanghai, China); phenylethyl alcohol (98%) Staufen, Germany) for 10 s at 13500 rpm. The homogenate was then
(TCI Co., Ltd., Shanghai, China); HCl (36.5%-38%), and guanidine hy­ added to a cold 7.5% trichloroacetic acid (TCA; 50 mL) containing 1 g of
drochloride (99%; Thermo Fisher Chemical™, Shanghai, China). 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).

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S. Al-Dalali et al. Food Chemistry 376 (2022) 131881

In a 90 ◦ C water bath, the screw-capped tube was incubated for 30 min. technical replicates), totalling 162 sub-samples.
The TBARSs were measured in triplicate using a micro-plate reader
(Shimadzu, Japan) at 532 nm against a blank. Calibration curves were 2.7. Analysis of volatile flavor profile
measured at the same wavelength as that for the samples. A series of
concentrations from 1,1,3,3-tetraethoxypropane were prepared and 2.7.1. Extraction of volatile flavor compounds
taken through the TBA procedure explained before for the samples. The Volatiles were extracted from beef samples at different periods of
results were reported in µg MDA/kg sample. All the TBARS values were frozen storage using headspace-solid phase microextraction (HS-SPME)
determined in triplicate using three 5 g sub-samples sectioned from the according to the literatures (Sun et al., 2020; Al-Dalali, Li, & Xu, 2021).
108 steaks (3 marinade solution treatments × 3 tumbling replicates × 4 In a 40 mL headspace vial, 5 g of sample was weighed, and 10 µL of 2-
frozen storage periods × 3 technical replicates), totalling 324 sub- methyl-3-heptanone (0.42 mg/mL) was added as an internal standard.
samples and 972 TBARS evaluations. 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
2.6.4. Protein oxidation 40 min under the same temperature by divinylbenzene-carboxen-
Protein oxidation of marinated raw beef meat during different times polydimethylsiloxane (DVB-CAR-PDMS; Supelco, PA, USA). At the
of frozen storage was determined by measuring carbonyl contents after splitless mode, volatiles were thermally desorbed for 5 min at 250 ◦ C in
derivatization with DNPH according to the method described by Berardo the injection port of GC–MS. Volatiles were extracted from injections in
et al. (2016). In brief, the protein was precipitated by mixing 3 g of beef two columns using three 5 g sub-samples sectioned from the 108 steaks
meat with 30 mL of phosphate buffer (20 mM, pH 6.5 containing 0.6 M (3 marinade solution treatments × 3 tumbling replicates × 4 frozen
NaCl), and then four aliquots of 0.2 mL were treated with 1 mL of TCA storage periods × 3 technical replicates), totalling 324 sub-samples and
(10%). The supernatant was removed after centrifugation, and two ali­ 648 extractions.
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) 2.7.2. Separation of volatile flavor compounds
as blank. Then, 0.5 mL of TCA (20%) was added after 1 h of reaction at Volatiles were individually separated using DB-Wax (30 m × 0.25
37 ◦ C in the dark. After the centrifugation of the samples, the superna­ mm × 0.25 μm) and DB-5MS (60 m × 0.25 mm × 0.25 μm) columns
tants were discarded. Excess DNPH was washed three times with 1 mL of (Agilent Technology, USA). Shimadzu GC–MS (GC–MS-QP2010 Ultra)
a solution containing absolute ethanol and ethyl acetate (1:1, v/v). was used. The carrier gas was helium at a flow rate of 2 mL/min, and the
Finally, 1 mL of guanidine hydrochloride (6.0 M) in phosphate buffer oven temperature was set at 40 ◦ C for 3 min, then raised to 200 ◦ C at
(20 mM) was added to dissolve the pellets. Carbonyl content (nmol/mg 5 ◦ C/min, then at 10 ◦ C/min elevated to 250 ◦ C in the DB-5MS column
protein) was calculated according to the absorbance (determined in and 230 ◦ C in the DB-Wax column and maintained for 3 min. The MS
triplicate) with a micro-plate reader (Shimadzu, Japan) at 280 and 370 detector conditions were as follows: the ion source and line transfer
nm as follows: 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
A370
Carbonylcontent(nmol/mgprotein)= ×106 ionization voltage.
εhydrazone370 ×(A280 − A370 ×0.43)
2.7.3. Identification and quantitation of volatile flavor compounds
where εhydrazone370 = 22,000 M− 1⋅cm− 1. Carbonyl content were Volatiles were identified after matching their retention indices (RIs)
determined in triplicate using three 3 g sub-samples sectioned from the and mass spectra (MSs) in the DB-Wax and DB-5MS columns, with those
108 steaks (3 marinade solution treatments × 3 tumbling replicates × 4 reported in the National Institute of Standard and Technology Library
frozen storage periods × 3 technical replicates), totalling 324 sub- (NIST 14). Some of them were positively confirmed by injecting their
samples and 972 carbonyl content evaluations. standard chemicals. Finally, the concentrations of volatiles were deter­
mined with the internal standard method, and the following equation
2.6.5. Fatty acids composition analysis was used:
The fatty acid composition of beef samples was assessed according to ( )
a previously described method (Gonzales-Barron et al., 2021) with volatile
Peakarearatio internal ×con.of internalstandard
minor changes. Lipid was extracted from 5 g of beef samples with a
standard
Con.(μg/kg)= ×1000
chloroform/methanol mixture (2:1; v/v) and ultrasonic shaking for 20 Sampleweight
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, 2.8. Electronic nose (E-nose) analysis
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 The odor profile of the beef samples at 0 and 6 months of frozen
saponified at 60 ◦ C for 30 min with 100 µL of KOH (2 M) in methanol, storage were differentiated using an Alpha-MOS Fox Electronic Nose (E-
and 3 mL of n-hexane was added. After the mixture was agitated for 1 nose Fox 4000, France), according to the method described by Zhao
min, 2 g of anhydrous sodium sulfate was added and centrifuged at 1008 et al. (2020). In a 10 mL headspace vial, an accurate weight of a minced
g for 5 min. The resulting fatty acid methyl esters (FAMEs) were beef sample (3 g) was placed. The airflow rate was fixed to 150 mL/min,
collected, filtered with a 0.22 µm filter membrane, and analyzed using and analysis was conducted for 160 s at 60 ◦ C. The intensities of 18 metal
gas chromatography (GC, 7890A, Agilent Technologies) equipped with a oxide sensors were measured and reported. E-nose analyses were
flame ionization detector. A DB-Wax column (30 m × 0.25 mm × 0.25 determined using four 3 g sub-samples sectioned from 54 steaks (3
μm, Agilent, USA) was used to separate the FAMEs. The oven tempera­ marinade solution treatments × 3 tumbling replicates × 2 frozen storage
ture was set at 50 ◦ C for 1 min, raised to 200 ◦ C at 25 ◦ C/min, ramped to periods × 3 technical replicates), totalling 216 sub-samples.
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 2.9. Statistical analysis
(He). The FAMEs were identified by comparing their retention times
with those of authentic standards. The FAMEs were quantified according All the data were presented as mean ± S.D. Three sub-samples were
to the percentage of fatty acid peak area. FAMEs were quantified using randomly obtained from one beef round primal cut for examination at
three 5 g sub-samples sectioned from 54 steaks (3 marinade solution four different times of frozen storage for each of three marinade treat­
treatments × 3 tumbling replicates × 2 frozen storage periods × 3 ments (physical and chemical measures were independently processed

3
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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

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

5
S. Al-Dalali et al. Food Chemistry 376 (2022) 131881

∑ ∑ ∑
increased in amount over the course of frozen storage (Table 1). Protein USFA ( MUFA and PUFA) increased during frozen storage from
oxidation has been linked to lipid oxidation in different meat products 5.785% to 12.710% (BS1), 6.295% to 9.650% (BS2), and 7.640% to
(Mercier, Gatellier, & Renerre, 1995; Shimizu et al., 2009; Soyer et al., 10.837% (BS3; Table 2). After five freeze–thaw cycles (in one cycle, the
2010). Given that the products of primary (hydroperoxides) and sec­ fresh-packed muscle samples were frozen at − 20.0 ± 0.5 ◦ C for 24 h and
∑
ondary (aldehydes) lipid oxidation can serve as substrates for protein thawed at 4 ± 0.5 ◦ C for 12 h), SFA content increased from 21.6% to
∑
oxidation, once lipid oxidation begins, protein oxidation occurs (Soyer 35.3 %, and MUFA content increased from 16.1% to 30.5 % (He et al.,
et al., 2010). Shimizu et al. (2009) reported that the carbonyl content of 2021), consistent with the findings of the present study. Oleic acid
the control and salted yellowtail meat samples increased significantly (C18:1, cis-9) and palmitic (C16:0) were the most abundant mono­
during the 20 weeks of frozen storage. Soyer et al. (2010) evaluated the unsaturated and saturated fatty acids in the beef samples, followed by
effect of frozen storage period and temperature in the protein oxidation stearic acid (C18:0), as shown in Table 2. The free fatty acid content in
of chicken meat. They reported that protein oxidation in breast and leg beef tissue and complicated lipid hydrolysis during the freeze–thaw
meat significantly increased following frozen storage period. They process can contribute to fatty acids in meat samples. The existence of
stated that the frozen temperature showed no difference during the first radicals, which are oxidation indicators, can cause oxidative changes in
three months. In contrast, a significant difference was found with frozen beef lipids (Dragoev, Kiosev, Danchev, Ioncheva, & Genov,
continued frozen storage over than 3 months, especially at a frozen 1998). In addition, the enzyme oxidation or auto-oxidation of oleic acid
temperature of − 7 ◦ C compared to of − 12 ◦ C and − 18 ◦ C. They proposed (C18:1, cis-9) and linoleic acid (C18:2, cis-9,12) is extensively explained
∑ ∑
that lipids and proteins are oxidized after being frozen for a long time. (Fig. S1). Finally, BS3 had the highest USFA and MUFA content,
The rate of protein oxidation significantly increased in chicken meat followed by BS2 and BS1. Their content increased with frozen storage
stored at − 7 ◦ C, indicating that meat frozen at moderate temperature is period. Fatty acid composition was influenced by the marination solu­
more susceptible to protein oxidation probably because of lipid oxida­ tions and frozen storage.
tion products or oxidation catalysts. Similarly, several studies reported
increasing carbonyl content during cold storage (Eymard, Baron, &
3.2. Identification of flavor profile and their potential sources in beef meat
Jacobsen, 2009) and frozen storage (Baron, Kjærsgård, Jessen, &
samples
Jacobsen, 2007). Increases in total carbonyls suggest that oxidative al­
terations occur in meat during frozen storage because carbonyls are the
The flavor profiles of marinated raw beef meat products were iden­
main autoxidation products. When reactive oxygen species attack pro­
tified using HS-SPME-GC–MS. A total of 28 volatiles were identified in
teins, carbonyl compounds are formed due to the interactions (Soyer
all the samples, 16 of which were positively confirmed by their authentic
et al., 2010).
chemicals. The remaining 12 volatiles were tentatively identified by
matching their MS and RIs in two different columns (i.e., DB-Wax and
3.1.5. Fatty acids composition
DB-5MS) with those reported in the literature and the NIST library. The
Table 2 shows the fatty acid composition (% of peak area) and
identified flavor chemicals were obtained from various chemical fam­
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
∑ ∑ metabolite interaction from these reactions create the majority of flavor
SFA content. The SFAs increased from 11.805%, 11.280%, and
components in meat and meat products (Ramalingam, Song, & Hwang,
10.430% at 0 months to 19.735%, 15.520%, and 17.992% at 6 months
2019). The potential precursors and metabolic pathways of the main
for the BS1, BS2, and BS3 samples, respectively (Table 2). Our findings
flavor components were hypothesized on the basis of the literature and
corroborated the results of Nazemroaya, Sahari, & Rezaei (2009). They
with MetaCyc platforms and used in exploring the mechanism of flavor
found that saturated fatty acid content increased after being frozen. The
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. Food Chemistry 376 (2022) 131881

raw beef meat were produced mostly through amino and fatty acid storage of beef meat samples were determined using HS-SPME-GC–MS.
metabolism. For instance, octanal, hexanal, nonanal, 1-octen-3-ol, 1- The marinated beef meat products were analyzed after being frozen and
heptanol, and 1-octanol are common found in meat products because of kept for 0, 2, 4, and 6 months, and a total of 28 volatiles were identified
their low thresholds (Yin et al., 2021). They were mostly derived from and quantified (Table S1 and Fig. 1). The total concentrations of the
the enzyme-oxidation or auto-oxidation of unsaturated fatty acids, such quantified chemical groups fluctuated between decrease, increase, and
as linolenic, linoleic, and oleic acids (see Fig. S1), which were the most then decrease. In general, their concentrations decreased with
abundant unsaturated fatty acids in beef meat, accounting for 0.14%, increasing frozen storage period (Table S1). The total concentrations of
0.48%, and 4.5% of the total percentage, respectively (Table 2). aldehydes increased with frozen storage period (from 13.36 µg/kg to
Monounsaturated fatty acids, such as oleic acid, can undergo enzyme- 33.55 µg/kg) from 0 and 4 months in BS1. In BS2, the concentration
oxidation or auto-oxidation and produce various kinds of hydroperox­ increased from 58.96 µg/kg at 0 months to 61.25 µg/kg at 2 months of
ides, such as 8-, 9-, 10-, 11- or 13-ROOH, which are then decomposed frozen storage and then decreased to 19.5 µg/kg at 6 months (Table S1
into aldehydes through homolysis by hydroperoxide lyase (Wu et al., and Fig. 1A). The total concentrations of alcohols decreased with
2020). The aldehydes can be partially reduced to matching alcohols by increasing frozen storage period up to 6 months in all beef samples. The
aldehyde reductase. For instance, octanal and nonanal may be produced total concentration decreased from 34.92 µg/kg to 16.55 µg/kg (BS1),
through the homolysis of 8- and 9-ROOH, respectively (Wu et al., 2020). 75.67 µg/kg to 16.40 µg/kg (BS2), and 79.51 µg/kg to 25.84 µg/kg (BS3)
Flavor compounds are being generated from polyunsaturated fatty at 6 months of frozen storage (Fig. 1A). Merlo et al. (2021) reported no
acids, such as linoleic, linolenic, eicosapentaenoic, and arachidonic apparent linear decreasing or increasing patterns in the volatiles. Similar
acids, by generating various hydroperoxides after homolysis (Benet variations in volatiles in meat products during refrigerated storage have
et al., 2015). Hexanal is largely driven by the alkoxy radical-scission of been reported (Zhang et al., 2020). Qi et al. (2021) stated that with
13-ROOH derived from linoleic acid. 1-Octen-3-ol is produced by the longer frozen storage times for raw meat, the overall amount of volatile
catabolism of arachidonic and linoleic acids (Wu et al., 2020). Hexanal is chemicals increased first, then decreased, and then increased, and the
often regarded as an excellent predictor of fat oxidation level (Yin et al., ketones and aldehydes fell in line. Meanwhile, the levels of hydrocar­
2021). The only precursor for 3-methyl-1-butanal synthesis is L-leucine bons, alcohols, esters, and furans increased, and then decreased.
through the decomposition into α-ketoisocaproic acid by transaminase, Lipolysis, proteolysis, and oxidation are the primary biochemical
which is further degraded by decarboxylase into 3-methyl-1-butanal and processes that occur during the post-mortem ripening of meat and affect
then to 3-methyl-1-butanol (Fig. S1) (Wu et al., 2020). The Maillard the juiciness, tenderness, and flavor of meat. Flavor precursors, such as
reaction produces aldehydes with branching or complex structures, such sugars, peptides, amino acids, organic acids, and adenine nucleotide
as benzaldehyde, which can be identified by a Maillard reaction system breakdown products, are the chemicals produced during post-mortem
model containing phenyl glycine (Whitfield, & Mottram, 1992). 1-Hex­ and influence the formation of meat flavor and during frozen storage
anol is produced through the reduction of hexanal or through linoleic (Kosowska, Majcher, & Fortuna, 2017). These precursors are degraded
acid oxidation (Merlo et al., 2021). Farnesyl pyrophosphate synthase is during frozen storage or participate in the Maillard reaction during the
an enzyme that catalyzes the conversion of prenyl diphosphate into cooking process and lipid degradation during frozen storage, forming
geranyl diphosphate. Then, geranyl diphosphate is converted to (3R)- flavor compounds. For instance, nonanal, pentanal, 1-octen-3-ol, octa­
linalool by linalool synthase (Jia, Crock, Lu, Croteau, & Chen, 1999). nal, octanol, hexanal, hexanol, heptanal, heptanol, and hexanoic acids
Benzene ring ethers (i.e., estragole and anethole) are essential to the are formed primarily as a result of lipid oxidation processes, and 3-
development of flavor and enhancement of a mild beef flavor (Zhou methyl-1-butanol forms as a result of the Strecker degradation of an
et al., 2021). The precursors of these compounds are phenylpropanoids amino acid (i.e., leucine). The flavor of non-ripened meat is weak and
derived from phenylpropanoid biosynthesis. Estragole (also called tasteless, but the flavor of matured beef is enhanced and intensified. The
methylchavicol) is produced through methyl group transformation fatty flavor, beef-like, broth-lik, sweet, and caramel flavors are enhanced
facilitated by s-adenosyl-L-methionine (SAMe) from chavicol to meth­ by ripening for up to 14 days during chilled storage (Kosowska et al.,
ylchavicol (i.e., estragole). While, anethole is produced through the 2017).
transformation of coumaryl acetate to trans-anol under the action of anol In addition, the marination process contributes to the number and
synthase, which is further transformed to anethole by the aid of SAMe content of identified compounds. Fig. 2B, E, and H illustrate that BS2
(MetaCyc platforms). Furans are typically generated during carbohy­ and BS3 had high content of volatile flavor compounds compared with
drate breakdown induced by the lipid oxidation and decomposition of BS1 at 0 month of frozen storage. Specifically, 1-hexanol, octanal,
amino acids. They may be produced by the rearrangement and thermal estragole, hexanoic acid, trans-2-octen-1-ol, tetradecanal, pentadecanal,
disintegration of cellulose and hemicellulose (Yin et al., 2021). Furaneol 1-heptanol, 3-allyl-2-methoxyphenol, and hexanal were detected only in
can be produced through the conversion of D-glucose into β-D-fructose BS2 and BS3 (Table S1), in which marinade content (i.e., salt) acceler­
1,6-bisphosphate by glycolytic enzymes (i.e., glucose-6-phosphate ated lipid oxidation and led to the formation of the compounds. Alco­
isomerase) and then transformed into furanones (Wein, Lewinsohn, & hols, aldehydes, and ethers were the chemical groups with the highest
Schwab, 2001). Furaneol can also be considered an intermediate for the concentrations in the beef samples.
formation of other flavor chemicals, such as furanthiols, thiophenes, and Ten alcohols were identified and quantified, including 2-ethyl-1-hex­
other sulfur compounds, and has been identified in sheep meat, fried anol, 1-hexanol, 1-octanol, 3-methyl-1-butanol, trans-2-octen-1-ol, 1-
prawn meat, roasted goose’s leg meat, and beef broth (Gąsior et al., octen-3-ol, 1-heptanol, 4-carvomenthenol, phenylethyl alcohol, and
2021). Most phenols are produced during the pyrolysis of lignin or linalool. Seven of these alcohols were detected in BS1, 10 were identified
through the decomposition activities of microorganisms (Gąsior et al., in BS2, and nine were identified in BS3. Moreover, 1-hexanol, trans-2-
2021), and they are known as the standard flavor characteristics of octen-1-ol, and 1-heptanol were detected because of lipid oxidation in
smoked food, which they are contributing to the woody and smoky notes BS2, in which added salt accelerated lipid oxidation and promoted the
(Yin et al., 2021). Additionally, Gąsior et al. (2021) reported that the formation of the compounds. As shown in Table S1, some alcohols
presence of phenolic chemicals in meat could become from beef given decreased with increasing frozen storage period. By contrast, 1-heptanol
grass and/or grain diets. 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
3.3. Dynamic changes in flavor compounds during the frozen storage of the major aroma components of cooked beef meatballs during storage,
beef meat especially 1-octen-3-ol, hexanol, and 1-octanol (Sun et al., 2020).
Eight aldehydes were identified and quantified at different times of
Dynamic changes in volatile flavor compounds during the frozen frozen storage, including benzaldehyde, benzeneacetaldehyde, octanal,

7
S. Al-Dalali et al. Food Chemistry 376 (2022) 131881

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 before they could accumulate was another aspect that may have
concentrations fluctuated during continuous frozen storage, where they inhibited the formation of volatiles during frozen storage periods, as
increased, then decreased, and then increased for some of them. Some shown in Table S1. Moreover, some volatile compounds disappeared
aldehydes disappeared after 2 months of frozen storage, such as ben­ over time for several reasons, including oxidation, volatilization due to
zeneacetaldehyde, octanal, tetradecanal, and hexanal. Aldehydes have low boiling points during the preparation and extraction of flavor
green, fatty, grassy, and fresh characteristics, and these compounds are compounds, Strecker degradation, and the chemical reaction among
produced from oleic acid. For example, octanal accounts for the green, different compounds.
meaty, fresh, fruity, and grassy notes, whereas nonanal contributes Fig. 2 illustrates the PCA analysis and VIP scores of marinated raw
fruity and sweet aroma notes (Merlo et al., 2021). Sun et al. (2020) beef meat at different times of frozen storage. Fig. 2C, F, and I show the
found that these aldehydes are the major components in beef, particu­ important flavors (VIP ≥ 1.0) screened by PLS-DA at different times of
larly hexanal, pentanal, benzaldehyde, heptanal, and (E)-2-heptenal. frozen storage. The sums of PC-1 and PC-2 in Fig. 2A, D, and G were
Regarding phenols, 3,5-di-t-butylphenol, 3-allyl-2-methoxyphenol, 93.9%, 89.1%, and 90.2%, respectively, which explained nearly 90.0%
and isoeugenol formed during frozen storage. 3,5-Di-t-butylphenol of the total variance in the effect of frozen storage periods on flavor
formed and had concentrations of 0.40, 0.30, and 0.49 µg/kg at 6 profile of individual marinated raw beef meat.
months of frozen storage in BS1, BS2, and BS3, respectively (Table S1). Fig. 2B, E, and H clearly show the correlation and distribution in each
3-Allyl-2-methoxyphenol was generated at 6 months of frozen storage in frozen storage period and corresponding chemicals in each marinated
BS2 and BS3, with content of 6.61 and 3.76 µg/kg beef, respectively. sample. According to the sum of the first two components, frozen storage
Isoeugenol was generated at 2 months of frozen storage in all the sam­ affected the aroma profiles of tested samples. These results were further
ples, and its content increased with frozen storage time. Phenols are explored through GC–MS. PCA analysis showed that 1-hexanol, 2-ethyl-
produced during the pyrolysis of lignin or through the decomposition 1-hexanol, 3-methyl-1-butanol, 1-octen-3-ol, phenylethyl alcohol, and
activity of microorganisms (Gąsior et al., 2021). linalool were present in all the samples at 0 month of frozen storage
Furans and acids disappeared during frozen storage and were only (Table S1). This finding was supported by the VIP results, as shown in
detected at 0 months. Furans can come from a variety of sources. Furans Fig. 2 and consistent with the findings of Merlo et al. (2021), who stated
are mainly synthesized through the lipid oxidation of free fatty acids that 1-octen-3-ol is the major alcohol in meat products.
(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) 3.4. Screening of potential flavor compound markers during frozen
reported these two acids in smoked bacon. They stated that carbohy­ storage
drate fermentation in microorganisms is the most likely source of
organic acids in fermented meat products. Our results were in line with The samples yielded a total of 28 volatile flavor components. The
those reported by Merlo et al. (2021), who stated that samples frozen for effect of frozen storage on each marinated sample was determined with
up to 30 days contained higher amounts of volatiles than the samples the VIP method, and potential marker flavor components were screened.
stored for longer periods. Key metabolic changes, such as lipid oxida­ When the VIP of a flavor component was equal to 1.0 or higher, it was
tion, which promotes the production of volatiles, may have occurred in used as a marker to distinguish the effect of frozen storage period
the first 2 months of storage and may be related to this behavior. Such (Huang et al., 2019). A high VIP increased the likelihood of a compound
findings are supported by the results of TBARS, which showed the to be recognized. Fig. 2C, F, and I summarize the important flavor
highest values at 2 months of frozen storage and then decreased with compounds identified through PLS-DA, where 9 markers have distin­
increasing frozen storage period (Table 1) (Merlo et al., 2021). Sun et al. guished the effect of freezing in the BS2 sample. Among them, 6 flavor
(2020) found that the odor-active components in beef meatballs dis­ compounds were screened at the frozen storage periods of 2 and 4
appeared after storage, and the flavor dilution factor changed as well. months, including tetradecanal, octanal, 2-ethyl-1-hexanol, benzenea­
The conversion of aldehydes (i.e., hexanal) into alcohols (i.e., hexanol) cetaldehyde, 1-heptanol, and isoeugenol (see Fig. 2F). Seven markers

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

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S. Al-Dalali et al. Food Chemistry 376 (2022) 131881

4-carvomenthenol (spicy and woody) were recognized as markers in explained 42% of the total variance, indicating that the top two fac­
BS1. Fig. S2 illustrates the PCA analysis results for marinated raw beef tors clearly determined the correlations among the variables. The sam­
samples with different marinade solutions. The sum of PC-1 and PC-2 in ples varied in terms of the variables. In the first factor, all the samples
Figs. S2A, C, E, and G were 100% for all of them, which explained all the subjected to frozen storage durations for 4–6 months were close
variances concerning the effect of the marination process on the flavor together, indicating similarity, as shown in Fig. 4A. These samples
profile of beef meat at different times of frozen storage. Figs. S2B, D, F, showed the characteristic flavor components belonging to alcohols, al­
and H clearly show the correlation and distribution in each marinade dehydes, ethers, and phenols and high carbonyl content and pH values,
solution and corresponding chemicals in each frozen storage. According confirming that most of the components were generated because of lipid
to the sum of the first two components, it can be concluded that the and protein oxidation during frozen storage. All the samples at 2 months
marination process contributed to the aroma profiles of the tested of frozen storage were located in the second factor, which presented
samples. high contents of lipid-derived components, such as hexanal, pentade­
canal, tetradecanal, benzeneacetaldehyde, octanal, benzaldehyde,
3.6. Electronic nose (E-nose) results nonanal, and 2-ethyl-1-hexanol and exhibited high TBARS levels
(Fig. 4B). These components were the major compounds generated
An E-nose is a rapid and effective tool for detecting a product’s through lipid degradation. All the samples at 0 months of frozen storage
aroma that does not require specific sample preparation. The metal were positioned at the top of the second factor, presenting high levels of
oxide sensors in an E-nose imitate the actions of a biological olfactory hexanoic acid, estragole, acetic acid, phenylethyl alcohol, and 2(5H)-
organism. Therefore, they can distinguish among the flavor profiles of furanone. This result might indicate that lipid and protein oxidation and
meat products by simulating the sense of smell of biological organisms the marinade solution contents were responsible for flavor formation
(Di Rosa, Leone, Cheli, & Chiofalo, 2017). Sensors P30/2, P40/2, P30/1, and changes of raw beef meat during different durations of frozen
PA/2, T70/2, P40/1, P10/2, P10/1, TA/2, and T30/1 clearly distin­ storage.
guished the differences among the different marinated samples at 0 and
6 months of frozen storage, especially the BS2 sample, which showed the 4. Conclusions
highest response intensities compared to the other samples (Fig. 3). The
response intensities of the metal oxides decreased with increasing frozen A total of 28 volatile flavor compounds were identified in all the
storage period of 6 months. This finding was in line with the GC–MS samples. The concentration of each identified compound fluctuated with
results; in which the concentrations of most quantified compounds frozen storage (increasing, decreasing, or increasing and then
decreased with increasing frozen storage. 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
3.7. Correlation analysis between lipid and protein oxidation and flavor
fied in BS1, 28 compounds were identified in BS2, and 27 compounds
changes through partial least squares discriminant analysis (PLS-DA)
were identified in BS3. Furthermore, lipid oxidation occurred during the
first 2 months of frozen storage. The TBARS values increased to
After data normalization, PLS-DA was used in elucidating the cor­
approximately 39.11 and 18.79 µg MDA/kg for BS1 and BS2, respec­
relations among lipid oxidation, protein oxidation, and flavor changes in
tively, and then slightly decreased with continued frozen storage dura­
the marinated samples at different times of frozen storage. The results
tion to 6 months. Carbonyl content linearly increased with frozen
clearly showed the correlation, as demonstrated in Fig. 4A and B, in
storage period, indicating that lipid degradation and marinade solutions
terms of volatile flavor components, TBARS, carbonyl content, and pH
were the main factors that influenced the aroma profile of raw beef
value. Factor-1 explained 74% of the total variance, and factor-2

LY2/LG
TA/2 LY2/G BS1-0
0.50 BS2-0
T40/1 0.45 LY2/AA
0.40 BS3-0
0.35 BS1-6
0.30
T40/2 0.25 LY2/GH BS2-6
0.20 BS3-6
0.15
0.10
0.05
P30/2 0.00 LY2/gCTl
-0.05
-0.10

P40/2 LY2/gCT

P30/1 T30/1

PA/2 P10/1
T70/2 P10/2
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.

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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.

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