Abstract
The quality and shelf life of beef are significantly affected by
storage conditions, emphasizing the urgent need for efficient
preservation technologies to extend shelf life and reduce waste. This
study evaluated the quality changes of beef during storage under
low-voltage electrostatic field (LVEF) combined with partial freezing
and under partial freezing alone. The results showed that during the
0–24 day storage period, LVEF effectively slowed the increases in pH,
thiobarbituric acid reactive substances, total volatile basic nitrogen,
and total viable counts, maintained tenderness, and delayed protein
oxidation. Metabolomics analysis indicated that LVEF inhibited the
formation of off-flavors such as hexanal, 1-octen-3-ol, and
benzaldehyde, and reduced the loss of inosine monophosphate, thereby
preserving beef quality. Metabolic network analysis further revealed
that these metabolites were closely associated with the metabolism of
fatty acids, phenylalanine, and proline. These findings suggest that
LVEF can effectively enhance the storage quality of beef.
Keywords: Beef, Low-voltage electrostatic field, Metabolomics, Flavor
quality
Graphical abstract
[33]Unlabelled Image
[34]Open in a new tab
Highlights
* •
Low-voltage electrostatic field can extend the shelf life of beef.
* •
Using metabolomics to reveal quality changes in beef during
storage.
* •
The main metabolic pathway during beef storage is arginine
biosynthesis.
* •
Octanal and benzaldehyde are closely associated with beef quality.
1. Introduction
Beef is high in protein, low in fat, and tasty, and is popular with
consumers. However, beef is susceptible to microbial contamination
during transportation, storage and marketing, leading to quality
deterioration and meat spoilage ([35]Casaburi et al., 2015). Proteins
will decompose under the action of microorganisms and enzymes to
produce metabolites such as trimethylamine and sulfide with a spoilage
odor ([36]Lin et al., 2022). Moderate fat oxidation helps to produce
volatile compounds with characteristic aroma. However, excessive lipid
oxidation produces odors such as rancidity. With the extension of
storage time, metabolites continue to accumulate and further generate
off-flavor volatile compounds, which significantly deteriorate beef
quality. Studies have shown that compounds such as benzaldehyde,
octanal, and nonanal are closely associated with meat spoilage
([37]Huang et al., 2022). According to statistics, the global waste
rate of fresh meat products is about 20–30 %. Therefore, developing
efficient preservation technologies to delay beef quality deterioration
is of significant practical importance for reducing resource waste and
ensuring meat quality.
Freezing is a common preservation method that can significantly extend
the shelf life of meat; however, ice crystals formed during the
freezing process can damage muscle tissue, resulting in moisture loss
and tissue softening, which in turn lead to a decline in product
quality ([38]Tan et al., 2021). During frozen storage, intrusion of ice
crystals into surimi tissues and mechanical damage can lead to thawing
losses, affecting the oxidation and aggregation of surimi proteins,
resulting in increased hardness and reduced palatability of surimi
([39]Yang, Jiang, et al., 2024). In addition, frozen storage may reduce
the tenderness of pork, increase total exudate, and decrease the
volatile flavor compounds of hand-pulled lamb([40]Luo et al., 2023;
[41]Medić et al., 2018). Due to the limitations of conventional
freezing storage, partial freezing storage—which involves storing
samples at temperatures 1–2 °C below their biological freezing
point—has attracted increasing attention for its ability to better
preserve the original quality of food. [42]Cao et al. (2023) studied
four low-temperature storage methods for beef and found that the shelf
life of partial freezing beef was approximately 18 days. [43]Liu et al.
(2013) found that partial freezing storage can slow down the changes in
pH, total volatile basic nitrogen (TVB-N), and TCA-soluble peptides in
fish fillets. [44]Qiu et al. (2020) found that partial freezing storage
can delay protein oxidation and inhibit the activity of tissue
proteases. However, partial freezing storage alone still faces the
problem of a relatively short shelf life, which limits its widespread
application.
Low-voltage electrostatic field (LVEF) is an emerging non-thermal
preservation technology characterized by low energy consumption,
environmental friendliness, and operational safety, and has thus
attracted widespread attention. This technology can resonate with water
molecules inside cells to interfere with the nucleation and growth of
ice crystals, affect biochemical reactions, and inhibit microbial
growth, thereby extending the shelf life of food ([45]Jha et al.,
2017). Currently, LVEF is mainly used for defrosting meat, e.g. beef,
pork, etc. However, LVEF has also been used for meat storage and
preservation. [46]Zhang et al. (2023) found that LVEF combined with
partial freezing could inhibit the increase of TVB-N and total viable
counts (TVC) and extend the shelf-life by 3 d. [47]Wu et al. (2024)
investigated the effects of LVEF-assisted freezing at different
temperatures on the physicochemical properties of pork and found that
LVEF-assisted freezing could inhibit ice crystal formation, maintain
the integrity of the muscle microstructure, and reduce protein
denaturation caused by freezing. [48]Yang, Wu, et al. (2024) found that
LVEF synergized at −12 °C could effectively inhibit the rise of
carbonyl content and surface hydrophobicity, maintain the integrity of
protein secondary and tertiary structures, and reduce the cross-linking
aggregation of proteins.
At present, research on the application of LVEF in meat preservation
remains limited. Existing studies have primarily focused on its effects
on the physicochemical properties of meat, with a lack of systematic
analysis of metabolic changes in beef during storage. Therefore, this
study employed a metabolomics approach to systematically evaluate the
quality changes in beef under LVEF combined with partial freezing
storage, with the aim of extending the shelf life of beef and reducing
resource waste. This work not only helps to broaden market strategies
and distribution channels for beef products, but also provides
theoretical and technical support for the practical application of LVEF
technology in meat preservation.
2. Materials and methods
2.1. Sample preparation
In this study, six Guizhou native yellow cattle with similar body
conditions, approximately 2 years of age and weighing around 300 kg,
were selected from the Guizhou Yellow Cattle Industry Group Co., Ltd.
To ensure consistency across experimental samples, all samples were
taken from the hind leg muscles. The cattle were slaughtered at a
certified slaughterhouse following standard operating procedures.
Immediately after slaughter, the beef was placed in insulated
containers with ice and transported to the laboratory. Under sterile
conditions, the samples were cut into small pieces weighing 500 ± 50 g
and sealed in sterile polyethylene bags. Subsequently, all samples were
randomly divided into two groups, with 18 samples in each group. LVEF
combined with partial freezing group (LP group): Samples were stored in
a − 5 ± 1 °C cold storage equipped with a LVEF device (Model: DENBA+,
Discharge plate model: DP-10, Waterproof discharge cloth model: DP-2,
AGUA business corporation, Japan). In addition, multiple electrode
plates were effectively installed around the cold storage room. The
electrostatic field generator produces 2500 V of output voltage, 220 V
of alternating voltage, 0.2 mA of current, and 50 Hz of frequency. The
LVEF generator was installed outside the cold storage chamber, while
the electrode plates were mounted in an opposing configuration on the
interior side walls. Upon activation of the power supply, an
electrostatic field environment was established between the electrodes.
Partial freezing group (PS group): Samples were stored in cold storage
at −5 ± 1 °C. On days 0, 6, 12, 18, and 24, samples were randomly
collected from each experimental group. After the samples were chopped,
visible connective tissue and fat were removed as much as possible, and
the meat was then ground using a meat grinder. Relevant physicochemical
parameters were measured subsequently. For each measurement, three
portions of beef from the LP and PS groups were taken, and the average
value was used as the representative measurement for that sample.
2.2. Determination of pH
The pH was determined following the method described by [49]Cao et al.
(2022), with minor modifications. A total of 5.0 g of beef was mixed
with 45 mL of physiological saline and homogenized for 1 min, after
which the pH was measured using a pH meter (PHS-3C, Shanghai Youping
Scientific Instrument Co., Ltd.). The pH meter was calibrated with
phosphate buffers of pH 4.00 and 6.86 prior to measurement.
2.3. Determination of TVB-N
TVB-N was determined using a volatile basic nitrogen rapid detection
kit (Xiamen Standao Scientific Instruments Co., Ltd.), following the
same method as described in the study by [50]Wang et al. (2025).
2.4. Determination of TBARS
TBARS was determined following the method described by [51]Jiang, Li,
et al. (2024), with minor modifications. Briefly, 5.0 g of beef sample
was homogenized with 25 mL of trichloroacetic acid solution, thoroughly
mixed, and then incubated in a thermostatic shaker at 50 °C for 30 min.
Then the mixture was filtered through qualitative filter paper.
Subsequently, 5 mL of the filtrate was transferred into a test tube and
mixed with 5 mL of thiobarbituric acid aqueous solution. The tube was
sealed with a stopper and incubated in a water bath at 90 °C for
30 min. After cooling to room temperature, the absorbance was measured
at 532 nm using a microplate reader (Multiskan SkyHigh, Thermo Fisher
Scientific, USA).
2.5. Determination of total viable counts (TVC)
TVC was determined following the method described by [52]Jiang, Liu, et
al. (2024), with minor modifications. Briefly, 2.5 g of beef sample was
aseptically weighed and mixed with 22.5 mL of sterile physiological
saline (0.85 %), followed by homogenization for 1 min. The homogenate
was then subjected to a series of tenfold serial dilutions using
sterile saline solution. An aliquot of 1 mL from the appropriate
dilution was inoculated onto plate count agar and incubated at
37 ± 1 °C for 48 ± 2 h in a constant-temperature and
humidity-controlled incubator. TVC was expressed as lgCFU/g.
2.6. Determination of shear force
Shear force was determined following the method described by [53]Cao et
al. (2022), with minor modifications. The beef samples were cut into
small cubes of 1 cm × 1 cm × 1 cm and measured along the vertical
direction using a digital muscle tenderness tester (C-LM3B, Beijing
Tianxiang Feidu Instrument Co., Ltd.).
2.7. Determination of total sulfhydryl and carbonyl
The total sulfhydryl content was determined following the method
described by [54]Lin et al. (2024) with minor modifications. A total of
10.0 g of beef was weighed and added to 40 mL of 0.1 mol/L sodium
chloride solution, homogenized for 1 min (5000 g), and then centrifuged
at freezing temperature for 15 min (5000 g, 4 °C). The supernatant was
discarded, and the process was repeated three times. After the third
centrifugation cycle, the supernatant was discarded, the mixture was
homogenized with sodium chloride solution, and filtered through four
layers of gauze. The precipitate obtained after centrifugation was the
myofibrillar protein. It was then diluted with a phosphate buffer
solution (pH = 6.5), stored at −80 °C, and set aside.
After pipetting 0.5 mL of the protein solution, 2.5 mL of
Tris-Glycine-8 M urea solution and 20 μL of Ellman's reagent were
added. The mixture was thoroughly mixed and incubated at room
temperature for 1 h. It was then centrifuged at 1000 g for 15 min, and
the supernatant was collected. The absorbance was measured at 412 nm.
Carbonyl content was determined using the instructions of Protein
Carbonyl Content Assay Kit (Soleberg Biotechnology Co., Ltd.).
2.8. Determination of volatile flavor compounds
Volatile flavor compounds were analyzed by a gas chromatography–mass
spectrometry (GC–MS) system (Trace1300-TSQ8000, Thermo Fisher
Scientific, USA). The volatile flavor compounds was determined
following the method described by [55]Wang et al. (2025) with minor
modifications. A total of 3.00 g of beef was weighed and mixed with
2 mL of saturated sodium chloride solution and 20 μL of the internal
standard methyl octanoate (30 μg/mL). The mixture was then transferred
into a 20 mL headspace vial, which was sealed with a
polytetrafluoroethylene-silica gel septum. The sample was equilibrated
at 60 °C for 20 min. Subsequently, the automatic extraction head coated
with fibers was inserted into the solid-phase microextraction device.
The device was then placed into the headspace vial and shaken at 60 °C
for 40 min to extract and adsorb the volatile flavor compounds. The
fibers were immediately transferred to the gas chromatography injection
port, where the adsorbed compounds were thermally desorbed and analyzed
at 250 °C for 5 min. Gas chromatography conditions: A CP-WAX 57 CB
capillary column (50 m × 0.25 mm × 0.2 μm, Agilent Technologies, USA)
was used. Helium was employed as the carrier gas at a flow rate of
1.0 mL/min in splitless mode. The chromatographic temperature program
was initiated at 40 °C and held for 2 min, then ramped at 5 °C/min to
120 °C, followed by a 3-min hold. The temperature was then ramped at
5 °C/min to 180 °C and held for 3 min. Mass spectrometry was performed
in electron ionization (EI) mode at an ionization energy of 70 eV. The
ion source and transfer line temperatures were set to 250 °C and
230 °C, respectively. The full scan mass range was from 30 to 550 m/z.
The obtained mass spectra were compared with the National Institute of
Standards and Technology (NIST) mass spectral database, and only
compounds with a matching score ≥ 700 were retained for further
analysis.
Quantitative analysis: 20 μL of 30 mg/mL methyl octanoate solution was
used as the internal standard. Volatile compound content is calculated
using the following formula:
[MATH: Ci=Cis×Ai
×VAis×m
:MATH]
Where C[i] is the mass concentration of any volatile flavor compound
(μg/kg); A[i] is the chromatographic peak area of a volatile flavor
compound; A[is], C[is], and V represent the peak area of the internal
standard, its concentration (μg/mL), and volume (μL), respectively; and
m is the sample mass (g).
2.9. Non-volatile metabolites
A total of 100 mg of ground beef sample was weighed and transferred
into an EP tube, and then 500 μL of aqueous methanol (80 %) was added.
The EP tube was vortexed, shaken, and then placed on ice for 5 min,
followed by centrifugation for 20 min (1500 g, 4 °C). The supernatant
was collected, diluted to 53 % methanol, and centrifuged again for
20 min (1500 g, 4 °C). The supernatant was then injected into the
Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LC-MS/MS)
system for analysis. The liquid chromatography conditions were
described by [56]Fu et al. (2024) with slight modifications. Liquid
chromatographic conditions: The Hypersil Gold C18 column (Agilent
Technologies, Santa Clara, CA, USA) was maintained at 40 °C. The flow
rate was 0.2 mL/min, the mobile phase A was 0.1 % formic acid, and the
mobile phase B was methanol. The chromatographic gradient elution
program was as follows: 2 % B, 0 min–1.5 min; 2 %–85 % B,
1.5 min–3 min; 85 %–100 % B, 3 min–10 min; 100 %–2 % B, 10 min–12 min.
Mass spectrometry conditions: scanning range from 100 to 1500 m/z,
spray voltage of 3.5 kV, sheath gas flow rate of 35 L/min, auxiliary
gas flow rate of 10 L/min, ion transfer tube temperature of 320 °C, RF
level for ion introduction set to 60, auxiliary gas heater temperature
of 350 °C, polarity set to both positive and negative modes, and MS/MS
performed in data-dependent acquisition mode.
2.10. Statistical analysis
Experimental data were performed in three independent experiments and
were expressed as mean ± standard deviation. Data were analyzed using
SPSS Statistics 26 (IBM Corporation, Armonk, NY, USA), and significance
was determined using one-way analysis of variance (ANOVA) and
Duncan-Watson test or two-tailed test. Principal component analysis
(PCA) and orthogonal partial least squares discriminant analysis
(OPLS-DA) were performed using SIMCA 14.1 software (Umetricus AB,
Sweden). Heatmaps were generated using TBtools, and volcano plots were
created via the online platform Bioinformatics
([57]https://www.bioinformatics.com.cn). Pathway enrichment analysis
and bubble plots were performed using Metabo Analyst 6.0
([58]https://www.metaboanalyst.ca). All other graphical visualizations
were generated using Origin 2021 (Origin Lab, Northampton, MA, USA).
3. Results and discussion
3.1. Changes in pH, TVB-N, TBARS, and TVC during beef storage
Changes in pH can reflect the process of muscle tissue breakdown. The
pH of fresh meat (FM) was 5.35, as shown in [59]Fig. 1a. During
storage, the pH of beef tended to decrease and then increase. After
beef slaughter, anaerobic metabolism of glycogen breakdown in muscle
produces lactic acid, which lead to a lower pH in early beef ([60]Lin
et al., 2022). And microorganisms in beef break down proteins into
alkaline substances such as ammonia and amino acids, which raises pH.
At 24 d of storage, the pH of LP and PS groups increased to 5.63 and
5.73, respectively. Notably, the pH of LP group was significantly lower
than that of PS group (p < 0.05), suggesting that LVEF effectively
inhibited pH elevation during beef storage, consistent with the
findings of [61]Ko et al. (2016).
Fig. 1.
[62]Fig. 1
[63]Open in a new tab
Changes in pH (a), TVB-N (b), TBARS (c), and TVC (d) of beef during
storage. Different capital letters (A–B) indicate significant
differences among storage methods at the same storage time, while
different lowercase letters (a–e) indicate significant differences
among storage times under the same storage condition (p < 0.05), as
below.
TVB-N refers to the microbial decomposition of proteins into ammonia or
amines in the case of spoilage of animal food, which reflects the
freshness of the food to a certain extent. The Chinese National Food
Safety Standard (GB 2707–2016) specifies a limit of 15 mg/100 g TVB-N
for fresh beef ([64]Fu et al., 2024). The TVB-N value of FM was
2.33 mg/100 g as shown in [65]Fig. 1b. The TVB-N values of beef
gradually increased with storage time. This was caused by
microorganisms and protein hydrolyzing enzymes breaking down the
proteins in meat and the gradual accumulation of alkaline nitrogenous
substances ([66]Alirezalu et al., 2021). Notably, LP group rose more
slowly and had a significantly lower TVB-N value of 10.37 mg/100 g at
24 d of storage than PS group (p < 0.05). The ozone generated by the
electrostatic field inhibited the growth of microorganisms and the
activity of enzymes, thereby suppressing the production of TVB-N
([67]Papachristodoulou et al., 2018).
Lipid oxidation generates aldehydes and ketones, which are associated
with unpleasant, sour odors and can diminish beef quality. As shown in
[68]Fig. 1c, the TBARS value for FM was approximately 0.16 mg/kg.
Throughout the storage period, the TBARS values of all beef samples
exhibited an upward trend, with LP group consistently showing
significantly lower TBARS levels compared to PS group (p < 0.05).
Increased oxidation of lipids and proteins in meat with longer storage
time, leading to increased levels of TBARS. At 24 d of storage, the
TBARS values for LP and PS groups were 0.35 mg/kg and 0.5 mg/kg,
respectively. This difference can be attributed to the electrostatic
induction effect of LVEF, which charges the food surface, reducing its
contact with oxygen and thereby slowing lipid oxidation ([69]Ko et al.,
2016).
TVC is a direct and effective indicator of meat freshness and shelf
life. [70]Chen et al. (2019) considered 6.00 lgCFU/g as the upper
acceptable limit for fresh beef. As shown in [71]Fig. 1d, the TVC
values of beef increased with storage time. This was due to the fact
that meat samples are rich in protein, which can be used as a medium
for microbial reproduction. The initial TVC in beef was 3.96 lgCFU/g.
At 18 d of storage, the TVC of LP group and PS groups were 4.87 and
6.07 lgCFU/g, respectively, and those of PS group exceeded the upper
limit for fresh meat. At 24 d of storage, the TVC of LP group was 5.92
lgCFU/g, still within the fresh meat range. [72]Zhang et al. (2024)
reported that high-voltage electric fields can slow the physicochemical
and microbial growth rates in fish during storage, effectively
preserving its original flavor and nutritional value. Overall, LVEF
inhibited the rate of increase in pH, TVB-N, TBARS and TVC during
storage, thereby effectively extending the shelf life of beef.
3.2. Changes in shear force during beef storage
The tenderness of meat plays an important role in the consumer's
perception of its palatability ([73]Lin et al., 2022). As shown in
[74]Fig. 2, the shear force of the beef continued to decrease as the
storage time increased. The shear force of FM was 65.72 N. At 24 d of
storage, the shear force of LP and PS groups decreased by 45.80 % and
63.99 %, respectively, and the shear force of LP group beef was
significantly higher than that of PS group (p < 0.05). LVEF accelerates
the freezing rate, resulting in smaller and more uniform ice crystals,
which minimizes damage to muscle tissue and effectively preserves the
shear force of beef ([75]Xie et al., 2021). The results showed that
LVEF storage delayed beef quality deterioration, thus maintaining the
texture of fresh meat.
Fig. 2.
[76]Fig. 2
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Variation of shear force during storage of beef.
3.3. Changes in total sulfhydryl and carbonyl during beef storage
Total sulfhydryl is one of the most functional groups in proteins and
can not only affect the enzymatic activity within the protein but is
also closely linked to protein denaturation. As shown in [78]Fig. 3a,
the total sulfhydryl content of myofibrillar proteins of FM was
104.79 μmol/g. The total sulfhydryl content of beef decreased
significantly with increasing storage time. The initial carbonyl
content in FM was 0.66 μmol/g. After 24 days of storage, the carbonyl
content increased to 2.32 μmol/g in the LP group and 3.35 μmol/g in the
PS group, with the LP group showing a significantly lower level than
the PS group (p < 0.05). Previous studies have shown that LVEF can
effectively inhibit sulfhydryl oxidation ([79]Yang et al., 2023). The
formation of carbonyl derivatives and disulfide bonds can cause
intramolecular and intermolecular cross-linking of proteins, which can
adversely affect beef quality. As shown in [80]Fig. 3b, Carbonyl
content in beef increased with storage time, indicating increased
oxidation of beef myofibrillar proteins. The carbonyl content of FM was
0.66 μmol/g, and after 24 days of storage, the carbonyl content of beef
from the LP and PS groups was 2.32 and 3.35 μmol/g, respectively. An
electric field can accelerate the freezing rate of water and promote
the formation of small ice crystals, thereby reducing damage to muscle
fibers and inhibiting the denaturation of myofibrillar proteins.
Meanwhile, because small ice crystals cause less damage to muscle
tissue and cellular structures, they can delay the release of lysosomal
enzymes and pro-oxidative factors within the cells, further suppressing
protein oxidation. Therefore, LVEF can effectively inhibit the
oxidation of beef myofibrillar proteins and extend their shelf life.
Fig. 3.
[81]Fig. 3
[82]Open in a new tab
Changes in total sulfhydryl (a) and carbonyl content (b) of beef during
storage.
3.4. Analysis of volatile flavor compounds during storage of beef
3.4.1. Analysis of the composition and content of volatile flavor compounds
in beef
During the storage period of beef samples, a total of 92 volatile
flavor compounds were identified, and their chromatograms are shown in
Fig. S1. These compounds included 22 alcohols, 22 aldehydes, 13
alkanes, 15 esters, 8 ketones, 3 acids, and 9 others. Among them, 74
compounds were identified in LP group, and 70 compounds were identified
in PS group. These volatile flavor compounds are generated during
storage through processes such as protein degradation, lipid and
carbohydrate metabolism, and microbial decomposition. As shown in
[83]Fig. 4a and b, a total of 40 volatile flavor compounds were
detected in the FM, with a total content of 854.88 μg/kg. Among them,
aldehydes and alcohols accounted for 47.21 % and 27.70 %, respectively,
indicating that these two types of compounds play a significant role in
the flavor of beef. This result is similar to the findings reported by
[84]Ba et al. (2012). From 0 to 24 d, the total content of volatile
flavor compounds in beef showed a trend of increasing and then
decreasing. The total volatile flavor compounds reached a maximum of
3540.28 μg/kg at 18 d for LP group and 3021.51 μg/kg at 12 d for PS
group. At 24 d of storage, the total content of volatile flavor
compounds in the LP group and the PS group was 2767.30 μg/kg and
1754.80 μg/kg, respectively, with the LP group showing a significantly
higher content than the PS group. This difference may be attributed to
the effect of the low-voltage electrostatic field (LVEF) on the
transmembrane potential and electron transfer of muscle cell membranes
during beef oxidation, which in turn altered the formation pathways and
rates of volatile flavor compounds, resulting in differences in their
content between the two groups ([85]Jiang, Li, et al., 2024).
Fig. 4.
[86]Fig. 4
[87]Open in a new tab
Changes in volatile flavor compounds during beef storage. a: Species;
b: Total content.
3.4.2. Analysis of differential volatile flavor compounds
PCA was used to analyze the differences in volatile flavor compound
content. As shown in [88]Fig. 5a, the first two principal components
(PC1 and PC2) account for 20.1 % and 14.9 % of the total variance,
respectively. In the score plot, the nine samples show a clear
separation, with the LP group clustered in the positive direction of
PC1 and the PS group in the negative direction, suggesting that LVEF
may have a significant influence on the volatile flavor compounds of
beef. To further investigate the impact of LVEF on beef flavor, OPLS-DA
was used to analyze volatile flavor compounds, and the reliability of
the model was verified through 200 permutation tests, as shown in
[89]Fig. 5b and c. Based on a VIP > 1 and p < 0.05 in the OPLS-DA
analysis, 21 differential volatile flavor compounds were identified in
the beef ([90]Fig. 5d). Odor activity value (OAV) is an important
indicator used to evaluate the contribution of volatile compounds to
the overall flavor. It is defined as the ratio of a compound's
concentration in the sample to its odor threshold. When OAV > 1, it
indicates that the concentration of the compound exceeds the human
sensory threshold and may contribute perceptibly to the overall aroma
of the sample. Therefore, we calculated the odor activity values (OAVs)
of 21 differential volatile compounds and identified 14 key ones
(OAV > 1).
Fig. 5.
[91]Fig. 5
[92]Open in a new tab
Analysis of volatile flavor compounds during beef storage. a: PCA; b:
OPLS-DA; c: substitution test; d: Thermogram of significantly different
volatile flavor compounds.
In this study, five significantly different alcohol compounds were
screened from beef samples, including 1-octen-3-ol, 1-hexanol,
1-heptanol, 1-octanol, and trans-2-octen-1-ol. 1-Octen-3-ol is formed
through lipid oxidation and imparts an undesirable mushroom-like odor
to beef. During storage, the content of 1-octen-3-ol showed a trend of
initially increasing and then decreasing. In FM, the concentration of
1-octen-3-ol was 120.14 μg/kg. After 12 days of storage, the
concentration of 1-octen-3-ol in the PS group reached a maximum of
636.26 μg/kg (OAV: 636.26), which was significantly higher than that
observed in the LP group (408.76 μg/kg, p < 0.05). These results
indicate that LVEF may exert a delaying effect on the formation of this
volatile compound. By day 24 of storage, the concentrations of
1-octen-3-ol in the LP and PS groups were 410.22 μg/kg and
109.58 μg/kg, respectively, with a significant decline observed in the
PS group. This reduction may be associated with the gradual depletion
of metabolic precursors within the beef matrix during storage. In
contrast, the application of LVEF combined with micro-freezing storage
effectively delayed the formation and accumulation of 1-octen-3-ol,
resulting in significantly higher levels in the LP group at the later
stage of storage compared to the PS group. 1-Hexanol has a grassy or
fruity aroma and a low sensory threshold, which contributes to
enhancing the fresh aroma of beef. In FM, the content of 1-hexanol was
24.15 μg/kg (OAV: 3.02). After 24 days of storage, the concentration of
1-hexanol in the LP group was 98.31 μg/kg (OAV: 12.29), which was
significantly higher than that in the PS group at 27.81 μg/kg (OAV:
3.28). Furthermore, studies have shown that 1-hexanol exhibits
antioxidant and antibacterial properties, which may help delay the
decline in beef quality ([93]Lu et al., 2018).
The 9 aldehyde differentiators were screened in beef and most of them
originated from lipid oxidation in meat, such as octanal, hexanal and
nonanal. High concentrations of octanal emit a pungent odor and are
considered a marker of lipid oxidation ([94]Huang et al., 2023). The
octanal content in FM was 39.68 μg/kg (OAV:56.67), and after 18 d of
storage, the octanal level in PS group beef reached 369.08 μg/kg (OAV:
527.26), significantly higher than that in LP group (133.65 μg/kg),
which may result in the generation of off-flavors. Hexanal is high in
beef and produces fruity and fatty flavors and is the main volatile
flavor compound in beef. During storage, the hexanal content showed an
increasing and then decreasing trend. In LP group, hexanal increased
from 115.24 μg/kg (0 d) to 965.24 μg/kg (18 d) and then decreased to
820.97 μg/kg (24 d, OAV:182.44). In PS group, hexanal increased from
115.24 μg/kg (0 d) to 527.36 μg/kg (6 d) and then decreased to
254.48 μg/kg (24 d, OAV,56.55). At 6 d of storage, the LP group hexanal
content was 267.13 μg/kg, which was significantly lower than that of PS
group (p < 0.05). Therefore, we speculate that LVEF treatment may
affect the formation and transformation of hexanal. With the increase
in storage time, the degree of lipid oxidation increases, leading to
higher levels of nonanal ([95]Fan et al., 2023). At 24 d of storage,
the nonanal content in LP group was 261.75 μg/kg, significantly lower
than in PS group (370.26 μg/kg, p < 0.05). Benzaldehyde produces an
unpleasant odor and can affect the aroma of beef. Throughout the
storage period, benzaldehyde was detected only in the later storage
stages (18–24 d) in the PS group, which could be related to the decline
in beef quality. After 24 days of storage, the benzaldehyde content in
the PS group was 19.12 μg/kg (OAV: 4.78). [96]Yu et al. (2025) found
that benzaldehyde was associated with quality deterioration during
frozen storage of meat.
Esters are generally formed either through the reaction of acids
produced by the degradation of fats or proteins with alcohols, or via
transesterification between fatty acids in triglycerides and ethanol.
They contribute desirable fruity aromas to meat products. In beef,
there were 2 esters of significantly different substances. Ethyl esters
can impart creamy, fruity, floral, and sweet aromas to beef. In FM,
Caproic acid vinyl ester was detected at 29.44 μg/kg (OAV: 29.43).
After storage, it was only detected on day 6 in the LP group at
93.82 μg/kg (OAV: 93.82). (E)-9-Octadecenoic acid ethyl ester was
detected only in the LP group after 12 days of storage, with a
concentration of 59.06 μg/kg. Ketone volatile flavor compounds are
products of lipid oxidation and can interact with myofibrillar proteins
in meat products, affecting the flavor of the meat. Acetoin and
2,3-octanedione have a butterscotch flavor. In the present study, LP
group detected a higher total content of 3-hydroxy-2-butanone and
2,3-octanedione than PS group. In the LP group, the content of
2-pentylfuran increased with the prolongation of storage time, reaching
46.37 μg/kg (OAV: 7.73) after 24 days. However, in PS group,
2-pentylfuran showed an increasing and then decreasing trend from
10.65 μg/kg (FM) to 87.99 μg/kg (12 d) and then to 10.55 μg/kg (24 d,
OAV: 1.76). 2-pentylfuran has a low threshold, exhibits a fruity flavor
and contributes to roasted meat aroma, and is a typical oxidative
breakdown product of linoleic acid. In summary, LVEF storage can
increase certain volatile flavor compounds during beef storage and
reduce the formation of off-flavors.
3.5. Analysis of nonvolatile metabolites during beef storage
3.5.1. Overview of non-volatile metabolomics analysis
To investigate the changes in non-volatile flavor compounds during beef
storage, the composition of metabolites was identified by LC-MS/MS, and
the chromatograms are shown in Fig. S2. Retaining the metabolites that
were full match in mzCloud and removing the metabolites that were not
queried in the chemistry book ultimately resulted in the screening of
313 effective non-volatile metabolites, including 56 amino acids and
their derivatives, 31 nucleosides, nucleotides, and analogs, 20 organic
acids and their derivatives, 99 lipids and lipid-like molecules, 28
organic oxygen compounds, 20 organic heterocyclic compounds, 38
aromatic compounds, and 21 other metabolites ([97]Fig. 6a). To further
compare the differences between the two groups of samples, the samples
were analyzed using OPLS-DA, and differential metabolites (fold change
(FC) > 2 or < 0.5, p < 0.05) were visualized as volcano plots, as shown
in [98]Figs. 6b and [99]7. In the volcano plot, gray dots represent
metabolites with no significant difference, while red and blue dots
indicate metabolites that are significantly upregulated and
downregulated, respectively. The results show that LP and PS groups
were completely separated at each time point, indicating that LVEF
treatment can cause differences in the levels of certain metabolites.
In the comparisons of LP-6d vs. PS-6d, LP-12d vs. PS-12d, LP-18d vs.
PS-18d, and LP-24d vs. PS-24d, 72, 8, 17, and 70 metabolites were
significantly upregulated, while 27, 17, 17, and 51 metabolites were
downregulated, respectively. At 24 d of storage, LP and PS groups
exhibited the richest differential metabolites.
Fig. 6.
[100]Fig. 6
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Proportion of non-volatile metabolite types (a) and OPLS-DA analysis
(b).
Fig. 7.
[102]Fig. 7
[103]Open in a new tab
Volcano plot of non-volatile metabolites.
3.5.2. Non-volatile differential metabolite analysis
To identify representative metabolites, differential metabolites were
further selected based on p < 0.05, FC > 2 or < 0.5, and VIP > 1
predicted by the OPLS-DA model. Most of the differential metabolites
between LP and PS groups during storage were amino acids and their
derivatives, lipids and lipid-like molecules.
Non-volatile compounds in fresh meat, such as amino acids and their
derivatives, are crucial for flavor. Proteins are easily degraded into
peptide chains and amino acids during storage due to microbial action.
At 24 d of storage, the number of significantly differential
metabolites in the amino acids and their derivatives category
increased. In LP group, the abundance of many amino acids and their
derivatives, such as argininosuccinic acid, L-cysteine-glutathione
gisulfide, and l-serine, was significantly lower than in PS group
(p < 0.05). Higher levels of argininosuccinic acid can cause meat to
have a bitter flavor ([104]Ramalingam et al., 2019). The abundance of
L-hydroxyproline and L-asparagine showed an increasing trend with
increasing storage time, and they may be potential markers of beef
deterioration during storage. Furthermore, throughout the entire
storage period, the carnosine content in the LP group remained
consistently higher. At 24 d of storage, the carnosine content in the
LP group was 12.65 %, significantly higher than the 3.83 % in the PS
group (p < 0.05). Carnosine is widely present in the muscles of most
animals and can inhibit lipid oxidation through a combination of free
radical scavenging and metal chelation. Therefore, carnosine helps slow
down lipid oxidation during beef storage ([105]Peiretti et al., 2012).
Nucleosides and nucleotides are essential in many physiological
processes. After 6 d of storage, the content of guanosine monophosphate
(GMP) in LP group was significantly higher than in PS group. GMP has a
distinctive mushroom flavor that interacts with disodium sarcosinate to
bring out freshness and thus add flavor to beef. Adenosine triphosphate
in beef is degraded to adenosine diphosphate, inosinic acid (IMP), and
hypoxanthine by enzymes. During storage, N2-methylguanosine and
xanthosine tended to increase, and IMP tended to decrease, but the IMP
of LP group decreased more slowly. IMP is the main flavor component of
meat, providing sweetness and pleasant flavor, unstable in muscle, can
be further degraded to hypoxanthine and xanthine, associated with meat
spoilage ([106]Yi et al., 2024).
During storage, lipids degrade into aldehydes, ketones, lower fatty
acids, and other compounds. In beef, a total of 29 differential lipid
and lipid-like molecules were screened. Acetylcarnitine content in beef
increased with storage time. At 24 d of storage, the LP and PS groups
acetylcarnitine contents were 11.39 % and 29.78 %, respectively.
[107]Jia et al. (2021) found that the accumulation of long-chain fatty
acyl carnitines promotes β-oxidation of fatty acids, leading to poor
meat quality. During the spoilage process of meat, changes in organic
acids and their derivatives, benzene and its derivatives, as well as
heterocyclic compounds, have a relatively minor impact on meat quality
([108]Fu et al., 2024). 5-hydroxyindole belongs to organic heterocyclic
compounds, and the content of 5-hydroxyindole in beef increased
significantly after 24 d of storage, and the 5-hydroxyindole in LP
group was significantly lower than that in PS group (p < 0.05).
Proteins are broken down by microorganisms into indoles, phenol,
putrescine, and various nitrogenous acids and fatty acids ([109]Liu et
al., 2022). This may be due to the fact that LVEF better inhibits
microbial growth and reproduction, thus reducing the production of
5-hydroxyindole.
3.6. Metabolic pathway and metabolic network analysis
To better visualize the intrinsic relationships among metabolites, the
detected metabolites were subjected to enrichment and pathway analysis
using Metabo Analyst 6.0. As shown in [110]Fig. 8a, a total of 58
metabolic pathways were enriched. Among them, pathways such as arginine
biosynthesis, alanine-aspartate-glutamate metabolism, the pentose
phosphate pathway, and the tricarboxylic acid (TCA) cycle exhibited
high enrichment ratios and strong statistical significance.
Furthermore, based on pathway impact values (PI>0.1) and statistical
significance (p < 0.05), eight key metabolic pathways were identified
([111]Fig. 8b), including arginine biosynthesis, the pentose phosphate
pathway, and the TCA cycle. These pathways are closely associated with
quality changes in beef during storage.
Fig. 8.
[112]Fig. 8
[113]Open in a new tab
Analysis of metabolic pathways during beef storage. a: Enrichment
analysis plot; b: Metabolic pathway bubble plot; c: Metabolic network
plot.
To gain deeper insights into the quality changes of beef during
storage, the metabolic network involved in flavor formation was
predicted based on the KEGG database and relevant literature, as shown
in [114]Fig. 8c. Proteins in beef are metabolized primarily by alanine,
aspartic acid, and glutamic acid to produce intermediate metabolites
that are then involved in the formation of other flavor compounds.
Hydrolysis of proteins by endogenous and microbial enzymes produces
peptides and free amino acids, which then produce alcohols and
aldehydes by transaminases and α-ketoglutaric acid enzymes. For
example, L-hydroxyproline can be generated through the metabolic
pathway of proline. As storage time increases, the concentration of
L-hydroxyproline in beef gradually accumulates. However, the
L-hydroxyproline content in samples treated with LP group was
significantly lower than PS group. LVEF treatment reduced proline
metabolism, a result consistent with the findings of [115]Fei et al.
(2024). L-glutamate, l-serine and d-glutamine are converted to pyruvic
acid, α-ketoglutaric acid, oxaloacetic acid, etc., and then further
converted to phosphoenolpyruvic acid, and finally glucose is
synthesized via the gluconeogenesis pathway. Their contents changed
dynamically during storage, but at the end of storage, LP group had
lower L-glutamate, l-serine and d-glutamine contents than PS group.
This suggests that LVEF treatment may slow down the degradation of
proteins and the metabolism of amino acids, thereby helping to delay
the deterioration of beef quality.
Carbohydrates go through the glycolytic pathway to produce pyruvate,
which in the production of acetyl-CoA, then enters the TCA cycle.
Citric acid and fumaric acid are critical intermediates in the TCA
cycle that promote lactic acid decomposition and have anti-aging,
appetite and fatigue-relieving effects. At the end of storage, the
citric and fumaric acid contents of LP group were significantly lower
than those of PS group. This suggests that LVEF treatment may have
reduced acetyl-CoA production, thereby affecting the TCA cycle. Malic
acid is produced through the TCA cycle and subsequently converted into
aspartic acid. Aspartic acid can then be converted into oxaloacetic
acid through the metabolic pathways of alanine, aspartic acid, and
glutamic acid, and further participate in the synthesis of various
other metabolites.
Lipids are hydrolyzed by lipases to produce free fatty acids, which are
then further degraded into various metabolites. Unsaturated fatty acids
can be converted into flavor compounds such as heptanol, octanol,
(E,E)-2,4-nonenal, and 2-pentylfuran by the action of enzymes. The LVEF
treatment increased the content of these flavor compounds resulting in
better beef flavor during storage. Lipid oxidation can promote the
breakdown of palmitic acid in meat, resulting in the formation of
capric acid. In addition, oxidation of fatty acids to produce
acetyl-CoA can further participate in fatty acid metabolism and
contribute to the formation of other flavor compounds. For example,
1-octanol and 3-octanone can be produced by the catalytic action of
acetyl-CoA carboxylase, β-ketoacyl synthase, and malonyl transferase
([116]Jiang et al., 2025). Whereas acetyl-CoA produces α-ketoglutarate
via the TCA cycle, it then undergoes the mangiferolic acid pathway to
produce phenylalanine, which is finally metabolized by phenylalanine to
produce benzaldehyde. During storage, benzaldehyde was detected only at
the late PS group stage, suggesting that LVEF treatment affected
phenylalanine metabolism. In summary, during beef storage, intermediate
metabolites of proteins, carbohydrates, and lipids can be
interconverted through various metabolic pathways, leading to the
formation of diverse flavor compounds. However, these metabolic
activities are also accompanied by continuous energy release and
nutrient depletion, ultimately affecting beef quality. LVEF treatment
can partially slow down these metabolic processes and help delay flavor
deterioration. The findings not only reveal the complex metabolic
network involved in flavor formation during beef storage but also
demonstrate that LVEF treatment plays a positive role in preserving
beef quality and enhancing flavor during storage.
4. Conclusion
This study demonstrated that LVEF treatment effectively slowed the
changes in pH, TVB-N, TBARS, TVC, and shear force in beef, and
inhibited protein oxidation. Ninety-two volatile flavor compounds were
identified using HS-SPME-GC–MS; alcohols and aldehydes were the
predominant compounds. LVEF promoted the formation of desirable
volatiles such as 1-hexanol, 1-heptanol, and hexanal, while reducing
undesirable compounds including 1-octen-3-ol, octanal, and
benzaldehyde. Furthermore, 313 non-volatile metabolites were identified
via LC-MS/MS. The results showed that LVEF significantly suppressed the
increase in the levels of L-hydroxyproline and L-asparagine and
mitigated the reduction in IMP during storage. Metabolic pathway
analysis indicated that LVEF modulated multiple pathways, slowing the
degradation of proteins, lipids, and carbohydrates, thereby enhancing
the stability of beef quality. In conclusion, LVEF holds promise for
delaying the deterioration of beef quality. Future research should aim
to further optimize LVEF processing parameters and improve equipment
stability. Additionally, the incorporation of sensory evaluation and
microbiome analysis is recommended to systematically assess its impact
on meat quality, thereby supporting its practical application in the
field of preservation.
The following are the supplementary data related to this article.
Supplementary Fig. S1.
[117]Supplementary Fig. S1
[118]Open in a new tab
Chromatograms of GC-MS
Supplementary Fig. S2.
[119]Supplementary Fig. S2
[120]Open in a new tab
Chromatograms of LC-MS/MS
CRediT authorship contribution statement
Yuxia Liu: Writing – original draft, Formal analysis, Data curation.
Wei Su: Writing – review & editing, Funding acquisition. Yingchun Mu:
Writing – review & editing, Supervision. Xiaomin Liu: Data curation,
Conceptualization. Kangli Yang: Formal analysis. Rongrong Mu:
Investigation.
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