Abstract
This study investigated the impact of steaming on the flavor and
metabolic profile of Antarctic krill, aiming to elucidate the pathways
responsible for flavor development and metabolic shifts during
processing. HS-SPME-GC–MS identified key volatile compounds, including
alcohols, aldehydes, ketones and so on. The results demonstrated a
significant increase in nonanal content from 2.23 ± 0.06 μg/kg to
8.14 ± 1.26 μg/kg after steaming. The formation pathways of two key
flavor compounds, nonanal and 1-octen-3-ol, were attributed to fatty
acid degradation. Hierarchical clustering and volcano plot showed
metabolic shifts between raw and steamed krill, with differential
metabolites like hydroquinone and gamma-aminobutyric acid emerging as
key contributors to flavor changes. Furthermore, metabolic network
further linked these shifts to reactions involving amino acids,
nucleotide and other compounds during steaming, impacting the overall
taste.
Keywords: Steaming, Antarctic krill, Flavor compounds, Formation
pathway, Untargeted metabolomics
Graphical abstract
[37]Unlabelled Image
[38]Open in a new tab
Highlights
* •
E-2-octenal, nonanal, 1-octen-3-ol are the key flavor compounds in
steamed krill.
* •
Steaming induces the formation of nonanal, 1-octen-3-ol via fatty
acid degradation.
* •
Lipid degradation and amino acid reactions contribute to the
development of flavor.
1. Introduction
Antarctic krill (Euphausia superba) has the advantages of large
biomass, rich nutrition, high protein and low fat ([39]Hamner & Hamner,
2000), and it is abundant in essential amino acids, as well as DHA and
EPA, along with other bioactive compounds ([40]Jiang et al., 2024). In
the waters off the western Antarctic Peninsula, krill make up 75–90 %
of the total biomass ([41]McBride et al., 2021), with estimates
suggesting that the krill reserves in this region could reach billions
of tonnes. Antarctic krill has broad application prospects and is
considered a potential future food source. Currently, Antarctic krill
products primarily include krill powder, dried krill, and krill oil,
which are produced through steaming processes ([42]Trathan et al.,
2021). However, the use of steam in heat processing has significant
impact on the volatile compounds in meat ([43]Khan et al., 2015).
The characteristic flavor of Antarctic krill meat was developed through
chemical reactions such as protein hydrolysis and fat oxidation ([44]Li
et al., 2022). Similarly, [45]Zhang, Cao, et al. (2019) identified and
quantified the characteristic flavor compounds in golden pomfret
fillets, analyzing the generation of volatile flavor compounds from the
perspectives of Strecker degradation, protein hydrolysis, and lipid
oxidative degradation. In another study, [46]Mall and Schieberle (2016)
used aroma extract dilution analysis to identify the primary aroma
compounds in raw and cooked shrimp, revealing that different heat
treatments caused variations in the types and quantities of aroma
compounds in shrimp meat. While the qualitative analysis of flavor
substances and the quantitative analysis of key volatile compounds in
food have become well-established, there is a lack of research into the
interactions between these flavor compounds and their formation
mechanisms.
The flavor is a key factor that influences people's preference for
food. Previous studies have investigated the volatile flavor compounds
and the impact of heat treatment on the flavor profile of Antarctic
krill. [47]Fan et al. (2017) used HS-SPME and GC–MS to analyze the
volatile flavor compounds in Antarctic krill, detecting a total of 42
volatile substances responsible for its unique flavor. Of these, seven
compounds made significant contributions to the flavor profile of
Antarctic krill. [48]Bai et al. (2022) conducted sensory evaluations
along with electronic tongue and nose analyses, as well as GC–MS, to
compare the flavor of Antarctic krill and white shrimp, showing notable
changes in the characteristic flavor compounds after steaming and
boiling. Additionally, [49]Zhang et al. (2018) investigated the effects
of different heating methods on the content of six non-volatile
reactive compounds (NRCs) in Antarctic krill and Pacific white shrimp,
demonstrating that adjusting heating conditions effectively controlled
the NRC levels.
However, researchers have primarily focused on the qualitative and
quantitative assessments of individual flavor compounds when studying
the flavor of steamed aquatic products, lacking a comprehensive
characterization of the multiple factors influencing flavor ([50]Zhang,
Ma, & Dai, 2019). Additionally, research on the interactions between
flavor components and the formation mechanisms of key compounds remains
limited ([51]Liang et al., 2022). Currently, the effects of steaming on
the flavor quality of Antarctic krill are not well understood, and the
mechanisms of flavor formation require further investigation.
Therefore, it is essential to explore the processes and mechanisms
responsible for the characteristic flavor of steamed Antarctic krill.
The present study aims to elucidate the formation pathway of key flavor
compounds during processing. The untargeted metabolomics approach was
employed to analyze the alterations of typical flavor compounds in
Antarctic krill pre- and post- steaming, thereby establishing a
foundation for quality control measures pertaining to Antarctic krill.
These results will establish a theoretical foundation for comprehending
the metabolic alterations and formation pathways of flavor compounds.
2. Materials and methods
2.1. Materials and reagents
Antarctic krill was supplied with the China National Aquatic Products
Corporation (Zhoushan, Zhejiang, China). All reagents are analytical
grade.
2.2. Preparation of taste extracts from Antarctic krill
The thawed Antarctic krill meat was homogenized with ultrapure water at
a ratio of 1:10 (w/v) and then centrifuged at 9000 rpm for 20 min at
4 °C. The resulting supernatant of raw Antarctic krill meat (RKM) was
collected as the taste extract. For the steamed samples, raw krill was
steamed for 5 min. The preparation of the natural flavor extract from
steamed krill meat (SKM) followed the same procedure as for the raw
krill meat.
2.3. GC–MS analysis of volatile flavor compounds
The volatile compounds of Antarctic krill meat were extracted using
HS-SPME ([52]Fan et al., 2017). The minced SKM or RKM samples (3.0 g),
along with 3 mL saturated NaCl solution and 2, 4, 6-trimethylpridine
were introduced into a 20 mL headspace bottle, respectively. An SPME
fiber (65 μm, PDMS/DVB, Supelco, Bellefonte, PA, USA) was inserted into
the headspace of the vial and extracted for 45 min at 65 °C. The fiber
was transferred to an injection port and desorbed for 5 min at 250 °C.
The volatile flavor compounds in SKM or RKM samples were identified
using GC–MS (7000 D, Agilent Technologies Inc., USA) with an HP-5MS
capillary column (30 m × 0.32 mm × 0.25 μm). The heating program was as
follows: the initial oven temperature was set at 40 °C for 3 min, then
increased to 250 °C at a rate of 5 °C/min, and held at 250 °C for
5 min. The sample was introduced into the GC at a flow rate of
1.0 mL/min using helium (99.999 %), with an injector temperature of
250 °C. The detector temperature was set at 250 °C, with a mass range
of 35–350 m/z.
The identification of volatile flavor compounds by GC–MS was performed
by searching the NIST20 mass spectrometry database. N-Alkanes (C8-C30)
were employed to calculate linear retention indices of volatile
compounds. The content of volatile compounds was calculated according
to the method of [53]Kaseleht et al. (2011). In brief, each sample was
spiked with 0.0465 μg of the internal standard 2,4,6-trimethylpyridine,
and the compounds were relatively quantified based on the content and
peak area of the internal standard.
2.4. Evaluation of odor activity values (OAV) of volatile compounds
OAV is employed to evaluate the contribution of a volatile flavor
compound to the overall flavor profile of a sample. The OAV is
calculated using the following formula:
[MATH: OAV=C/OT :MATH]
where OAV represents the odor activity value; C denotes the content of
the volatile compound detected in the sample (μg/kg); OT refers to the
literature-based odor threshold of this compound (μg/kg).
2.5. Fatty acids analysis using GC–MS
Antarctic krill meat (15 g) was combined with 200 mL of chloroform and
100 mL of methanol, then stirred thoroughly and placed at 4 °C
overnight. The filtrate was collected and mixed with 0.9 % sodium
chloride solution (5:1, v/v). After standing for 12 h, the lower
organic phase was dried. The dried sample (20 g) were combined with
2 mL of 10 % concentrated sulfuric acid in methanol and reacted at
60 °C for 15 min. The cooled samples were then mixed with 2 mL of
n-hexane, and the upper solution was filtered through a non-polar
0.22 μm filter after standing for 10 min.
The fatty acid composition of raw and steamed krill meat was analyzed
using GC–MS (7000 D, Agilent Technologies Inc., USA) with an HP-INNOWAX
quartz capillary column (30 m × 0.32 mm × 0.25 μm) following the method
of [54]Valasi et al. (2021). The sample was introduced into the GC with
helium at a flow rate of 1.0 mL/min. The heating program was as
follows: the initial oven temperature was set at 120 °C, then increased
to 210 °C at a rate of 5 °C/min and maintained at 210 °C for 10 min.
The mass spectrometer operated in electron ionization mode with a mass
scan range of 50 to 500 m/z in full-scan mode. The electron energy was
set at 70 eV, with the ion source temperature at 230 °C and the
quadrupole temperature at 250 °C.
2.6. Analysis of peptides in krill taste extract
The Antarctic krill meat (6 g) was homogenized with a solid-liquid
ratio of 1:4, and then centrifuged at 9000 rpm for 30 min at 4 °C. The
resulting supernatant was mixed with acetonitrile to precipitate
macromolecular proteins. After centrifugation, the supernatant was
obtained and then freeze-dried. The resulting powder was dissolved
(10 mg/mL), desalted, and then analyzed by an ultra-performance liquid
chromatography (Dionex UltiMate 3000, Thermo Fisher Scientific Inc.,
Waltham, USA) coupled with a quadrupole Orbitrap mass spectrometer (Q
Exactive, Thermo Fisher, USA).
The separation conditions for UHPLC were as follows: Mobile phase A
consisted of 0.1 % formic acid in water, while mobile phase B was 0.1 %
formic acid in acetonitrile. The analysis was performed using an
Agilent Advanced Bio Peptide Map C18 column (150 mm × 2.1 mm, 2.7 μm
particle size). The flow rate was set at 0.25 mL/min with an injection
volume of 30 μL. The total run time was 46 min, and the column
temperature was maintained at 40 °C.
The mass spectrometer operated in electrospray positive ion (ESI+) mode
with an electrospray voltage of 5500 V. The scanning range for mass
spectrometry was 300–1500 m/z. The atomization gas pressure was set to
60 psi, the auxiliary heating air pressure to 50 psi, the air curtain
pressure to 35 psi, and the ion source temperature was kept at 525 °C.
The flavor peptides and amino acids were assessed based on the BIOPEP
database ([55]https://biochemia.uwm.edu.pl/biopep-uwm/), followed by
the prediction of their flavor characteristics.
2.7. Analysis of amino acid cleavage sites of peptide chain in krill taste
extract
A 0.2 g portion of the lyophilized sample underwent derivatization
according to the method of [56]Shi et al. (2013). The derivatized
sample was then vacuum-dried and dissolved. The solution was incubated
in a water bath at 54 °C for 45 min and then vacuum-dried. The dried
sample was dissolved in 80 % ethanol and extracted with n-hexane. The
lower phase was filtered through a 0.22 μm filter membrane.
Free amino acids (FAA) were analyzed using HPLC with an Agilent 1260
Infinity system (Agilent Technologies Co. Ltd., California, USA),
equipped with a UV-detector. The analysis was conducted using a ZORBAX
Eclipse XDB-C18 column (4.6 × 250 mm × 5 μm) at 254 nm. Mobile phase A
consisted of 0.1 mol/L sodium acetate-acetonitrile (97:3, v/v, pH 6.5),
while mobile phase B was 80 % acetonitrile in water. The samples were
eluted at a flow rate of 0.8 mL/min with the following gradient:
0–15 min, 95–84 % A; 15–35 min, 84–50 % A; 35–38 min, 50–95 % A;
38–40 min, 95 % A.
2.8. The approach of non-targeted metabolomics based on LC-MS/MS
2.8.1. Liquid chromatography conditions
The liquid chromatography analysis was performed using a Vanquish UHPLC
System (Thermo Fisher Scientific, USA) with a method slightly modified
from [57]Zelena et al. (2009). Chromatography was conducted using an
ACQUITY UPLC® HSS T3 column (150 × 2.1 mm, 1.8 μm) (Waters, Milford,
MA, USA), with the column temperature set at 40 °C.
The Antarctic krill sample was mixed with 0.75 mL of
methanol-chloroform solution (9:1, v/v) and 0.25 mL of water. The
mixture underwent ball milling homogenization at 50 Hz for 120 s,
followed by ultrasonication for 30 min and ice bath treatment for an
additional 30 min. The homogenate was then centrifuged at 12,000 rpm at
4 °C for 10 min. The resulting supernatant was collected and
concentrated to dryness. Subsequently, 200 μL of
2-chloro-L-phenylalanine solution (4 ppm in 50 % acetonitrile) was
added accurately to the dried residue. The mixture was filtered and
prepared for LC-MS analysis.
2.8.2. Mass spectrum conditions
Mass spectrometric detection of differential metabolites was done using
an Orbitrap Exploris 120 (Thermo Fisher Scientific, USA) with an ESI
ion source according to the method of [58]Want et al. (2013). Detection
was conducted in Full MS-ddMS2 mode (data-dependent MS/MS).
2.8.3. Data analysis
It was employed for multivariate data analysis and model construction
([59]Thévenot et al., 2015). After scaling the data, orthogonal-partial
least-square discriminant analysis (OPLS-DA) was used to build the
models. Metabolic profiles were visualized using score plots, where
each point represented a sample. Corresponding S-plots were generated
to highlight the metabolites that influenced the clustering of the
samples. The P-value, VIP from OPLS-DA, and fold change (FC) were
applied to identify variables contributing to classification. Finally,
metabolites with P-value <0.05 and VIP > 1 were considered
statistically significant.
2.8.4. Pathway analysis
Differential metabolites were subjected to pathway enrichment analysis
using MetaboAnalyst, which combines both pathway enrichment and
topology analyses ([60]Xia & Wishart, 2011).
2.9. Statistical analysis
All experiments were performed at least three times and the data were
expressed as the mean value ± standard deviation. Statistical analysis
was performed using SPSS 19.0 software.
3. Results and discussion
3.1. Effects of steaming on the relative content of volatile flavor compounds
in Antarctic krill
The exquisite flavor of krill meat primarily depends on the composition
of volatile compounds. In this study, HS-SPME combined with GC–MS was
used to analyze the types and concentrations of volatile flavor
compounds in steamed krill. The RKM and SKM samples were detected to
contain a total of 39 volatile flavor compounds, including alcohols,
aldehydes, esters, and so on ([61]Table 1). The species and
concentrations of these volatile compounds differed between SKM and
RKM. Alcohols and aldehydes were formed through lipid degradation, the
Maillard reaction, Strecker degradation, and the interaction of their
metabolites ([62]Al-Dalali et al., 2022).
Table 1.
Analysis of volatile compounds in Antarctic krill.
Classification Compounds RI Identifiction Content (μg/kg)
__________________________________________________________________
Odor description
RKM SKM
Alcohols
1 2,6-nonadiene-1-ol – MS N 3.03 ± 0.23 green, vegetable
2 1-octene-3-ol 978 MS, RI N 0.49 ± 0.04 mushroom, green
3 2-ethyl-1-hexanol 1029 MS, RI N 14.68 ± 4.12 mushroom
4 2,6-dimethyl cyclohexanol 1108 MS, RI N 1.15 ± 0.03 flower
5 α-terpineol 1192 MS, RI N 0.40 ± 0.10 flower
6 5-nondecene-1-ol 1206 MS, RI N 0.80 ± 0.12
7 phytol 2114 MS, RI N 1.25 ± 0.53 flower
Aldehydes
1 E-2-octenal 972 MS, RI 6.09 ± 0.97 3.53 ± 0.21 nutty
2 phenylacetaldehyde 1043 MS, RI N 2.18 ± 0.65 green, honey
3 nonanal 1104 MS, RI 2.23 ± 0.06 8.14 ± 1.26 fat, citrus, green
4 2,4-dimethyl benzaldehyde 1178 MS, RI 0.44 ± 0.09 N almond
5 hexadecaldehyde 1817 MS, RI N 1.25 ± 0.33 fat
Ketones
1 1-phenyl-2-pentanone – MS 0.20 ± 0.01 N
2 6-methyl-5-heptene-2
-one 986 MS, RI 4.54 ± 0.43 2.28 ± 0.43 fruity
3 3-ethyl-4-heptanone 1048 MS, RI N 0.35 ± 0.09
4 heptanophenone 1065 MS, RI N 0.88 ± 0.02 fruity
5 acetophenone 1066 MS, RI N 0.89 ± 0.01 sweet, fruity
6 2,5-hexanedione 1244 MS, RI N 0.29 ± 0.07 smelly
7 benzophenone 1633 MS, RI N 0.22 ± 0.03 rose, sweet
Esters
1 2-methyl-butanoic acid methylester – MS 1.09 ± 0.08 N fruity
2 methyl acetate 1173 MS, RI N 0.54 ± 0.09 aroma
3 butyl benzoate 1376 MS, RI N 0.28 ± 0.03 fruity
4 dibutyl phthalate 1883 MS, RI 3.50 ± 0.30 N
Hydrocarbons
1 ethyl benzene 853 MS, RI 26.33 ± 0.48 1.08 ± 0.10 aromatic
2 α-pinene 930 MS, RI 13.17 ± 0.39 N citrus
3 γ-terpinene 1008 MS, RI 6.91 ± 0.21 N rosin，resin
4 D-limonene 1028 MS, RI 17.45 ± 1.08 N orange, lemon
5 undecane 1099 MS, RI 0.72 ± 0.06 N alkane odor
6 dodecane 1101 MS, RI 1.00 ± 0.08 N alkane odor
7 2-methyl-naphthalene 1295 MS, RI N 2.56 ± 0.35 naphthalene odor
Others
1 dimethyl disulfide – MS 0.36 ± 0.04 N onion, cabbage
2 methoxy-phenyl oxime 905 MS, RI 14.27 ± 0.02 18.79 ± 0.59
3 2-methyl-benzofuran 1199 MS, RI 3.26 ± 0.30 N
4 benzothiazole 989 MS, RI N 2.52 ± 0.26 caramel
5 2,4-di-tert-butylphenol 1225 MS, RI N 0.23 ± 0.05 potion
6 butyl hydroxytoluene 1513 MS, RI 0.38 ± 0.01 0.69 ± 0.05
7 tetradecanoic acid 1517 MS, RI 0.51 ± 0.09 2.17 ± 0.17
8 2-pentadecyl furan 1865 MS, RI N 0.20 ± 0.01
9 N-hexadecanoic acid 1961 MS, RI 1.89 ± 0.06 2.50 ± 0.11
[63]Open in a new tab
Note: - indicates not calculated; N means not detected; Odor
description refers to literature.
The alcohols produced by steaming in this study predominantly exhibited
green, floral, and vegetal notes, which may significantly influence the
flavor profile of Antarctic krill meat ([64]Lorenzo et al., 2013). A
notable change in alcohol concentrations was observed after steaming.
As shown in [65]Table 1, seven alcohols were detected in Antarctic
krill post-steaming, most of which contributed green, vegetal, and
floral flavors ([66]Xu et al., 2021). Among these, 2,6-nonadien-1-ol
imparted a green and vegetal fragrance, while α-terpineol provided a
floral aroma ([67]Xu et al., 2021). 1-Octen-3-ol, with the higher odor
activity value (OAV) in Antarctic krill, was characterized by a
mushroom and green aroma and is also considered a key contributor to
the earthy flavors found in aquatic products. Five aldehydes were
detected in Antarctic krill, including phenylacetaldehyde and
hexadecanal, which were found in steamed Antarctic krill, with a
significant increase in nonanal content. Phenylacetaldehyde imparted
green and honey-like aromas, while acetaldehyde contributed a fatty
note, both potentially playing a key role in the flavor profile of
Antarctic krill meat ([68]Serot et al., 2001). The content of nonanal
in Antarctic krill increased from 2.23 to 8.14 μg/kg after steaming,
contributing a fatty aroma. This increase is believed to result from
the thermal oxidation or degradation of unsaturated fatty acids,
particularly linoleic or linolenic acids ([69]An et al., 2020; [70]Cao
et al., 2014).
Ketones are typically produced through the oxidation of lipids and the
Strecker degradation of amino acids ([71]Zhang et al., 2020). In total,
seven ketone compounds were detected in Antarctic krill, contributing
to the fruity aroma of krill products ([72]Sarnoski et al., 2010).
Ester compounds, which are esterification products of carboxylic acids
and alcohols formed during lipid metabolism, play a key role in
determining the flavor characteristics of Antarctic krill
([73]Carrapiso et al., 2002). Steaming led to the formation of methyl
acetate and butyl benzoate, both with fruity aromas, as well as
benzothiazole, which imparted a caramel-like flavor.
The OAV is an important indicator for assessing the overall
contribution of a single compound to the odor profile of Antarctic
krill meat ([74]Zhang, Ma, et al., 2019). The impact of volatile
compounds on the sample's flavor depends not only on their
concentration but also on their odor threshold value ([75]Zhu et al.,
2016). Generally, compounds with an OAV greater than 1 are typically
regarded as active and make a significant contribution to the overall
flavor of the sample ([76]Hu et al., 2021). As shown in [77]Table 2,
among the aldehyde compounds, the content of E-2-octenal decreased
after steaming, but its OAV remained significantly greater than 1. The
OAV of phenylacetaldehyde and nonanal in SKM were 2.18 and 8.14,
respectively, indicating that aldehydes played a crucial role in the
characteristic flavor of Antarctic krill. Overall, E-2-octenal,
phenylacetaldehyde, and nonanal were identified as key contributors to
the flavor of steamed Antarctic krill, imparting grassy, citrus, and
floral notes to SKM ([78]Zhao et al., 2020).
Table 2.
Odor activity evaluation and odor description of volatile compounds in
Antarctic krill.
Classification Compounds Threshold
(μg/kg)^a OAV
__________________________________________________________________
Odor description^b
RKM SKM
Alcohols 1-octene-3-ol 1 N 0.49 mushroom, green
α-terpineol 330 N 0 flower
Aldehydes E-2-octenal 3 2.03 1.18 nutty
phenylacetaldehyde 1 N 2.18 green, honey
nonanal 1 2.23 8.14 fat, citrus, green
Ketones 6-methyl-5-heptene-2-one 50 0.09 0.05 fruity
Hydrocarbon D-limonene 10 1.75 N orange, lemon
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Note: N means not detected; a threshold reference; b odor Description:
refer to previous literature.
3.2. Effects of lipid oxidative degradation on flavor formation in steamed
Antarctic krill
The lipids in Antarctic krill serve as essential precursors for the
formation of volatile flavor compounds, which are subsequently
converted into fatty acids during steaming ([80]Matsui et al., 2003).
The volatile compounds produced after steaming are mainly the result of
lipid oxidation and the interactions between products of lipid
oxidation and those of the Maillard reaction ([81]Wang et al., 2022). A
total of 19 fatty acids were identified in RKM and SKM samples
([82]Fig. 1A). After steaming, the total contents of saturated fatty
acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated
fatty acids (PUFA) increased significantly, indicating that the fats in
Antarctic krill were degraded by the steaming process. Lipid oxidative
degradation is a key pathway for flavor formation, and certain
unsaturated fatty acids, such as oleic and linoleic acids, are
particularly prone to oxidation into peroxides during steaming due to
the presence of double bonds in their molecules. The further
decomposition of these peroxides generates ketones, aldehydes,
alcohols, and other compounds. Previous studies have shown that
hydroperoxides can also form from SFA, MUFA, and esters ([83]Abdelrazig
et al., 2020).
Fig. 1.
[84]Fig. 1
[85]Open in a new tab
Effects of oxidative degradation of fat and proteolysis on flavor
compounds. (A) Fatty acid composition of steamed Antarctic krill.
Different letters showed significant differences between RKM and SKM
for the same fatty acids (P < 0.05). (B) Possible pathways for the
formation of nonanal and 1-octen-3-ol during the steamed in Antarctic
krill. (C) Analysis of amino acid break sites of steamed Antarctic
krill.
The flavor components of Antarctic krill, including ketones,
hydrocarbons, and aldehydes, are primarily generated through lipid
degradation and oxidation during steaming ([86]Matsui et al., 2003).
Linear fatty aldehydes and alkanes are typical lipid oxidation products
([87]Farvin et al., 2012). Nonanal is a product formed through the
oxidation of oleic acid ([88]Cao et al., 2014), while the odor-active
compound 1-octen-3-ol, found in krill meat samples, is formed through
the oxidative degradation of linoleic acid ([89]Fig. 1B).
3.3. Analysis of polypeptides in flavor extract of steamed Antarctic krill
Polypeptides can exhibit taste characteristics such as bitterness,
umami, sweetness, and sourness, playing a crucial role in enhancing
food flavor ([90]Warren et al., 2017). Many known umami peptides are
derived from hydrolyzed animal proteins ([91]Zelena et al., 2009). For
instance, [92]Want et al. (2013) identified umami peptides such as SEE,
EEE, EDE, and DES from fish protein hydrolysate. As shown in [93]Table
3, several peptide sequences were identified from taste extracts of
heat-treated Antarctic krill.The peptides EDPIVP and EDPLVP had
relatively higher sweetness and umami scores compared to others
([94]Table 3). It has been reported that the taste characteristics of
peptides may be related to the types of amino acid residues within
their sequence, with peptides containing more umami amino acid residues
tending to have a stronger umami taste ([95]Horai et al., 2010).
Table 3.
Cleavage sites and taste of peptides in heat-induced Antarctic krill.
Sequence Cleavage sites Confidence Taste prediction (scores)
__________________________________________________________________
Sour Sweet Bitter Salty Umami
ETGLKYFPSQGNFIVIDFGIDGDEVFQYLL KETGLKYFPSQGNFIVIDFGIDGDEVFQYLLS >95 %
0.2667 0.3000 0.9000 0.1333 0.3000
PAGMVVNPITSTQQGNLSTQQPSIIAH PPAGMVVNPITSTQQGNLSTQQPSIIAHN >95 % 0.0370
0.3333 0.3704 N 0.0370
LFDYPIFFALGLFSSTFGFIMLGAM VLFDYPIFFALGLFSSTFGFIMLGAMG >95 % 0.0400
0.2400 1.0000 0.0400 0.0400
DPSGDRLRQWLDIDKDIFYNPNK NDPSGDRLRQWLDIDKDIFYNPNKI >95 % 0.3043 0.2174
0.6957 0.2174 0.2609
DAERIHGISTELAMEQGVSLQEVL YDAERIHGISTELAMEQGVSLQEVLA >95 % 0.2917 0.2917
0.5833 0.0417 0.4167
AGFAGDDAPRAVFPSIVGRPR KAGFAGDDAPRAVFPSIVGRPRH >95 % 0.1429 0.5714
1.0000 0.1429 0.2381
SGISNKIASLLKQDWGSLE RSGISNKIASLLKQDWGSLEE >95 % 0.2105 0.2632 0.6316
0.0526 0.1053
KSVVKEVTSAGGDIVLFI LKSVVKEVTSAGGDIVLFID >95 % 0.2778 0.6111 0.7778
0.0556 0.2222
DPSQSLSETEFVGL WDPSQSLSETEFVGLA >95 % 0.2857 0.2143 0.7143 0.0714
0.4286
ENVLITTPKTN QENVLITTPKTNN >95 % 0.2727 0.2727 0.6364 N 0.0909
TAVHHPLIDIVA KTAVHHPLIDIVAD >95 % 0.0833 0.4167 0.7500 0.0833 0.0833
DQVLLSGVQGIT ADQVLLSGVQGITD >95 % 0.0833 0.3333 0.6667 0.0833 0.0833
QDVIIKELQGS IQDVIIKELQGSE >95 % 0.3636 0.2727 0.6364 0.0909 0.2727
PIDEPVIP QPIDEPVIPG >95 % 0.3750 0.5000 0.8750 0.2500 0.3750
ELPDPVIP RELPDPVIPE >95 % 0.2500 0.5000 1.0000 0.1250 0.3750
EDPIVP EEDPIVPP >95 % 0.5000 0.5000 0.6667 0.3333 0.5000
EDPLVP SEDPLVPS >95 % 0.5000 0.5000 1.0000 0.3333 0.5000
[96]Open in a new tab
Note: N means not detected; The BIOPEP database for peptides and amino
acids ([97]https://biochemia.uwm.edu.pl/biopep-uwm/).
The study revealed that the umami taste enhancement thresholds of
ALPEEV and EAGIQ were 1.52 mmol/L and 1.94 mmol/L, respectively
([98]Sud et al., 2007). These peptides, known to possess umami taste,
were predicted and scored using the same method applied in this study.
The results showed umami scores of 0.8333 for ALPEEV and 0.4000 for
EAGIQ. In comparison, the umami scores of EDPIVP and EDPLVP, identified
in this study, were both 0.5000, falling between the values of 0.4000
and 0.8333. This suggests that the umami threshold of EDPIVP and EDPLVP
might range between 1.52 and 1.94 mmol/L, confirming the reliability of
the prediction method used. Therefore, the polypeptides
AGFAGDDAPRAVFPSIVGRPR, ELPDPVIP, EDPIVP, and EDPLVP, isolated from
steamed Antarctic krill, exhibit both umami and sweetness.
Additionally, these peptides showed relatively high bitterness scores,
likely due to the presence of hydrophobic amino acids in their
sequences.
3.4. Analysis of peptide chain amino acid cleavage site of steamed Antarctic
krill
The principle of the Edman degradation method involves modifying the
N-terminal of water-soluble small peptides in the sample and
subsequently cleaving them. This allows the determination of the amino
acid cleavage sites at the N-terminus of larger peptide chains during
the formation of smaller peptide hydrolysis products, based on changes
in free amino acid content. After steaming, a significant increase in
the content of certain amino acids indicates the specific cleavage
sites in the peptide chain. For instance, the contents of E, R, T, A,
Y, and V increased significantly after steaming ([99]Fig. 1C),
suggesting that the peptide chain cleaved at these sites during the
steaming process.
3.5. Non-targeted metabolomics analysis of Antarctic krill during steaming
3.5.1. Multivariate statistical analysis
Non-targeted metabolomics was used to identify and analyze metabolites
in Antarctic krill before and after steaming, using both positive
(left) and negative (right) ion scanning modes. The base peak
chromatogram ([100]Fig. 2A) of the samples showed that the trends
across different groups of Antarctic krill samples were similar,
indicating good repeatability. Quality control (QC) procedures
([101]Fig. 2B) were performed to remove characteristic peaks that did
not meet the requirements (RSD > 30 %), resulting in a higher-quality
dataset ([102]Fig. 2C). The PCA score plot demonstrated that the QC
samples clustered together, confirming the reliability and
reproducibility of the analysis. Following LC-Orbitrap-MS analysis, a
total of 7572 metabolites were detected. As shown in [103]Fig. 3A,
while the content of most metabolites decreased, some compounds
increased in SKM. This indicates that steaming caused significant
changes in the metabolite content of Antarctic krill. [104]Fig. 3B
showed the hierarchical clustering tree diagram of samples before and
after steaming. The samples were classified into two distinct
categories, indicating significant changes between SKM and RKM samples.
OPLS-DA (Orthogonal Partial Least Squares Discriminant Analysis) is
effective for filtering irrelevant information and accurately analyzing
the differences and associations of metabolites across different
samples ([105]Heidrich et al., 2021). As shown in the scores plot
([106]Fig. 3C), the separation between RKM and SKM reflects notable
changes in the metabolites of Antarctic krill after steaming. The
samples within the RKM group showed good aggregation, while some
separation within the SKM group could be attributed to thermal
induction. The OPLS-DA analysis yielded key parameters: R^2X = 0.536,
R^2Y = 0.999, and Q^2 = 0.964, all exceeding 0.5, indicating that the
model is robust for identifying differential metabolites. The OPLS-DA
S-plot ([107]Fig. 3D) highlights the differential metabolite profiles
crucial for distinguishing between RKM and SKM.
Fig. 2.
[108]Fig. 2
[109]Open in a new tab
(A) Base peak chart of typical samples. (B) Principal component
analysis score plot of quality control. (C) Relative standard deviation
distribution of quality assurance.
Fig. 3.
[110]Fig. 3
[111]Open in a new tab
Multivariate statistical analysis. (A) Hierarchical clustering heat map
of metabolites. (B) Clustering analysis. (C) OPLS-DA Score Plot. (D)
OPLS-DA s-plot.
3.5.2. Detection and identification of differential metabolites
Differential metabolite screening was conducted using VIP values, which
reflect the importance of each variable in the first principal
component of OPLS-DA. A higher VIP value indicates a greater
contribution of the variable to the grouping. Additionally, a t-test
was conducted to further evaluate the significance of metabolites
between groups. Metabolites with VIP values greater than 1 and P-values
less than 0.05 were considered as potential biomarkers. The analysis
identified several potential biomarkers in both the RKM and SKM groups,
including hydrocarbons, amino acids and their derivatives, organic
acids, organic bases, aldehydes, esters, and fatty acids. Specifically,
the expression of 21 metabolites was up-regulated, including
acetylcholine chloride, niacinamide, pyridoxine, methyl jasmonate,
dTMP, and cis-4-hydroxy-L-proline. Conversely, the expression of 136
metabolites was down-regulated, including L-aspartic acid, citric acid,
guanosine monophosphate (GMP), L-lysine, L-methionine, and
L-phenylalanine.
Hierarchical clustering analysis (HCA) is a widely used method in data
mining and statistics to explore the diversity in metabolite profiles
across different experimental samples. In this study, HCA was employed
to reveal variations among the metabolite profiles of the samples. As
shown in [112]Fig. 4A, the color gradient from blue to red represents
the range of metabolite expression abundance, with blue indicating low
expression and red indicating high expression. The color gradient
clearly illustrates the differences in metabolite levels between RKM
and SKM samples. Notably, the expression of differential metabolites
was significantly down-regulated in steamed Antarctic krill, consistent
with the previously observed results.
Fig. 4.
[113]Fig. 4
[114]Open in a new tab
Detection and identification of differential metabolites. (A)
Hierarchical clustering heatmap of differential metabolites. (B)
Volcano Plot. (C) Z-score Plot. (D) Differential metabolic matter
charge ratio and P-Value Scattering Plot. (E) Analysis of differential
metabolite formation pathways.
The standard score (Z-score) map provides a visual comparison of the
relative contents of differential metabolites between the RKM and SKM
samples at the same level. From the differential metabolite volcano
plot ([115]Fig. 4B), it is evident that the expression fold difference
between the RKM and SKM samples is most pronounced. The four
metabolites showing the most significant differential expression are
hydroquinone, 5-hydroxypentanoic acid, gamma-aminobutyric acid, and
L-2,4-diaminobutyric acid. [116]Fig. 4C presents the standard scores of
differential metabolites in Antarctic krill samples before and after
steaming. The higher the value on the ordinate, the greater the
metabolite content. This visualization provides an intuitive comparison
of the relative contents of 157 differential metabolites between the
RKM and SKM groups, highlighting that thermal induction caused
significant changes in the samples. [117]Fig. 4D displays a scatter
plot of the mass-to-charge ratio versus P-value for the metabolites,
offering a clear and intuitive view of the distribution of key
differential metabolites. Metabolites with significant differential
expression (P < 0.05) include methyl jasmonate,
3-dehydro-scyllo-inosose, uracil, and pyridoxine.
3.5.3. Analysis of differential metabolite formation pathways in steamed
Antarctic krill
The related metabolic network ([118]Fig. 4E) illustrated the
correlation of differential metabolites in Antarctic krill before and
after thermal induction, and explored possible metabolic pathways of
these metabolites. Amino acids emerged as the most frequently involved
metabolites, forming a prominent cluster that includes phenylalanine,
glycine and serine, key differential metabolites affected by steaming.
Additionally, several amino acids, such as aspartic acid, methionine,
lysine and leucine, which played important roles in the flavor of
Antarctic krill, were significantly downregulated after steaming.
The observed reduction in amino acid levels may result from their
participation in the Maillard reaction, where they react with glucose
or other carbohydrates during heat processing, leading to a decrease in
their content. This aligned with the previous findings that these
changes in amino acids not only directly influenced the flavor
characteristics of Antarctic krill but also synergize with nucleotides
such as GMP and AMP, further enhancing or modifying the umami profile
([119]Zhong et al., 2021). The identification of various nucleotide
derivatives underscores their importance in flavor formation. Previous
research ([120]Chen et al., 2021) had similarly demonstrated the role
of GMP and other nucleotides in enhancing the umami taste of seafood.
Therefore, the flavor changes in Antarctic krill during steaming were
the result of a multifaceted interplay between regulated metabolic
pathways.
The mechanisms underlying flavor formation in steamed Antarctic krill
are highly complex and involve the interplay of lipid oxidation, amino
acid metabolism, and nucleotide interactions, among other biochemical
processes. Amino acid metabolism and nucleotide metabolism jointly
serve as key contributors, with the reduction of certain amino acids
and the formation of nucleotide derivatives significantly shaping the
flavor profile. The oxidation and degradation of lipids serve as
primary pathways for flavor development. During steaming, unsaturated
fatty acids, such as oleic and linoleic acids, undergo oxidation due to
their chemical susceptibility (double bonds), producing hydroperoxides.
These hydroperoxides subsequently decompose into various flavor-active
compounds, including ketones, aldehydes, and alcohols. For instance,
nonanal is a key product derived from oleic acid oxidation, while
1-octen-3-ol, a crucial odor-active compound, originates from linoleic
acid degradation. Saturated and monounsaturated fatty acids also
contribute to flavor by forming linear aldehydes and hydrocarbons
([121]Farvin et al., 2012; [122]Matsui et al., 2003). These lipid
oxidation products form the backbone of the characteristic aroma of
steamed Antarctic krill. They are further enriched through interactions
with Maillard reaction intermediates.
The interplay between lipid oxidation and the Maillard reaction plays a
pivotal role in flavor complexity. Lipid oxidation products, such as
aldehydes, can react with Maillard reaction intermediates to generate
secondary volatile compounds, including furans and alcohols. These
cross-reactions enhance the sensory attributes of steamed krill by
adding depth to its roasted and savory flavor. Aromatic amino acids,
such as phenylalanine, also participate in metabolic pathways linked to
secondary metabolite biosynthesis. These processes contribute to the
chemical diversity of flavor compounds formed during steaming.
In summary, the flavor formation in steamed Antarctic krill is a result
of a multifaceted interplay between lipid oxidation, amino acid
metabolism, and Maillard reactions. Lipid-derived volatiles, amino acid
degradation products, and nucleotide interactions collectively shape
the distinctive umami and aromatic profile of krill. This study
advances the understanding of molecular mechanisms involved in seafood
flavor development and offers valuable guidance for optimizing
processing techniques in the food industry.
4. Conclusion
This study provided a detailed analysis of the volatile compounds,
metabolites, and taste characteristics of Antarctic krill before and
after steaming. HS-SPME-GC–MS identified various volatile compounds,
including alcohols, aldehydes, and ketones, which contribute to the
krill's flavor profile. Notably, the content of nonanal and
1-octen-3-ol significantly increased after steaming due to lipid
degradation. Metabolite profiling revealed substantial shifts in
metabolite content, with significant downregulation of L-aspartic acid,
citric acid, GMP, L-lysine, L-methionine and L-phenylalanine during
steaming. Hydroquinone, gamma-aminobutyric acid, and
L-2,4-diaminobutyric acid were identified as the most significantly
altered metabolites. Metabolic network further linked these shifts to
reactions involving amino acids, nucleotide and other compounds during
steaming. The results highlighted that steaming significantly affects
both the chemical composition and flavor of Antarctic krill, offering
valuable insights into the metabolic transformations during food
processing.
CRediT authorship contribution statement
Tingting Yang: Writing – original draft, Software, Methodology,
Investigation, Data curation. Yang Liu: Software, Methodology. Jing
Bai: Writing – review & editing, Methodology. Yan Fan: Writing – review
& editing, Visualization. Ye Chen: Methodology, Investigation. Ping
Dong: Writing – review & editing. Xia Yang: Writing – review & editing,
Software. Hu Hou: Writing – review & editing, Funding acquisition.
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.
Acknowledgements