foods

Review
Metabolomics Approaches for the Comprehensive Evaluation
of Fermented Foods: A Review
Yaxin Gao 1 , Lizhen Hou 1 , Jie Gao 1 , Danfeng Li 1 , Zhiliang Tian 1 , Bei Fan 1,2 , Fengzhong Wang 2,3, *
and Shuying Li 1, *

1 Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, No. 2 Yuan Ming Yuan
West Road, Beijing 100193, China; [email protected] (Y.G.); [email protected] (L.H.);
[email protected] (J.G.); [email protected] (D.L.); [email protected] (Z.T.); [email protected] (B.F.)
2 Key Laboratory of Agro-Products Quality and Safety Control in Storage and Transport Process, Ministry of
Agriculture and Rural Affairs, Chinese Academy of Agricultural Sciences, Beijing 100193, China
3 Key Laboratory of Agro-Products Processing, Ministry of Agriculture and Rural Affairs, Chinese Academy of
Agricultural Sciences, Beijing 100193, China
* Correspondence: [email protected] (F.W.); [email protected] (S.L.); Tel.: +86-010-62815977 (F.W.);
+86-010-62810295 (S.L.)

Abstract: Fermentation is an important process that can provide new flavors and nutritional and
functional foods, to deal with changing consumer preferences. Fermented foods have complex
chemical components that can modulate unique qualitative properties. Consequently, monitoring the
small molecular metabolites in fermented food is critical to clarify its qualitative properties and help
deliver personalized nutrition. In recent years, the application of metabolomics to nutrition research
of fermented foods has expanded. In this review, we examine the application of metabolomics





 technologies in food, with a primary focus on the different analytical approaches suitable for food
Citation: Gao, Y.; Hou, L.; Gao, J.; Li, metabolomics and discuss the advantages and disadvantages of these approaches. In addition,
D.; Tian, Z.; Fan, B.; Wang, F.; Li, S. we summarize emerging studies applying metabolomics in the comprehensive analysis of the
Metabolomics Approaches for the flavor, nutrition, function, and safety of fermented foods, as well as emphasize the applicability of
Comprehensive Evaluation of metabolomics in characterizing the qualitative properties of fermented foods.
Fermented Foods: A Review. Foods
2021, 10, 2294. https://doi.org/ Keywords: fermented foods; metabolomics; metabolomic approach; qualitative property
10.3390/foods10102294

Academic Editors: Mohamed Farag

and Ludger A. Wessjohann 1. Introduction
Fermented foods have been produced and consumed since around the Neolithic period
Received: 7 September 2021
Accepted: 22 September 2021
and have been embedded in dietary cultural norms around the world [1,2]. Fermentation
Published: 28 September 2021
preserves food and provides new flavors and textures from traditional foods that contribute
to pleasant gastronomic experiences and health benefits [3,4]. Globally, a wide variety
Publisher’s Note: MDPI stays neutral
of fermented foods commonly present in daily diets, which include those derived from
with regard to jurisdictional claims in
beans, milk, cereals, vegetables, fruits, meat, and seafood [5]. Some commonly consumed
published maps and institutional affil- fermented foods include miso, soy paste, natto, and vinegar from East and Southeast
iations. Asia and yogurt, cheese, and kefir from Europe and America [6–8]. Other traditionally
fermented foods include pickled fruit and vegetables and fermented beverages (e.g., beer,
wine, and tea) from cereals and plants [9,10].
Fermentation is a metabolic process wherein microorganisms enzymatically convert
Copyright: © 2021 by the authors.
substrates in raw materials into new products, which results in the unique final flavor, nutri-
Licensee MDPI, Basel, Switzerland.
tional, functional, and safety characteristics of fermented foods [5,11]. The microorganisms
This article is an open access article
used in fermented foods mainly include fungi (e.g., Saccharomyces spp., Mucor spp., and
distributed under the terms and Rhizopus spp.) and bacteria (e.g., Bacillus subtilis spp., Lactobacillus spp., and Bifidobacterium
conditions of the Creative Commons spp.) [12,13]. Fermented foods have been recognized as key ingredients to develop novel
Attribution (CC BY) license (https:// functional foods in the food industry. Fermentation can foster the improvement of food
creativecommons.org/licenses/by/ properties, including the transformation of the nutrient profile and the formation of novel
4.0/). flavor, as well as functional and health properties, because microorganisms can generate

Foods 2021, 10, 2294. https://doi.org/10.3390/foods10102294 https://www.mdpi.com/journal/foods

Foods 2021, 10, 2294 2 of 18

enzymes that degrade macromolecular compounds and nonnutritive factors (e.g., tannin,
protease inhibitor, and soybean agglutinin) in raw materials, to improve the digestion and
absorption characteristics [10,14–17]. In addition, food safety and shelf life can be enhanced
by preventing growth of pathogenic microorganisms [18]. Therefore, the actions of microor-
ganisms on the primary and secondary metabolites generated during the fermentation
process modify the properties of fermented foods. Recently, researchers have preferred
using metabolomics analysis to provide detailed information regarding metabolites (amino
acids, lipids, and phytochemicals) and thus effectively evaluate food properties.
Currently, metabolomics is an emerging field but is also considered an established but
evolving analytical technology widely involved in many fields such as food science, food
quality, and food safety [19,20]. With the continuous advancement of separation techniques
and high-resolution mass spectrometry, and improvements in database and data process-
ing software, metabolomics is gradually gaining acceptance as a method to empirically
delineate the properties of fermented foods and probe functional metabolomes [21,22].
The concept of metabolomics was first proposed by Nicholson in 1999, as a component
of systems biology. It extends the principles of genomics and transcriptomics, enabling
qualitative and quantitative analysis of small molecular metabolites (<1500 Da) in biological
systems [23,24].
Metabolomics allows for the collection of all metabolites in cells, tissues, or organ-
isms under specific physiological periods or conditions [25]. Metabolomic signatures
(fingerprints) can be obtained using different approaches, such as nuclear magnetic res-
onance (NMR) spectroscopy, gas chromatography–mass spectrometry (GC-MS), liquid
chromatography–mass spectrometry (LC-MS), and capillary electrophoresis–mass spec-
trometry (CE-MS) [26–28]. MS-based metabolomics is divided into two types based on
whether they are targeted. Non-targeted metabolomics is biased toward high-resolving
power, detecting as many metabolites as possible without quantification; involves detectors
including time-of-flight (TOF), Orbitrap, and Fourier-transform ion cyclotron resonance
(FT-ICR); can identify several thousand metabolites. In contrast, targeted metabolomics
aims to achieve highly sensitive detection and quantification, usually targeting and quanti-
fying a selected group of compounds with a triple quadrupole (QQQ) detector [19,28]. The
sensitivity, resolution, and metabolite coverage vary among these approaches, based on the
corresponding analytical instruments, which often complement each other. However, the
primary disciplines utilizing the applications of quantitative and qualitative metabolomics
include microbiology [29], plant physiology [30], medicine [31], human health [32], and
food and nutritional research [33].
In this review, we assess the latest applications of metabolomics in fermented food
research. We discuss several metabolomics approaches and outline the perspectives on the
current trends toward applying metabolomics to probe the flavor, nutrition, function, and
safety properties of fermented foods. Further, this review provides a reference for quality
control and identification associated with fermented foods.

2. Methodology
Studies describing food metabolomics have been published in many journals in the
same field. Multiple databases were used to conduct a comprehensive search on related
topics, such as Science Direct, National Center for Biotechnology Information, Web of
science, and Google Scholar. The keywords included metabolomics, food, fermentation,
detection methods, flavor, nutrition, function, safety, research, and were combined in
different ways to effectively search relevant and up-to-date literature.

3. Metabolomics Workflow
The basic metabolomics workflow was gradually standardized during application de-
velopment (Table 1). It comprises three key steps: sample preparation, analytical approach,
and data processing and biological interpretation (Figure 1, from http://www.google.com,
accessed on 30 June 2021) [23,34]. Effective sample preparation is a critical step for subse-
 3. Metabolomics Workflow
The basic metabolomics workflow was gradually standardized durin
development (Table 1). It comprises three key steps: sample preparation, a
Foods 2021, 10, 2294
proach, and data processing and biological interpretation 3 of 18
(Figu
http://www.google.com, accessed on 30 June 2021) [23,34]. Effective sampl
is a critical step for subsequent analysis [35]. The methods for collecting an
samples
quent analysis vary
[35]. Thebased
methods onforthe samples
collecting andor research
processing questions,
samples andonmainly
vary based the inv
samplespendent sampling
or research toand
questions, reflect theinvolve
mainly temporal dynamicssampling
time-dependent and microbial
to reflect succession
the
temporal dynamics and microbial successions involved in fermentation [36].
fermentation [36]. Metabolite separation and detection (e.g., NMR and GC-M Metabolite
separation and detection
recognized (e.g.,
as key NMRinand
steps GC-MS) have
metabolic been recognized
profiling as key steps studies,
[37]. Metabolomics in d
metabolic profiling [37]. Metabolomics studies, distinguished by different separation and
by different separation and detection methods, have been widely used in foo
detection methods, have been widely used in foods. They can effectively discriminate and
predicteffectively discriminate
potential metabolites and predict
and explain potential
the biological metabolites
processes and
studied, thus explain the b
reflecting
cesses
the food studied,
properties, suchthus reflecting
as flavor, the food properties, such as flavor, nutrition.
nutrition.

Figure 1.Figure
Schematic overview
1. Schematic of the
overview most
of the mostimportant platforms
important platforms used
used for metabolomics
for metabolomics studies. studies.

3.1. Metabolomic Approaches Based on Different Separations and Instrumentation

3.1. Metabolomic
3.1.1. NMR-Based Approaches Based on Different Separations and Instrumentation
Metabolomics
NMR3.1.1. NMR-Based
is one Metabolomics
of the most frequently used analytical tools in metabolomic fingerprinting
and profiling [38]. Several recent publications have described NMR-based metabolomics
NMR is one of the most frequently used analytical tools in metabolomi
in plants, animal tissues, foods, and beverages [39]. NMR, which relies on bulky and ex-
pensive ing and profiling
instruments (magnets [38].
and Several recent
electronics), publications
can interpret havedynamics
biomolecular described withNMR-ba
lomics in
stereochemical plants,
detail, animal
and detect all tissues,
compounds foods,
with and beverages
abundant protons,[39].
such NMR, which re
as sugars,
polyphenols, amino acids, vitamins, nucleotides, and other organic
and expensive instruments (magnets and electronics), can interpret biomolecompounds [40–42].
Beyond the initial installation costs, the running costs are relatively inexpensive [43]. More-
ics with stereochemical detail, and detect all compounds with abundant pro
over, the non-destructive characteristics of the technology have ensured wide adoption
amongsugars, polyphenols,
current metabolomic amino acids,
approaches, vitamins,
since sample nucleotides,
preparation and other
is relatively simple organic
and does[40–42]. Beyond
not involve the initial
chemical installation
extractions, costs, thetorunning
which contributes the abilitycosts are relatively
to analyze
a single[43]. Moreover,
sample in multipletheconsecutive
non-destructive characteristics
experiments of thewith
[44,45]. Moreover, technology
minimal have e
sampleadoption
preparation, NMR current
among technology enables simultaneous
metabolomic identification
approaches, and absolute
since sample preparatio
quantification of compounds in a sample based on the direct relationship between molar
simple and does not involve chemical extractions, which contributes to the a
concentration and the intensity of the NMR resonances, achieving high-throughput de-
tectionlyze
[46]. aHowever,
single sample
the lowerinsensitivity
multiple and consecutive
large sample experiments [44,45]. Moreover,
quantity requirements are
sample
the principal preparation,
drawbacks of NMRNMR [47,48].technology enables simultaneous
In addition, high-resolution identification
NMR spectroscopy
(HRMAS-NMR) has become
quantification a rapid and accurate
of compounds in a samplealternative
based technique
on the at the molecular
direct relationship be
level, following the development of high-field NMR spectrometers [49]. In recent years,
concentration and the intensity of the NMR resonances, achieving high-th
NMR spectroscopy has been widely applied to analyze the metabolomes of traditional
tection
foods such [46].
as soy sauceHowever, the lower
[50], fermented sensitivity
fish sauce and large
[51], and alcoholic sample
beverages quantity requ
[49].
the principal drawbacks of NMR [47,48]. In addition, high-resolution NMR
(HRMAS-NMR) has become a rapid and accurate alternative technique at t
level, following the development of high-field NMR spectrometers [49]. In
NMR spectroscopy has been widely applied to analyze the metabolomes
foods such as soy sauce [50], fermented fish sauce [51], and alcoholic bevera
Foods 2021, 10, 2294 4 of 18

3.1.2. FT-IR-Based Metabolomics

Infrared (IR) spectroscopy is considered an effective tool for classifying, character-
izing, and identifying small molecules, and FT-IR spectroscopy has rapidly become a
valuable metabolic fingerprinting tool [52–54]. FT-IR enables the simultaneous and accu-
rate measurement of major and minor components, such as carbohydrates, polysaccharides,
polyphenols, proteins, amino acids, and fatty acids [55–57]. Similar to NMR spectroscopy,
FT-IR spectroscopy can also be performed in a rapid and non-destructive manner without
sample extraction and derivation on either liquid or solid samples [58]. It is often applied
for rapid detection of adulteration and evaluation of the maturity of foods (e.g., sesame
oil, mango) [59–61]. However, its major disadvantage is the inability to distinguish the
chemical constituents of a mixture [62]. Thus, FT-IR is generally combined with other
analytical techniques to provide more accurate information on elemental composition and
molecular structure, resulting in the identification of the main metabolites (e.g., sugars and
organic acids) found in fermented foods [54,63,64].

3.1.3. GC-MS/LC-MS-Based Metabolomics

Over the past decades, MS-based chromatographic systems (GC or LC) have rapidly
developed into the most commonly used analytical approaches in metabolomic studies,
partly because they yield higher sensitivity than the previously discussed techniques [35,65,66].
The crucial component for GC-MS and LC-MS operation is the mass detector, including
QQQ, TOF, and electrospray-ionization (ESI), each of which have advantages depending on
the specific applications and sample types [67,68]. The choice of chromatographic separa-
tion for metabolite analysis is related to the physicochemical properties of the compounds,
and both approaches have their own related advantages and disadvantages.
GC-MS is the preferred analytical technique for the study of volatile compounds and
is mainly employed for evaluating primary metabolites such as amino acids, fatty acids,
organic acids, alcohols, and sugars or sugar alcohols [45,69]. GC-MS-based metabolomics
provides high sensitivity and highly repeatable fragmentation, and takes advantage of well-
established spectral databases, such as the National Institute of Standards and Technology
(NIST), Wiley, and the Automated Mass spectral Deconvolution and Identification System
(AMDIS), allowing for the identification of important biomarkers [70]. However, one
drawback of GC-MS-based metabolomics is the complex pre-treatments required before
analysis, followed by difficult sample processing and derivatization required to modify
polarity and facilitate effective separation [71,72]. Thus, the long detection time required
for this approach may not meet the requirements for the rapid detection of many analytes.
In contrast, LC-MS is the most widely used technique in metabolomics for profiling soluble
and/or unknown metabolites and is commonly used for evaluating secondary metabolites
such as flavonoids, saponins, alkaloids, phospholipids, and polyamines [45]. Similar to
GC-MS, LC-MS-based metabolomics is also characterized by high sensitivity, robustness,
and versatility. Notably, volatility is not required in this approach, allowing for more
extensive metabolite detection. The sample derivatization process can be omitted, reducing
background signal noise and providing higher quality data [73,74]. Owing to higher
separation and sensitivity, GC-MS/LC-MS-based metabolomics has been widely applied
to study various complex food matrices, such as dry-cured hams [75], tea [76], honey [77],
as well as fermented foods [78,79].

3.1.4. CE-MS-Based Metabolomics

CE-MS, another approach in metabolomics, is now a widely used analytical separation
technique, notable for the separation of highly polar and charged metabolites, based on
varying mass-to-charge ratios, such as amino acids, organic acids, and nucleotides [80,81].
The most attractive aspect of CE-MS is that the proposed workflow only requires small
amounts of organic reagents. This facilitates convenient analysis of volume-limited bio-
logical samples, providing rapid yet comprehensive profiling of compounds from small
inorganic ions to large proteins [82]. However, the low sample throughput in CE-MS results
Foods 2021, 10, 2294 5 of 18

in relatively poor concentration sensitivity, leading to lower detection sensitivity and repro-
ducibility. In this sense, compared with other analytical separation techniques, metabolome
profiling studies using CE-MS lack representativeness, which limits its application [83].
In contrast with GC-MS and LC-MS approaches that require interaction with a stationary
phase, separation by CE relies on a small electrolyte-filled capillary. Most metabolites
are too polar or ionic to be retained by the columns used in GC- and LC-MS [37]. Often,
samples that cannot be separated by GC- or LC-MS can be easily resolved by CE-MS [84].
Thus, CE-MS with higher separation is considered a complementary tool to traditional chro-
matographic techniques, thus improving the efficiency and abundance in the metabolite
detection spectra [21]. Over the past few years, recent developments in CE-MS instrumen-
tation for sample throughput and quality control have been achieved. The usefulness of
CE-MS as an independent or supplementary technique is gradually gaining traction in the
fields of biomedical, clinical, microbial, plant, and food metabolomics [85].

3.1.5. Electronic Nose-Based Metabolomics

Another important detector with applications in metabolomics research is the elec-
tronic nose (E-nose), which deploys electronic sensors to mimic the biological olfactory
mechanism to obtain the overall odor of products [86,87]. The common E-nose comprises an
aroma extraction system, sensor array, analog-to-digital system, and a method for pattern
recognition, allowing it to recognize simple or complex, natural or synthetic organic odor
compounds but not specific metabolites [88,89]. In this context, the volatile compounds
detected by an E-nose exhibit a unique composition, such that they can be used for quality
change detection and identification. The E-nose approach has been extensively used in
many industries, such as grain [90], dairy [91], and pharmaceuticals [92]. In comparison
with traditional high-throughput metabolomic techniques, E-nose technology is easy to
operate, offers a lower cost alternative, and non-destructive detection; however, E-nose-
based metabolomics technology is still considered to be in its infancy [93]. Furthermore,
the use of E-nose technology can quickly achieve real-time monitoring of the quality of
agricultural products compared to GC-MS [94]. Combining E-nose technology with MS
has been successfully used to distinguish the quality, aroma, and origin of fermented foods,
including apple cider vinegar and soybean paste [95,96].

3.2. Metabolomic Analyses Based on Data Interpretation and Multivariate Statistics

Metabolomics generates extensive datasets that require effective data processing
methods to successively interpret their biological implications. Bioinformatics and multi-
functional tools for performing compound identification, statistics, enrichment, pathway
prediction, and visualization provide high-quality and comprehensive metabolomics infor-
mation (Table 1).
Before pattern recognition analysis (multivariate statistical analysis), raw metabolomics
data must be pre-processed, and a series of adjustments such as baseline correction, peak
recognition, peak alignment, and normalization must be performed using the appropriate
software, such as MestreNova (Mestrelab Research, Santiago de Compostela, Spain) and
Progenesis QI (Waters Corp., Milford, MA, USA) [97,98]. Subsequently, metabolite identifi-
cation and characterization are completed based on accurate mass, secondary fragments,
and isotope distribution, through gradually standardized online software or websites (e.g.,
the Human Metabolome Database, HMDB, https://www.hmdb.ca, accessed on 30 June
2021; METLIN Database, https://metlin.scripps.edu/, accessed on 30 June 2021) [98]. The
software used may have a great influence on data processing and results; it is important to
select an appropriate software package according to different metabolomics methods and
research objectives.
In addition, the application of multivariate statistical analysis can effectively reduce the
complexity of a reliable metabolomics dataset containing qualitative and quantitative infor-
mation in interpreting complex biological systems or related biological processes [99,100].
To date, the multivariate statistical tools developed, mainly through SIMCA-P software
Foods 2021, 10, 2294 6 of 18

(Umetrics, Sweden), comprise three major techniques: unsupervised, supervised, and

pathway analysis [101]. Principal component analysis (PCA), the most commonly used
unsupervised tool, converts a large number of related datasets into a limited number
of variables to visualize the most important trends after dimensionality reduction [102].
Partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares
discriminant analysis (OPLS-DA) are usually used to assess obvious differences between
the groups (variables) within datasets and identify the biomarkers through the value of
variables of importance in projection (VIP) [102,103]. Regarding pathway analysis, pathway
enrichment analysis of differential metabolites is mainly based on the Kyoto Encyclopedia
of Genes and Genomes (KEGG, https://www.genome.jp/kegg/, accessed on 30 June
2021) database or MetaboAnalyst (https://www.metaboanalyst.ca/, accessed on 30 June
2021), which helps understand the mechanisms of metabolic pathway changes in a sam-
ple [99]. Therefore, a reasonable and effective data processing method for metabolomics
can maximize the interpretation of the biological significance of the sample and predict the
properties of fermented foods [104,105].

Table 1. Computational tools for metabolomics analysis.

Biobanks Role References

Data processing prediction, publication, verification; data
MestreNova [97]
storage and retrieval
Data processing and normalization; qualitative,
Progenesis QI quantitative and identification of small molecules with [98]
significant changes
Multivariate statistical analysis; pattern recognition of
SIMCA [97,99–101]
PCA, PLS-DA, OPLS-DA
Multivariate tool; heatmap of metabolites and their
RStudio [97]
concentration changes
Physicochemical and biological properties; biomarker
HMDB [97–101]
discovery; metabolic pathway information
Metabolite identification; metabolite structure; links to
METLIN [98,100,101]
other databases
Biological properties of organic small molecule;
PubChem [100]
metabolite structure; links to other databases
Data analysis, visualization, and functional annotation;
MetaboAnalyst multivariate analysis; metabolite significance; pathway [98,99,101]
identification
Metabolic pathway; metabolite interactions; delivery of
KEGG [98,99,101]
gene to metabolite function
Hierarchical cluster analysis; heatmap of metabolites and
Mev [100]
their concentration changes
Interaction network visualization: correlation analysis
Cytoscape [99]
linked to gene, protein, and metabolite expression
Note and Abbreviations: SIMCA, soft independent modeling of class analogy; Mev, Multiple Experiment Viewer.

4. Applications of Fermented Foods

With increased consumption and a societal increase in health consciousness, the gen-
eral population is more concerned about whether food and nutrition play an important
role in maintaining health and reducing the risk of various diseases. The rise in health
awareness has driven tremendous public interest in fermented foods, which have multiple
health benefits, including antioxidant, anti-cancer, anti-inflammatory, and cardioprotective
Foods 2021, 10, 2294 7 of 18

properties [106,107]. Fermentation can influence the improvement of organoleptic char-

acteristics, nutritional and functional properties, and safety considerations with a final
positive effect on human health, mainly because of the secondary metabolites generated
from the interaction of raw materials and probiotics [108,109]. Metabolomic profiling of
fermented foods has been used to monitor metabolites representative of qualitative proper-
ties during fermentation to predict nutritional, functional, flavor, and safety characteristics
of the final products [109,110].

4.1. Flavor
The flavor of fermented foods is mostly generated through microbial metabolism and
enzymatic biochemical reactions [111]. It is a key indicator of sensory qualities, which often
directly determines acceptance by consumers [112]. Characterization of fermented food
flavor also contributes to determining the maturity of a product and the differences among
products obtained from different production locations or fermentation strains [112,113].
It mainly depends on the composition of volatile flavor compounds, which constitute a
preferred flavor, or an unpleasant flavor based on different composition conditions. Flavor
components are usually identified by GC-MS [114,115]. The most relevant information
obtained from metabolomic studies on the flavors of fermented foods is presented in
Table 2.
Distinctive flavors are essential factors in alcoholic beverages, including baijiu, huangjiu,
beer, and red wine, which are all commonly consumed in Chinese society. Yang et al. [116]
applied GC-GC-TOF-MS to analyze baijiu made from yellow, white, and black daqu, and
captured 401 volatile compounds, mainly aromatic compounds and pyrazines. Among
them, white daqu has the most types of esters and alcohols, but the levels of esters and
alcohols in yellow and black daqu were higher than in white daqu, which may be re-
lated to microbial metabolism during fermentation. The flavor of Chinese huangjiu from
Shanxi, a yellow rice wine with zao-aroma, was also analyzed based on GC-MS and GC-
olfactometry (GC-O) techniques, and ethyl cinnamate and ethyl 3-phenylpropionate were
demonstrated to be key aroma compounds, which indicated differences from others such
as Zhejiang Shaoxing and Fujian Hongqu huangjiu [112,115]. Pu-erh tea is a unique tea
obtained from the fermentation of Monascus tea, and the understanding of the formation
and evolution mechanism of its flavor-active compounds can improve its quality. Deng
et al. [117] summarized the metabolic evolution of key flavor-active compounds during
Pu-erh tea fermentation systematically based on headspace solid phase microextraction
(HS-SPME)-GC-MS. The volatile fingerprint and flavor profiles of fermented soybean foods
have been investigated using metabolomics technologies. The flavor of fermented soy
foods significantly reduces the proportion of beany flavor substances in the soybean itself,
which is an unpleasant flavor affecting its consumption [118]. Wangzhihe red sufu, a
Chinese traditional fermented condiment, was analyzed using metabolomics during the
fermentation process, and four typical commercial red sufu products were detected by GC-
MS, GC-O/MS, and E-nose [119]. The most prevalent volatile components (e.g., phenolics,
esters, and alcohols) varied in the molded phetze, salted phetze, and post-fermentation
stages of red sufu, and there were differences in the overall volatile profiles of the four
sufu products, which were conducive to effective discrimination of the extent of sufu
maturity and the type of sufu products. Notably, the flavor of products related to natto,
and soybeans fermented by B. subtilis, are usually undesirable to the Chinese population,
whereas natto is very popular in Japan where it is an important side dish. The volatile
metabolite composition of natto products has been investigated through GC-MS and NMR,
and it has been shown that ammonia, 2,5-dimethylpyrazine, isovalerate, isobutyrate, and
2-methylbutyrate are increased in amount and proportion in natto, which may explain
the undesirable flavor [120–122]. Gao et al. [122] studied soymilk fermented by B. sub-
tilis through NMR and not only found undesirable flavor compounds but also explained
the changes in nutrients and functional components in fermented soymilk. In addition,
metabolomics has played a key role in determining the maturity of cheese and other dairy
Foods 2021, 10, 2294 8 of 18

products [113], the analysis of the correlation between flavor components and microbial
communities in the types of fermented mandarin fish [123], and the elucidation of dy-
namic changes in flavor composition during the fermentation of rose vinegar and shrimp
paste [124,125].

Table 2. Metabolomics applied to different fermented foods on the flavor property.

Metabolomic Analysis
Fermented Foods Microorganisms References
Compounds/Properties
Techniques
Analyzed
Yeast, Aspergillus, Mucor,
Alcohols, esters, phenolic acids,
Dajiang Rhizopus, Lactobacillus, HS-SPME/GC-MS [78]
aldehydes, ketones
Tetragenococcus
Monascus purpureus,
GC-MS, GC-MS-O,
Red sufu Aspergilus oryzae, Amino acids, organic acids [119]
E-nose
Actinomucor elegans
Amino acids, organic acids,
Natto Bacillus subtilis GC-MS; NMR [120–122]
pyrazines; ammonia
HS-SPME/GC-MS, Lactose, lactate and citrate,
Cheese Lactic acid bacteria [113]
FT-IR, E-nose amino acids, fatty acids
Esters, linalool, neroidol, geranyl
Huangjiu Yeast GC/GC-MS, GC-O acetone, 2-pentyl-furan, [112,115]
methanethiol
Yeast, Lactobacillus,
Baijiu GC×GC-TOF/MS Aromatic compounds, pyrazines [116]
Acetobacter
HS-SPME-Arrow-GC- Piperitone, mintlactone, menthyl
Red wine Yeast [79]
MS/MS acetate, neomenthyl acetate
Ethyl acetate, phenylethyl
Vinegar Acetobacter, Lactobacillus HS-SPME/GC-MS [124]
alcohol, acetoin, acetic acid
β-damascenone,
Monascus purpureus,
methoxybenzene,
Pu-erh tea Bacillus, Rasamsonia, HS-SPME/GC-MS [114,117]
2,4-nonadienal, terpinene,
Lichtheimia, Debaryomyces
linalool
Psychrilyobacter, Alcohols, hydrocarbons,
Siniperca chuatsi HS-SPME/GC-MS [123]
Fusobacterium, Vibrio nitrogen compounds
Salimicrobium, Lentibacillus,
Alcohols, aldehydes, nitrogen
Shrimp paste Lactobacillus, HS-SPME/GC-MS [125]
compounds
Tetragenococcus

4.2. Nutrition and Function

Consumer interest “beyond nutrition” and “functional” foods has surged dramatically,
and fermented foods occupy a significant space in the functional foods market [126,127].
Controlling the growth of natural microorganisms or employing probiotics during fermen-
tation, can improve the nutritional and functional value of food [128]. The functions of
fermented foods are mainly related to the biological reactions caused by enzymes produced
by microorganisms. On the one hand, the basic nutrients of the substrate (e.g., proteins,
carbohydrates, and lipids) are degraded into more basic molecular components (e.g., pep-
tides, amino acids, monosaccharides, and fatty acids) or undergo configuration changes
(e.g., glycoside to aglycon). However, specific functional components (e.g., menadione and
nattokinase) of microbial metabolism are produced. The metabolites (such as genistein, tea
polyphenols, selenium) constitute the dietary components of fermented foods, which will
affect the epigenetic mechanisms (such as DNA methylation, histone modification, and
demethylation) driven by intestinal microbiota, lymphocytic cells, and cancer cells in vivo,
Foods 2021, 10, 2294 9 of 18

conferring fermented foods with antioxidant, anti-inflammatory, antihypertensive, anti-

cancer, and neuroprotective properties [129–131], thus extending their use as nutraceuticals.
The application of metabolomics to decipher nutritional and functional components has
recently increased (Table 3). Currently, attention on fermented functional foods is mainly
focused on fermented bean products, fermented dairy, and alcoholic beverages.
Fermented soy products, including miso, meju, soybean paste, tempeh, and natto, are
a staple in daily cuisine in East Asian countries. Based on metabolic characterization, it was
found that during fermentation, formation and abundance of small peptides, amino acids,
and fatty acids through the degradation of macromolecular substances such as protein and
fat have an important influence on the nutritional quality of fermented soy foods [132,133].
With the extended fermentation time, the composition ratio of the main metabolites will
change, and the level of functional active ingredients (e.g., γ-aminobutyric acid (GABA)
and isoflavones) will increase, improving the nutritional value and biological activity,
potentially benefiting human health [21,122,134]. Kim et al. [135] applied GC-MS and ultra-
performance liquid chromatography (UPLC)-Q-TOF-MS to identify doenjang metabolites;
they determined that amino acids and sugars are the main nutrients, while increasing
levels of isoflavones and soyasaponins after the configuration changes exerted antioxidant
properties and improved digestibility. Subsequently, some researchers attempted metabolic
tracking of isoflavones in healthy adults after consumption of fermented soy foods [136].
The study used NMR and UPLC-MS to monitor the changes in isoflavones in the food,
plasma, and urine after consumption of fermented or non-fermented soybeans, confirming
that the bioavailability of isoflavones in fermented soybeans increased with a positive
impact on health. Another study used metabolomics based on ultra-high performance
liquid chromatography (UHPLC)-Q-TOF-MS/MS to identify possible antihypertensive
compounds in Korean fermented soybeans [131], analyzed changes in the levels of amino
acids and lipids, and determined that levels of phenolic compounds, GABA, antioxidants,
and antihypertensive peptides after fermentation were high.
Fermented dairy products in the market are mainly yogurt and cheese, both of which
are fermented by lactic acid bacteria, such as Lactobacillus bulgaricus and Streptococcus ther-
mophilus [137,138]. The probiotic effects of fermented foods have been reported; different
fermentation strains and conditions affect the nutritional and functional quality of the
final products [139]. The ripening process of cheese has been metabolically fingerprinted
using NMR [137]. As the ripening period increases, the metabolic profile showed a distinct
pattern of change. Carbohydrates, organic acids, amino acids, and derivatives mainly
contribute to the nutritional value of cheese. In addition, fermentation temperature has a
significant impact on yogurt quality, which affects microbial growth [140]. Metabolomic
profiles were evaluated for yogurt at different temperatures based on UPLC-QQQ-TOF-MS.
Lipids and lipid-like molecules were the main metabolites, and amino acids and peptides
were the second most abundant; both of these were generated by lipid metabolism and
protein degradation. These are more conducive to digestion and absorption and confer
health benefits by reducing blood pressure and regulating immunity [139,141,142].
Alcoholic beverages are prepared from plant sources such as grapes and cereals, the
characteristics are mainly affected by yeast fermentation. Sugars represent the main groups
of metabolites in wines, while organic acids, amino acids, and phenolic compounds rep-
resent other minor metabolites, which together constitute the basic nutritional quality of
wines fermented with yeast strains [143]. Grapes and red wines are the major dietary
sources of stilbenes, which influence human health through antioxidant, anti-obesity,
anti-tumor, and neuroprotective effects, with resveratrol as a typical representative com-
pound [144,145]. HPLC-MS is the most commonly applied technique for determining
phenolic compounds, particularly stilbenes [146]. Tabago et al. [49] successfully sum-
marized the potential of NMR metabolomics in terms of geographical origin, variety,
vintage, fermentation, and quality evaluation of wine and alcohol, to distinguish wines
under different fermentation conditions such as bacterial and yeast strains and agricultural
cultivars [147].
Foods 2021, 10, 2294 10 of 18

In addition, metabolomics has been applied to cabbage vinegar [148], Chinese fer-
mented fish sauce [23], Chinese Pu-erh tea [105], and pickles [149] to characterize the
key chemical components related to nutrition and function, providing a reference for the
development of functional foods.

Table 3. Metabolomic analysis techniques applied to different fermented foods considering nutritional and functional
properties.

Metabolomic Analysis
Fermented Foods Microorganisms References
Techniques Compounds
Bacillus sp., Mucor sp., Small peptides, amino acids,
Meju UPLC-Q/TOF-MS [132]
Aspergillus sp. GABA
Amino acids, organic acids,
Penicillium glabrum, GC-TOF-MS,
Doenjang sugars and sugar alcohols, [133–135]
Aspergillus oryzae UPLC-Q/TOF-MS
isoflavones
Bacillus subtilis, Mucor
Cheonggukujang (or sp., Bacillus sp., Phenolic compounds,
UPLC-Q/TOF-MS [131,136]
miso, natto) Aspergillus sp.; E. peptides, GABA
faecium
Lactose, uridine
Cheese Lactic acid bacteria NMR diphosphate-hexose, amino [137]
acids, organic acids,
Lipids, lipid-like molecules,
Yogurt Lactic acid bacteria UPLC-Triple/TOF-MS small peptides, amino acids, [98,139]
GABA
NMR, FT-IR;
Yeasts, Lactobacillus, Polyphenols, amino acids,
Wines GC/GC-TOF/MS; [49,116,146,147]
Acetobacter ethanol, resveratrol, stilbenes
HPLC-MS;
Organic acids, alcohols,
sulfides (dimethyl sulfide,
Cabbage vinegar Lactobacillus, Acetobacter NMR, GC-MS [148]
dimethyl disulfide, and
dimethyl trisulfide)
Aspergillus pallidofulvus,
Phenolic compounds, amino
Pu-erh tea Aspergillus sesamicola, UHPLC-Q/TOF-MS [105]
acids
Penicillium manginii
Firmicutes,
Fermented fish sauce Proteobacteria, UHPLC-Q/TOF-MS Small peptides, amino acids [23]
Fusobacteria
Organic acids, GABA,
Latilactobacillus curvatus,
Pickle nozawana NMR, GC-MS choline, 2,3-butanedione, [149]
Levilactobacillus brevis
acetoin, ethyl acetate

4.3. Safety
Consumers not only pay attention to the sensory and nutritional value of food but
now also have an increasing awareness of food safety, encouraging the food industry to
establish and maintain product characteristics [150]. Highly complex biological changes
are usually encountered during the fermentation process, and metabolomic analyses by
NMR or LC-MS can explain safety properties of fermented products indicating whether
fermentation improves the safety or generates toxic compounds. The most relevant studies
on the safety of fermented foods based on metabolomic analyses are presented in Table 4.
The improvement in food safety is mainly reflected in the changes in the nonnutri-
tive factors or toxic compounds of the raw materials (e.g., soyasaponins and alkaloids)
before and after fermentation, which improve digestion and absorption. Seo et al. [151]
performed MS-based metabolomics on koji fermentation with different substrates (soy-
Foods 2021, 10, 2294 11 of 18

bean, rice, and wheat) and microbial inoculation. The breakdown of glycosides to free
compounds, including soyasaponins and flavonoids, is obvious during fermentation,
revealing the nutritional, functional, and consumer safety aspects of fermented foods.
Kasprowicz-Potocka et al. [152] concluded that the reduction of alkaloids occurs in fer-
mented lupin meal by yeasts. Nevertheless, a few compounds are not conducive to food
quality during fermentation. Biogenic amines and ethyl carbamate carry a certain toxicity
in fermented foods (e.g., miso soy paste and fermented fish) and have been detected with
LC-MS, CE-MS, and GC-MS-based metabolomics [21,23]. However, probiotics influenced
the levels of methylamine, putrescine, ethyl paraben, and ethyl carbamate [37,153]. In
addition, metabolomics approaches have also been widely used in the detection of chemi-
cal preservatives in fermented foods, including shrimp paste, pickles, soy sauce, and fish
sauce [154]. Metabolites from pathogenic microorganisms can be rapidly determined using
metabolomic approaches, whereas the development is still in the early stages [155].

Table 4. Metabolomic analyses applied to different fermented foods considering safety properties.

Metabolomic Analysis
Fermented Foods Microorganisms References
Compounds/Components
Techniques
Analyzed
Bacillus subtilis,
Doenjang Rhizopus, Mucor, GC-MS Soyasaponins [134]
Aspergillus sp.
GC-TOF-MS,
Cheonggukjang Bacillus sp. Soyasaponins [21]
CE-TOF-MS
Aspergillus oryzae,
GC-TOF-MS,
Koji Bacillus Soyasaponins [151]
UHPLC-MS/MS
amyloliquefaciens
Bacillus subtilis, Benzoic acid, sorbic acid,
Vinegar Rizhopus, Mucor, GC-MS dehydroacetic acid, ethyl [153]
Aspergillus sp. paraben
Firmicutes,
Trimethylamine N-oxide,
Fermented fish sauce Proteobacteria, UHPLC-Q/TOF-MS [23]
putrescine, cadaverine
Fusobacteria
Lentibacillus,
Lactobacillus, Benzoic acid, sorbic acid,
Shrimp paste, Pickles GC-FID [154]
Latilactobacillus, propionic acid
Tetragenococcus
GC/LC-MS, NMR, Pathogenic bacteria and its
Cheese Lactic acid bacteria [155]
FI-TR metabolites

5. Conclusions and Future Prospects

As the population and health awareness continue to increase, there will be an increas-
ing need for functional as well as nutritious, safe, and palatable foods to meet changing
consumer preferences. Fermentation technology can become a source of such functional
foods, including the industrial production of traditional fermented foods, the development
of fermented foods based on new raw materials, and the improvement of existing food
fermentation technologies. Currently, fermented foods usually contain one or more pro-
biotics beneficial to health. Fermentation is a highly complex biological process, which
is essential for imparting new flavor characteristics to fermented foods, improving the
nutritional and functional properties of raw materials, and improving the biological safety
of fermented products. The industrial production and promotion of fermented foods is
Foods 2021, 10, 2294 12 of 18

dependent on understanding its qualitative properties, which may require the introduction
of new application technologies for characterization.
The characterization should mainly include the main metabolites and secondary
metabolites in the fermented foods, which directly reflect the quality characteristics, such
as flavor and nutrition. Based on the established analytical techniques and knowledge of
systems biology, metabolomics may prove to be an indispensable technique as it allows
simultaneous qualitative and quantitative analysis of all low-molecular-weight metabolites
in a certain organism or cell during a specific physiological period, it is also a manifestation
of protein and gene level results. Thus, it can help consumers have more direct access to
products with dietary nutrients that they desire. Thus, the application of metabolomics
combined with multivariate statistical analysis has evolved into a useful tool in food
science.
This review shows the importance of applying metabolomic analysis to identify the
quality characteristics of fermented foods. It was possible to perceive from the latest
research in this field that the existing techniques used in metabolomic studies for investi-
gating the molecular fingerprints of fermented foods include NMR, FT-IR, LC-MS, GC-MS,
CE-MS, and E-nose. Each technique has its advantages and disadvantages in terms of
sensitivity, reliability, and sample preparation; therefore, the use of a combined method is
recommended to provide a more comprehensive understanding of the metabolomes of fer-
mented foods. The application of metabolomics in characterizing the qualitative properties
of fermented foods, specifically flavor, nutrition, function, and safety was also summarized
in this review, and may assist in the discovery of biomarkers that discriminate maturity,
variety, region, and promotion of value-added fermented foods. Nevertheless, consider-
ing the complexity of fermented foods and metabolomics, the verification of functional
biomarker compounds in fermented foods, development of metabolomics technology for
rapid and accurate detection, and integration with different techniques will be required in
further research. Moreover, the development and improvement of a reliable database of
fermented food metabolites is necessary for practical and industrial applications.

Author Contributions: Conceptualization, F.W. and S.L.; writing—original draft preparation, Y.G.;
writing—review and editing, supervision, Y.G., D.L., Z.T.; investigation, L.H., J.G.; validation, Y.G.,
S.L.; funding acquisition, F.W. and S.L.; project administration, F.W., B.F. and S.L. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by China Agriculture Research System (CARS-04), Central
Public-interest Scientific Institution Basal Research Fund (S2021JBKY-02) and Central Public-interest
Scientific Institution Basal Research Incremental Fund (Y2020PT10).
Data Availability Statement: Not applicable.
Acknowledgments: The authors thank the funds and the Knowledge Innovation Program Funding
of Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences.
Conflicts of Interest: The authors declare no conflict of interest.

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