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com
ScienceDirect
Applications of plant-based fermented foods and
their microbes
Sander Wuyts, Wannes Van Beeck, Camille Nina Allonsius, Marianne
FL van den Broek and Sarah Lebeer
Plant-based fermentations and their microbes provide an
underexplored source for novel biotechnological applications.
Recent advances in DNA sequencing technologies and
analyses of sequencing data highlight that a diverse array of
lactic acid bacteria (LAB) frequently dominate these plant
fermentations. Because of the long history of safe LAB use in
fermented foods, we argue here that various novel probiotic,
synbiotic and a range of other industrial applications can be
produced based on new insights in the functional and genetic
potential of these LAB. To aid in this quest, comparative
genomics tools are increasingly available enabling a more
rational design of wet-lab experiments to screen for the most
relevant properties. This is also true for the exploration of useful
enzymatic and (secondary) metabolic production capacities of
the LAB that can be isolated from these plant-based
fermentations, such as the recent discovery of a cellulase
enzyme in specific Lactobacillus plantarum group members.
Address
Environmental Ecology and Applied Microbiology (ENdEMIC),
Department of Bioscience Engineering, University of Antwerp,
Groenenborgerlaan 171, 2020 Antwerp, Belgium
Corresponding author: Lebeer, Sarah (sarah.lebeer@uantwerpen.be)
Current Opinion in Biotechnology 2020, 61:45–52
This review comes from a themed issue on Food biotechnology
Edited by Mark Blenner and Jan-Peter van Pijkeren
For a complete overview see the Issue and the Editorial
Available online 13th November 2019
https://doi.org/10.1016/j.copbio.2019.09.023
0958-1669/ã 2019 Elsevier Ltd. All rights reserved.
Introduction
Fermented foods have been an important component of
human diets all around the world. They are still widely
consumed, but refrigeration and other habits in industrial
food processing have put the consumption of these foods
under pressure [1]. There exists a huge variety in fermen-
ted products, due to diversity in raw materials or substrates
on which the fermentation is performed or different process
parameters. This variety is also reflected by the diversity of
microorganisms that drive the fermentation processes as
main biological actors [2]. Organisms from all three
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Domains of life have been found to be crucial to the taste,
texture and smell of the fermented end product. For
example, strains from the yeast Saccharomyces cerevisiae
are indispensable for baking bread and brewing beer.
Lactic acid bacteria (LAB), contrastingly, are the key
bacteria in the fermentation process of cheeses, yoghurts
and fermented vegetables [3,4]. Even Archaea have been
found to be involved in the fermentation of high-salt
fermented seafood [5]. While the use of microbes in food
fermentations can be regarded as one of the oldest applica-
tions within biotechnology, today these microorganisms are
also widely exploited for the production of pharmaceuti-
cals, biofuels and bioplastics as well as the purification of
industrial oils and water. Furthermore, selected microbes
isolated from fermented food are exploited as probiotics to
promote the health of humans, animals and plants [6]. To
further improve the effectiveness of probiotic products
however, there is a constant need for previously unchar-
acterized strains and strain combinations that perform
better or show novel characteristics.
Because of their large variety and a high — often still
unknown — microbial richness, spontaneously fermented
foods are a promising resource for the discovery of previ-
ously uncharacterized microorganisms. Furthermore, fer-
mentation processes are relatively easy to set-up, maintain
and reproduce (e.g. the reproducibility of spontaneously
fermented carrot juices [7]). For this reason, the microbes
residing in them have a higher potential to be easily
cultured, compared to, for example, the strictly anaerobic
gut bacteria [8], which allows for easier manipulation and
more in-depth study.
Compared to dairy-based food fermentations, fermen-
tations that use plant materials as main substrate are
underexplored, except for a few notable examples like
sauerkraut and kimchi [9]. They thus remain an inter-
esting source of LAB, which usually drive these
fermentation processes, as schematically visualized
in Figure 1. Here, we describe how plant-based fer-
mented foods can be screened for these LAB and
provide insights on how one could assess their poten-
tial as probiotics and industrial microbes.
From plant-based fermented foods to useful
microbes
Studying plant-based fermented foods is of interest,
not only because of the probiotic and industrial
Current Opinion in Biotechnology 2020, 61:45–52
46 Food biotechnology

Figure 1

PRODUCES FERMENTED PRODUCTS

Lactobacillus genus complex Others
Lactiplantibacillus
(= L. plantarum group) Leuconostoc Bacillus
Levilactobacillus (B. subtilis & B. amyloliquefaciens)
(= L. brevis group)

Lactiplantibacillus
Levilactobacillus
Latilactobacillus
(= L. sakei group) Weisella Streptococcus
Fructilactobacillus (S. thermophilus)
(=L. fructivorans group)
Limosilactobacillus
(L. fermentum group)
Lactiplantibacillus

Oenococcus Pediococcus Enterococcus

(E. faecium & E. Faecalis)

Root
Cereal
Vegetables
Grapes
Legume
Abundance

Fermentation time

Residing non-LAB community Potential useful LAB/microbes

Current Opinion in Biotechnology

Schematic overview of bacterial diversity and community dynamics within different examples of plant fermentations. Bacterial diversity at genus
level is visualized for plant fermentation classified in distinct groups (roots, vegetables, legumes, cereals and fruits such as grapes) based on
[2,3,4,7,9]. It should be noted that the genera mentioned in this figure represent a non-exclusive list and the new genus names for the members
of the Lactobacillus genus complex are used, as proposed in Zheng et al., 2019 (Submitted). Fructilactobacillus (L. fructivorans group) was added
for the cereal fermentations especially due to the activity of Fructilactobacillus sanfranciscensis (L. sanfranciscensis).

potential of their microbes, but also because they have
been recently proposed as alternative non-dairy food
matrices for probiotic administration [10]. In contrast
to their dairy alternatives, plant-based fermentations
are suitable for lactose-intolerant, milk-allergic or
vegan people, and are appealing because vegetables
are a source of essential nutrients, vitamins, minerals,
anti-oxidants, and fibers, and they have a low sugar
content [1,11]. The food product itself could thus be
used as a new carrier for traditional (dairy-based)
marketed probiotics, enabling access to a new con-
sumer market.

Current Opinion in Biotechnology 2020, 61:45–52 www.sciencedirect.com


Figure 2
CULTIVATED FERMENTED
FOOD STRAINS
PROBIOTIC & INDUSTRIAL POTENTIAL
WETLAB EXPERIMENTS
• Antimicrobial activity IMPROVED
• Immunomodulation RATIONAL
• Enzymatic activity
EXPERIMENTAL
• (Secondary) metabolite production DESIGN
• EPS production
Schematic overview of various genomic and functional screenings that can be performed on promising isolates from plant-based fermented foods.
The genomic and phenotypical assays mentioned are examples and form a non-exclusive list. More information about the different approaches is
discussed in the text.
Furthermore, plant-based fermentations are of interest
because of the microbes that drive these fermentation
processes (Figure 1). Especially fermented foods of which
the microbial communities are poorly characterized
remain a rich source of untapped microbial diversity.
However, recently due to a more widespread availability
of DNA sequencing technologies, a shift from culture-
based to sequencing-based techniques can be observed in
studies that explore this diversity. While these culture-
independent techniques have their own advantages, for
the selection and characterization of new industrially-
relevant strains, it is of course necessary to obtain physical
isolates. For example, for the cultivation of LAB, a
bacterial group that has many examples of industrialized
probiotic strains, usually MRS-medium (de Man, Rogosa
and Sharpe medium) is used, with or without the addition
of vitamin solutions (e.g. Ref. [7]). However, tailored
media and growth conditions are often needed, including
adaptation of the growth temperature, carbon source,
nitrogen sources, pH and oxygen level. Although still
laborious, culturomics approaches, in which a multitude
of culture conditions are tested, could help with this
optimization, as it is also increasingly applied for the
cultivation of human microbiota members [12] and
plant-associated prokaryotes [13].
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Exploring probiotics from fermented plants Wuyts et al. 47
STRAIN IDENTIFICATION
• Marker gene sequencing (16SrRNA)
• MALDI-TOF MS
• Whole genome sequencing
GENOMIC TOOLS & DATABASES
• Bacteriocins: Bagel 4
• Secondary metabolites: AntiSMASH
• Virulence factors: Vfanalyze
• Antibiotic resistance: CARD
• Adhesion factors: HMMER, CW-PRED
BLAST, InterProScan
• Glycosyl transferases
& hydrolases: HMMER + dbCAN
Current Opinion in Biotechnology
Once isolated, a next step is identification of the bacterial
isolate (Figure 2). A proper identification is important from a
safety and regulatory point of view, especially if the strains
are to be used in industry. Traditionally, PCR-based tech-
niques (e.g. denaturing gradient gel electrophoresis;
DGGE) or marker gene sequencing (e.g. Sanger sequenc-
ing of the 16S rRNA gene) are used for identification. More
recently, matrix assisted laser desorption ionization-time of
flight mass spectrometry (MALDI-TOF MS) [14] and
whole-genome sequencing (WGS) [15] have emerged as
tools for microbial identification. The latter has risen in
popularity as sequencing costs are decreasing and WGS
captures much more information on the strain of interest
compared to sequencing of a single marker gene. There-
fore, more advanced computational analyses are being
developed that leverage this genome information obtained
through WGS, such as we have recently implemented for
species identification in the Lactobacillus genus complex
[16]. Although WGS-based identification is more expensive
than traditional methodologies and requires considerable
bio-informatic expertise, having access to draft or full
genome sequences is instrumental to explore the functional
potential of the isolated microbes. Furthermore, several
industrially relevant traits could be species-specific prop-
erties. For example, only after an in-depth comparative
Current Opinion in Biotechnology 2020, 61:45–52
48 Food biotechnology
genomics approach, leading to several reclassifications, we
detected that all members of the Lactobacillus casei species,
harbor catalase genes, probably making them more resistant
oxidative stress [17]. Such information about potential
characteristics can be important for industrial application.
Exploring the probiotic potential: starting with
the genome
Genome analyses can be the first step in the exploration of the
probiotic potential of newly isolated bacterial strains.
Although, evidently, not all genome-based predictions are
correct, as many of the predicted functions of genes need to be
experimentally validated, it is a useful tool to provide the first
insights in the functional potential of isolates. Such initial
analyses could guide the researchers towards selecting the
more promising isolates for subsequent wet-lab experiments.
This is especially useful when a large amount of strains has
been isolated requiring a more rational design of wet-lab
experiments to screen for the most relevant properties.
Currently, standard pipelines for the genomic screening
of potential probiotic strains do not exist, similarly as it is
still very hard to define which functional properties
should be screened for in the lab to predict probiotic
efficacy [18,19]. However, as an ‘overarching’, required
property of a suitable probiotic, specific aspects related to
safety should be checked, such as the absence of known
virulence properties and toxins, as well as known antibi-
otic resistance genes on mobile elements. On a genomic
level, absence of known virulence factors can be evalu-
ated with databases such as the virulence factors database
(VFDB) [20] and their associated web services (Figure 2).
However, they should be analyzed with caution as the risk
for false positives is high since probiotics could use
virulence-like properties to allow survival in a certain
host environment (e.g. adhesion to target cells). In addi-
tion, false negative results are also possible as not all
genetic elements associated with the variety of bacterial
pathogenic mechanisms are yet known or are limited to
selected genes. Similarly, the presence of known antibi-
otic-resistance genes can be screened with web-based
platforms and databases such as the Comprehensive
Antibiotic Resistance Database (CARD; Figure 2) [21],
but they should be experimentally confirmed, which can
easily be done via minimal inhibitory concentration
(MIC) determination assays [22]. In Europe, the directive
of the European Food Safety Authority (EFSA) states
that the MIC should not surpass the cut-off values for
nine specific antibiotics, which should be evaluated for
the strain of interest [23]. In addition, it is also advisable to
check whether antibiotic resistance genes are located on
mobile elements, because this is especially a risk factor,
but hereto, closed genomes or genomes sequenced via
long-read sequencing should ideally be available.
Another property that could be defining for a probiotic is
its capability to outcompete or kill pathogens. For many
Current Opinion in Biotechnology 2020, 61:45–52
LAB, pathogen inhibition could be the result of the
production of lactic acid, which has been shown to be
active against a range of pathogens [24–26]. In addition,
some LAB strains are characterized by more specific
methods of actions for pathogen inhibition, which can
be screened for at genomic level. For example, recent
bacteriocin gene-trait matching across the complete
Lactobacillus pan-genome did find that lactobacilli from
the animal and human microbiota encode over twice as
many bacteriocins as those from other sources [27].
Web-based tools, such as BAGEL 4 [28] and antiSMASH
[29], dedicated to search for bacteriocins and secondary
metabolite gene clusters, respectively, are used for the
detection of genes involved in the production of such
antimicrobial compounds (Figure 2). If hits are found, the
activity of these predicted compounds can be explored in
literature or relevant databases. Again, the activity of
these predictions should be validated and confirmed
using laboratory experiments (see below).
Furthermore, colonization or retention of the putative
probiotic strain in the human host by means of adhesion
to host cells, is often seen as a beneficial property, because it
increases the time potential probiotic strains have to exert a
beneficial effect [30]. It is therefore interesting to explore
whether the isolates show the potential to adhere to
(human) host mucosal surfaces. In Gram-positive
bacteria, host adhesion is mostly attributed to sortase-
dependent cell surface proteins that can be screened for
using similarity searches with tools such as BLAST or
HMMER (Figure 2) and well described genes as reference,
for example, the spaCBA genes from Lactobacillus rhamnosus
GG [30,31] or lectins that are known to adhere to different
glycan structures (e.g. Ref. [32]). Alternatively, CW-PRED
[33] or InterProScan [34] can be used to identify the
conserved consensus motifs from these known genes in
the genome(s) of interest as demonstrated for the members
of the Lactobacillus Genus Complex [35]. Recently, a com-
bination of both approaches (similarity searches and con-
served consensus motif search) was used to explore host
adhesion and host interaction of the strains in a commer-
cially available probiotic product [36]. In addition, since
glycosylation of surface molecules affects the host-microbe
interaction, genomes of interest can also be screened for
the presence of clusters of glycosyltransferases with
HMMER and glycosyltransferase reference profiles from
the dbCAN database [37]. This analysis led to the identifi-
cation of a putative glycosylated surface adhesin in L. casei
AMBR2 [17]. Yet, ultimately, all these predicted genomic
functions still need to be substantiated in the laboratory, in
animal models and — most importantly — in clinical trials.
Phenotypically substantiating potential
probiotic functions: where to start?
The above-described genome analysis could give some
initial insights into interesting properties of the strains
under study. However, many of these traits should be
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further investigated in the wet-lab. For example, many
LAB in fermented foods are able to outcompete potential
spoilage and pathogenic bacteria. While lactic acid is key
in this activity, other more specific molecules could be
involved. Therefore, we believe that one of the most
interesting properties to investigate in the wet-lab fol-
lowing the genomic predictions are bacteriocins and other
secondary metabolites as described above (Figure 2).
Initial screening of these compounds is usually performed
by evaluating the activity of live cells or cell-free super-
natant for the induction of inhibition zones in agar-based
assays, such as well-diffusion assays with the pathogens of
interest, including neutralization of the effect of lactic
acid. These assays are not trivial, because the culture
medium and conditions can have a large impact on the
antimicrobial activity, as well as the presence of other
stimuli to induce the expression of the antimicrobial
compound. For example, it is well documented that many
Lactobacillus plantarum strains need the presence of
‘inducer bacteria’ to induce plantaricin production [38].
Further characterization requires purification of the bac-
teriocin or other antimicrobial compounds and identifica-
tion of the structure via mass spectrometry approaches. Of
note, the antimicrobial activity of lactic acid can be
enhanced when combined with other antimicrobial mole-
cules. For the fungal pathogen Candida albicans, for
example, we recently found that lactobacilli from the
L. rhamnosus/casei group show the capacity to inhibit
hyphae morphogenesis by the additive effects of lactic
acid and secreted enzymes with chitinase activity [39].
Another property of the diverse LAB in fermented foods
is their strain-specific capacity to exert immunomodula-
tory effects beyond their nutritional value and enzymatic
capacities [6]. Many of the immunomodulatory molecules
of LAB are cell surface molecules such as peptidoglycan
[40], lipoteichoic acid [41], glycoproteins such as S-layer
proteins [42] and pili appendages [43], which are thought
to exert immunomodulatory effects via pattern recogni-
tion receptors such as Toll-like receptors and C-type
lectins [44]. Often strain-specific variations exist in the
molecular structure of these molecules, resulting in
altered signalling through pattern recognition receptors.
Experimental validation of the immunological effects of
the individual strains and their surface molecules is thus
crucial, as it is yet very difficult to predict the immuno-
modulatory capacity of isolated LAB based on the
presence of certain genes in their genomes, such as
glycoproteins [45] and pili [46] or other adhesins. Indeed,
in addition to cell surface compounds, also other (second-
ary) metabolites such as polyketides, ornithine and
D-phenyllactic acid (D-PLA), can activate the immune
system, for example, via the Aryl Hydrocarbon Receptor
[47,48,49]. D-PLA is an anti-bacterial and immunomo-
dulating metabolite found in high concentrations in LAB-
fermented food such as sauerkraut [49]. Therefore, the
ultimate immunological host response will depend on the
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Exploring probiotics from fermented plants Wuyts et al. 49
integrated sum of all individual microbial molecules
interacting with their cognate host receptors [44]. Specific
interesting properties such as induction of regulatory
T-cells or anti-inflammatory cytokines remain to be sub-
stantiated in vitro and in vivo, preferably in humans.
Nevertheless, considering the large diversity of LAB in
plant-based fermented foods (Figure 1), they are an
attractive source of immune-modulatory microbes.
Moreover, since they are isolated from plant-based and
fibre-rich material, these LAB are expected to have a
considerable glycosyl hydrolase and carbohydrate-
degrading capacity (Figure 2), such as we have recently
shown for Lactobacillus mudanjiangensis [50]. This pro-
vides unique opportunities for specific prebiotic and
synbiotic development, if the selected LAB are able to
selectively degrade specific polysaccharides. These poly-
sacharides could then be combined into a synbiotic with
their cognate microbe, if the potential probiotic proper-
ties of these microbes are substantiated.
From plant-based fermented foods to
industrial applications
In addition to the development of probiotics (and prebi-
otic/synbiotic combinations), bacterial isolates originating
from fermented foods can also be of interest for a wide
array of other industrial applications. Of course, the food
industry itself could be interested in these strains and
exploit them as starter cultures or for the production of
enzymes, food additives and antibiotics. For example,
exopolysaccharides (EPS), that is, extracellular polysac-
charides that are secreted in the environment or cova-
lently bound to the bacterial cell surface [51], are often
used in the food industry due to their positive contribu-
tion to textural properties [52]. Interestingly, these mole-
cules are also used for, for example, reduction of blood
viscosity in the pharmaceutical industry and as a size-
exclusion chromatography matrix in analytical chemistry
[53]. The key enzymes involved in their biosynthesis are
glycosyl transferases. From a genomic point of view,
dbCAN, as mentioned earlier, can be a useful resource
for detecting these glycosyl transferases as well as the
glycosyl hydrolases described above, and thus
provide insights in whether the strains under study
are able to produce EPS or not (Figure 2). Furthermore,
glycosyl hydrolases are a widespread group of enzymes
that hydrolyze the glycosidic bond in complex
carbohydrates and thus play an important role in a
microorganism’s capability to metabolize different
carbohydrate sources [35,54]. Insights into their glycosyl
hydrolases-repertoire will thus open the door for the
exploration of possible other industrial applications.
Recently, a comparative genomics approach which
included a scan for glycosyl hydrolases, discovered the
presence of a cellulase gene in L. mudanjiangensis [50], a
species that has only been isolated from fermented vege-
tables [55]. This is an example of a microbial glycosyl
hydrolase that can be deployed to hydrolyze cellulose
Current Opinion in Biotechnology 2020, 61:45–52
50 Food biotechnology
with potential applications in the paper and pulp industry,
usage in food and feed technology, coffee extraction, bio-
bleaching, detergent production, and biofuel production
[50]. This analysis thus made the isolated strains possibly
attractive for industrial applications.
In addition to their large enzymatic repertoire, the (second-
ary) metabolites of the LAB from fermented foods can have a
wide variety of applications. These include biogenic amines
such as produced by Lactobacillus brevis in fermented carrots
[7] or other microbes in sauerkraut [56]. In fermented foods,
these biogenic amines are unwanted because they can cause
malodor and headaches, but they could also be used as
pharmaceutical compounds, for example to promote cardio-
vascular health and longevity [57], although this is still
controversial. However, Lactobacillus reuteri-derived hista-
mine, an example of a biogenic amine, was found to sup-
presses pro-inflammatory TNF production which could
ultimately lead to improved anti-inflammatory strategies
for chronic immune-mediated diseases [58]. Similarly, the
bacteriocins or isolated secondary antimicrobial metabolites
described above could also be used as purified molecules
instead of live probiotics, which could be a more attractive
valorization route with regards to formulation and regulation.
Conclusions
Although consumption of especially plant-based fermen-
ted foods has dropped in the Western world, it is currently
regaining popularity since an increasing number of con-
sumers start to appreciate their rich flavors and potential
health-promoting properties. Simultaneously, the scien-
tific interest in the study of these microbial ecosystems
has boosted recently. Here, we argue that plant-based
fermented foods are an interesting source for the detec-
tion of novel interesting strains with large potential for
various probiotic and industrial applications.
Conflict of interest statement
Nothing declared.
Acknowledgements
The authors would like to thank the whole Laboratory of Applied
Microbiology and Biotechnology of the Department of Bioscience
Engineering of the University of Antwerp for all fruitful scientific
discussions and interactions. Work in the Lebeer lab on fermented foods is
funded by IWT SBO funding (150052 ProCure project, www.
procureproject.be) and UAntwerpen funding (Dehousse scholarship
Wannes Van Beeck). Camille Allonsius holds a personal FWO-SB grant.
Icons were made by Freepik, Nikita Gobulev (vegetable icon, Figure 1),
Good Ware (root icon, Figure 1) from www.flaticon.com.
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