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Bioprotective mechanisms of lactic acid bacteria
against fungal spoilage of food
Solvej Siedler1, Rafik Balti2 and Ana Rute Neves1
In modern societies, conscious consumers demand healthy,
fresh and natural foods devoid of added chemical
preservatives and stabilizers. The use of lactic acid bacteria
(LAB) to preserve food is one of the oldest and best
characterized approach. Production of organic acids is the
main feature LAB use to outcompete spoilage organisms, but
several other mechanisms have been demonstrated. In this
review, a critical overview of the mechanisms used by LAB to
inhibit spoilage organisms will be presented. Discrepancies
between the concentrations of compounds resulting from LAB
activity and their inhibitory amounts are discussed. Technical
limitations hindering discoveries in this field as well as future
trends in the application of LAB solutions to food bioprotection
will be covered, including antifungal peptides and competitive
exclusion.
Addresses
1
Bacterial Physiology, Discovery, R&D, Chr. Hansen A/S, Bøge Allé 10-
12, 2970 Hørsholm, Denmark
2
Unité de Physiologie Fonctionnelle et Valorisation des Bio-Ressources
(UR17ES27), Higher Institute of Biotechnology of Beja, University of
Jendouba, PB 382, Habib Bourguiba Avenue, 9000 Beja, Tunisia
Corresponding author: Siedler, Solvej (dksosi@chr-hansen.com)
Current Opinion in Biotechnology 2019, 56:138–146
This review comes from a themed issue on Plant biotechnology
Edited by Rute Neves and Herwig Bachmann
https://doi.org/10.1016/j.copbio.2018.11.015
0958-1669/ã 2018 Elsevier Ltd. All rights reserved.
Introduction
Lactic acid bacteria (LAB) have been used for thousands
of years to protect a range of foods, including bread,
vegetables and dairy products against spoilage organisms.
In recent years, there has been an increased emphasis on
identifying novel LAB strains with antimicrobial activity
for food bioprotection [1–3], focusing on the characteri-
zation of the antimicrobial spectrum and not the under-
lying mechanisms [4–9]. However, there is also an
increasing interest in understanding the principles behind
the antifungal activity, including the identification and
quantification of bioactive compounds produced by bio-
protective LAB [10,11]. There are two main approaches
Current Opinion in Biotechnology 2019, 56:138–146
to identify bioactive compounds, first, high resolution
analytical methods (e.g. GC-MS, HPLC-MS, HPLC-
MS, MS/MS) to identify novel compounds, validating
their bioactivity afterwards, and second, bioassay guided
approaches, where fractions are analyzed regarding the
remaining bioactivity, followed by the identification of
novel bioactive compounds in the corresponding fraction.
This review will have a closer look at compounds result-
ing from LAB activity that have been reported to have an
inhibitory effect on yeast and mold growth. Discrepancies
between measured and inhibiting concentrations of these
compounds will also be addressed. Furthermore, we
discuss the difficulties in identifying and validating
new antimicrobial compounds due to low production
titers and potential additive and synergistic effects
between them.
Compounds derived from bacterial
metabolism
In the past, several antimicrobial compounds produced by
LAB have been identified and characterized individually
for their inhibitory potential against spoilage organisms
(Figure 1a, Table 1). These compounds range from
simple organic acids and primary metabolic products to
very complex compounds derived from bioconversions or
peptide synthesis as well as protein cleavage. The most
important and best characterized antimicrobials produced
by LAB are lactic and acetic acid, which are bioactive in
their protonated form at low pH [12] and may act syner-
gistically [13]. Other LAB metabolic products contribute
to the overall antimicrobial capacity of LABs, although
their specific effects are difficult to quantify [14]. While
most of the organic acids are produced through central
carbon metabolism, other inhibitory compounds are pro-
duced via secondary metabolism or bioconversion
(Figure 1b, Table 1).The mechanisms behind the inhi-
bition of some single compounds have been studied in
detail, and encompass membrane destabilization [15],
proton gradient interference [16–18], enzyme inhibition
[14], to creation of reactive oxygen species [14,19]
(Figure 2). Yet, these studies focus only on individual
compounds neglecting synergistic and/or additive effects.
Synergistic/additive effects and concentration
efficacy
The identification of novel bioactive compounds is chal-
lenging, and the development of identification methods
with higher sensitivity and more efficient separation (e.g.
GC–MS, HPLC-MS/MS) was key to enable detection of
low concentrations of organic compounds in complex
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 Bioprotective lactic acid bacteria Siedler, Balti and Neves 139

Figure 1

(a)

Acids derived from amino acids Organic acids

Acetic acid Hydrocinnamic acid

2-Hydroxy-3- Lactic acid
Indolelactic acid Vanillic acid
methylbutanoic acid Succinic acid

Propionic acid Benzoic acid p-Coumaric acid

2-Hydroxy-4- Formic acid
Phenyllactic acid methylpentanoic acid

Azelaic acid

2-Hydroxy-(4-
4-Hydroxy-
phenyllactic acid
LAB Salicylic acid

methylthio) butanioic
acid Cyclic dipeptides

Others
Volatiles Acroleim

Cyclo(L-Phe-L-Pro)
Mevalonolactone Ethanol
Fatty acids Reutericyclin
Cyclo(L-Phe-4-OH-L-Pro)
Diacetyl
H2O2 Methylhydantoin
CO2
3-Hydroxydodecanoic acid

δ-Dodecalactone
3-Hydroxy-5-cis-dodecanoic acid

(b)
Lactose or Glucose Peptides Amino acids Hippurlc acid

Cyclic
dipeptides Amino acids Hippurlc acid
Glucose-6-P CO2

Reutericyclin
Fructose-1,6-BP D-Leucine α-Keto acid Benzoyl-CoA
Xyl-5-P

DHAP GAP-3-P
Acetyl-P Fatty-acids
Benzoic acid
Hydroxy-acid
Glycerol-3-P Pyruvate
Acetate

Acetoacetyl-CoA
Glycerol Glycerol Acetyl-CoA Salicylic acid
α-acetolactate
Mevalonolactone
Reuterin (3-HPA) Malonyl-CoA
Formate

Acrolein Diacetyl Ethanol

Current Opinion in Biotechnology

(a) Chemical structures of various antimicrobial compounds found in the supernatant of LAB, (modified from Ref. [10]). Sources are indicated:
central carbon metabolism or fatty acid synthesis (blue); compounds derived from amino acids (green); compounds derived from amino acids and

www.sciencedirect.com Current Opinion in Biotechnology 2019, 56:138–146

140 Plant biotechnology

Table 1

List of compounds derived from LAB metabolic activity and their antimicrobial spectrum

Compound Producing Microorganisms Pathway a Target Ref.

Lactic acid all LAB C yeasts, Gram 
Acetic acid heterofermentative LAB C yeasts, Gram 
Benzoic acid L. plantarum S,B fungi, Gram [55]
Azelaic acid L. reuteri 5529, P. freudenreichii L. saci68 and L. bacteria [26]
spicheri O15
Propionic acid L. buchneri, L. diolivorans C fungi [24]
Cinnamic acid derivatives L. amylovorus DSM 19280 O fungi [21,23]
Salicylic acid L. amylovorus DSM 19280 O,B fungi [20,23]
Vanillic acid Lactobacillus O [21]
p-Coumaric acid b L. reuteri, L. plantarum b O [20,22]
Diacetyl
Lactococcus, Leuconostoc, Lactobacillus and
Acetaldehyde C yeasts, Gram  [56]
Acetoin Pediococcus
Hydrogen peroxide all LAB C yeasts, Gram  [14]
Carbon dioxide heterofermentative LAB C [14]
Ethanol heterofermentative LAB C [57]
Reuterin/Acrolein L. reuteri C fungi, protozoa, Gram  [58,59]
3-Hydroxy fatty acids C [60]
Reutericyclin L. reuteri S Gram + [18]
Cyclic dipeptides L. plantarum, L. pentosus S fungi [25,61,62]
3-Phenyllactic acid L. plantarum, Lb alimentarius, L. rhamnosus, S
4-Hydroxyphenyllactic acid Lb sanfransiscensis, L. hilgardii, Leuconostoc citreum, S fungi [61,63]
L. brevis, L. Acidophilus, Leuconostoc mesenteroides
2-hydroxy-4-methypentanoic acid L. plantarum, L. paracasei S fungi [20,53,64]
2-hydroxy acids L. paracasei S fungi [53]
Methylhydantoin S
Mevalonolactone L. plantarum S fungi, Gram [25]
d-Dodecalactone L. plantarum S fungi, Gram  [65]
a
Abbreviations: C, central carbon metabolism; S, secondary carbon metabolism: B, bioconversion; O, other/no de novo synthesis.
b
Hypothetic release from grass after addition of LAB [20].

solutions. Several studies have identified 10 potential
antimicrobial compounds in supernatants of LAB [20–23].
However, the individual contribution is difficult to quan-
tify, as the reported levels of antifungal compounds
produced by LAB are often considerably below the con-
centrations exerting inhibitory effects when assayed indi-
vidually in the laboratory (Table 2). These high minimal
inhibitory concentration (MIC) values suggest that inhi-
bition is not occurring in the original material, but the
compounds might contribute to the overall inhibition by
having a synergistic or additive effect. In fact, sometimes
only certain combinations of compounds, acting synergis-
tically, show an inhibitory effect [24]. In our opinion,
these high MIC values hinder identification of the com-
pounds with bioassay-guided fractionation techniques, as
their additive effect would be lost, and/or their concen-
tration would be too low to allow detection of their
antimicrobial activity.
Corsetti et al. reported on the fungal inhibitory effects of a
LAB isolate from sourdough. They found a mixture of
organic acids to be synergistically responsible for the
inhibitory effect of the LAB strain Lactobacillus sanfran-
ciscensis CB1, that is, acetic acid, caproic acid, formic acid,
propionic acid, butyric acid and n-valeric acid. The acids
tested individually at concentrations produced by L.
sanfranciscensis did not inhibit Fusarium graminearum
623, but their combination resulted in strong inhibition
of the mould [24]. However, these results have not been
verified in other studies. In 1999, Niku-Paavola et al.
analysed active fractions of a culture filtrate from L.
plantarum, identifying benzoic acid, methylhydantoin,
mevalonolactone and cyclo(glycyl-L-leucyl). While the
compounds alone only resulted in an inhibition of 10–
15% compared to the growth in reference media, combi-
nations of the compounds together with 1% lactic acid
could prevent growth of the bacterium Pantoea agglomer-
ans completely [25]. In a recent study, Le Lay et al.
analyzed the compounds produced by Leuconostoc citreum
L123, Lactobacillus brevis Lu35, Lactobacillus reuteri
5529 and Lactobacillus spicheri O15 in wheat flour hydro-
lysate broth and compared it to the compounds produced

(Figure 1 Legend Continued) fatty acid synthesis (purple); compounds derived from bioconversion from extracellular compounds-no de novo
synthesis (red); unknown biosynthesis (black); and most likely not synthesized by LAB but found in supernatant after fermentation for example,
released from grass (orange). (b) Overview of the known biosynthetic pathways of bioactive compounds in lactic acid bacteria. The bioactive
molecules are highlighted with a red border. Direct reactions are given with a bold arrow, multiple reactions are indicated by a dashed line.

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Figure 2
Cell wall instability / permeability
Fatty acids
Peptides
CO2
yeast
Oxidative stress
Acrolein
• H2O2
OH
Different inhibitory mechanisms of the described compounds against spoilage organisms include membrane destabilization [15], proton gradient
interference [16–18], enzyme inhibition [14], and creation of reactive oxygen species [14,19]. Not all mechanisms are known in detail and some are
still under investigation.
by Lactobacillus casei Lu53 as a negative control [26].
Various combinations of the identified compounds were
used to evaluate their effect on conidial germination and
growth of the molds Penicillium corylophilum and Eurotium
repens. Some combinations presented the same activity as
the bacterial culture supernatant thus confirming the
involvement of the identified molecules in the antifungal
activity. In short, the combination of lactic acid and acetic
acid together with ethanol and hydrogen peroxide was
sufficient to restore the inhibition phenotype. Interest-
ingly, further addition of the other identified compounds
(e.g. phenyllactic acid, hydroxyphenylllactic acid and
azelaic acid) to the mixture decreased the antifungal
activity, suggesting that these compounds play no role
in the inhibitory mechanism. Omitting acetic acid from
the mixture resulted in even lower antifungal activity
[26]. Acetic acid was found in concentrations close to its
MIC values [27], while the other compounds were
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Bioprotective lactic acid bacteria Siedler, Balti and Neves 141
Proton gradient interference
Lactic acid
Acetic acid
Propionic acid
Reutericyclin
Peptides
Enzyme inhibition
Hydroxy acids, CO2
Enzyme Enzyme Enzyme
Competitive Non-competitive
inhibition inhibition
Current Opinion in Biotechnology
produced in concentrations lower than their MIC value
[26]. Combination of compounds as a solution to inhibit
spoilage organisms has also already drawn commercial
interest. For example, the combination of subinhibitory
concentrations of diacetyl (0.05% w/v) and cinnamic
aldehyde (0.0025% w/v) for inhibiting the growth of yeast,
moulds and bacteria has recently been patented (WO
2011095372 A1). Curiously, studies aiming at understand-
ing the mechanisms underlying the combinatorial effects
of antimicrobial compounds produced by LAB are scarce.
Moreover, considering that many of these compounds are
weak organic acids it is even more surprising that the
effects are rarely examined under controlled conditions of
pH. MIC values can be used to indicate inhibition poten-
tial, but direct comparison is often hindered as MICs are
not measured under standardized conditions against the
same yeast or mould strains (Table 2). Even in the same
species, large variations in sensitivity towards a certain
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142 Plant biotechnology

Table 2

Compounds produced by lactic acid bacteria and their individual minimal inhibitory concentration (MIC) values against yeast, mould and
bacteria as well as the reported production concentration in LAB. The concentrations produced are highly dependent on the conditions
used and should only be considered as an indicator

Compound MIC yeast (mM) MIC mould (mM) MIC bacd Reference Produced (mM) Reference
(mM)
Lactic acid 278 274–405 2–11 [13,27,66] 55 [26]
Acetic acid 100 38–41; 0.5–14 [13,24,27,66,67] 5–40 [24,26,56]
80
8.3
Benzoic acid 10 1.27 a [68] 0.19 [69,70]
Azelaic acid 4.5 [71,72] 0.01 [26]
10.6–21.3
Succinic acid 200–500 [73] 29–300 [73,74]
Propionic acid 8.1 [24] 54 [75]
Formic acid 21–43 19.5 0.1–1.5 [24,66,76] 1.5–27 [24,39,74]
Ethanol 434 [77] 80 [57]
Diacetyl 1.45–2.9 0.005 0.005–0.011 [56] 0.15–0.7 [56]
0.7
Hydrogen peroxide 4.4–80 1–2 [78,79] 0.0006 [26]
Reuterin/Acrolein 8–50 [58]
a a
Reutericyclin 0.3–2.8 [18] 14 [80]
Cyclo(L-Phe-L-Pro) 0.08 [61]
3-Phenyllactic acid 50–500d, 55 [61,73] <7 [73,81]
0.57
4-Hydroxyphenyllactic acid 50-500 b [73] <7 [73]
3-Hydroxy fatty acids <0.46 <0.46 [15] 0.009 [15]
Indolelactic acid 24 [53] 0.02 [53]
2-Hydroxy-(4-methylthio)butanoic acid 66 [53] 0.029 [53]
2-Hydroxy-3-methylbutanoic acid 42 [53] 0.2 [53]
2-Hydroxy-4-methypentanoic acid 38 [53] 0.49 [53]
d-Dodecalactone 3.2–4.2 1.7–3.2 3.9–31 [65]
c
MIC50.
a
mM.
b
pH-dependent.
d
Bacteria.

molecule can occur, which makes the reproducibility of
the assays in different laboratories very difficult [28].
Especially, spoilage organisms obtained from a food
source often have higher resistance towards for example,
lactic acid than another differently isolated ancestor [29].
To get a better understanding of the mechanisms behind
the bioactive compounds and their synergistic or additive
effects, understanding of the physiology of common
spoilage yeast and mould strains is needed. Additionally,
for food applications other factors such as the impact of
the antimicrobial compound on the organoleptic proper-
ties of the fermented product need to be considered. For
example, acetic acid is a well-accepted food preservative,
but it is unlikely that consumers will indulge in a fer-
mented milk with a vinegar-like taste.
Proteinaceous compounds
Proteinaceous compounds produced by lactic acid bacte-
ria comprise ribosomal (RSPs) and non-ribosomal (NRPs)
peptides as well as peptides derived from enzymatic
hydrolysis of proteins. In this review, we focus on anti-
fungal peptides (AFPs) derived from the cleavage of food
proteins, since ribosomal synthesized peptides, such as
bacteriocins, have been extensively reviewed elsewhere
[30,31]. AFPs are a subset of the larger group of antimi-
crobial peptides (AMPs) [32]. AFPs are specific and active
against fungal infections, and the target pathogens do not
develop resistance [33,34]. Until now, several AFPs have
been purified and identified from food protein hydroly-
sates [35]. While some AFPs where derived from fermen-
tation products, such as kefir and sour cream [36–38],
many AFPs have been identified from sourdough fer-
mented wheat germ [39], and Chinese traditional fermen-
ted meat Dong (Nanx Wudl) [40]. Recently, Song et al.
reported the purification of an active antifungal peptide
TFNTPAMYVAIQAVLSLYASGR from spiny head
croaker (Collichthys lucidus) by-product fermentation
[41]. The theoretical mass of this peptide was
2373.75 Da and it exerted a noticeable antifungal effect
against Pestalotiopsis sp. with MIC value of
0.82 mg mL 1. Extensive studies have been done on
the antifungal activity of milk derived peptides and AFPs
have been mostly identified in peptide fragments from
lactoferrin (Lf) [42]. Lactoferricin (Lfcin), the major
peptide derived from Lf, possesses an 18-residue loop
region with a disulphide bridge and many positively

Current Opinion in Biotechnology 2019, 56:138–146 www.sciencedirect.com


charged and hydrophobic residues [43]. Bellamy et al.
reported that the primary antifungal mechanism of action
of Lfcin appears to be through direct interaction with the
fungal surface and disruption of the fungal membrane
[44]. In addition, Lfcin causes dissipation of the proton
gradient across the cell membrane [43]. Another impor-
tant peptide is the Lf(1–11) peptide, which comprises the
first 11 amino acid residues of the N-terminal region of Lf
and exhibits antifungal activity against Candida albicans
and Aspergillus fumigatus [45]. The mechanism of action of
Lf(1–11) also appears to rely on interactions with the
fungal membrane, with important structural features
including its highly cationic nature and the presence of
hydrophobic valine (V6) and tryptophan (W8) residues
[46]. Isracidin (R1PKHP-IKHQG-LPEQV-LNENL-
LRF23) produced by the digestion of as1-casein with
chymosin at pH 6.4 is potently antimicrobial against both
Gram-positive and Gram-negative bacteria [47]. Also,
Lahov and Regelson reported that isracidin protects mice
against C. albicans by stimulation of both phagocytosis and
immune responses [48]. While many peptides derived
from milk proteins have been identified by cleavage with
human enzymes found in the digestive system, for exam-
ple trypsin and chymosin [49], not many studies have
been examined the proteases from LAB that contribute to
AFP production. A recent study has identified a metallo-
protease from Lactococcus lactis involved in the production
of a bioactive peptide SSSEESII from as2 of casein [50].
Furthermore, a bioactive peptide (DMPIQAFLLY)
derived from cleavage of b-casein has been recently
identified in sour cream containing L. rhamnosus and L.
paracasei [38].
In addition to AFPs from milk and dairy products, AFPs
were identified in bovine blood and egg proteins. The
peptide VNFKLLSHSLLVTLASHL (1992.401 Da) iso-
lated from the a-subunit of bovine hemoglobin strongly
inhibited the growth of C. albicans [51]. Secondary structure
derivatives of peptide fragments at the catalytic site of
chicken and human lysozymes were synthesized and tested
for antifungal activity against C. albicans [52]. These pep-
tide fragments exhibited variable fungicidal activity. Lyso-
zyme, the full helix-loop-helix (HLH) domain (HP-full) of
human lysozyme and its C-terminal a-helix (HP-H2) all
showed a strong fungicidal activity. On the other hand, the
full HLH domain (CP-full) of chicken egg-white lysozyme
was inactive against this fungus.
Competition for nutrients
Production of antimicrobial compounds is presumably the
most widely used mechanism to inhibit spoilage organisms.
However, considering that the concentrations of the antimi-
crobial compounds are often order of magnitudes below their
individual inhibitory concentration, it is plausible to specu-
late that other mechanisms play a role. One such mechanism
is competition for nutrients, where spoilage organisms and
LAB compete for the available nutrients, either as a
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Bioprotective lactic acid bacteria Siedler, Balti and Neves 143
mechanism alone or in combination with bioactive com-
pounds. LAB might be able to inhibit the growth of spoilage
organisms by efficiently using nutrients which would limit or
even deplete the pool of essential nutrients in the growth
medium. Honoré et al. measured the metabolite consump-
tion by three L. paracasei strains and correlated the differ-
ences to growth inhibition [53]. The consumption of glucose
and glutamine by these Lactobacilli correlated inversely to
the growth of spoilage mould. In the human body, competi-
tive exclusion has been described as ‘nutritional immunity’,
where depletion of iron and zinc in specific body niches
results in inhibition of pathogenic fungal growth [54]. While,
the latter mechanism might not be active in lactic acid
bacteria, competition for other essential nutrients cannot
be ruled out even in nutrient rich raw materials. To what
extent competitive exclusion plays a role in the different
food matrices has not been studied in detail and its role on the
overall bioprotective mechanism remains to be elucidated.
Conclusion
LABs can produce a variety of compounds with antimi-
crobial activity. Of note, the amounts of individual com-
pounds produced by LAB are often insufficient to sustain
the inhibitory phenotype, which most likely arises from
synergistic or additive bioactivity from the combination of
compounds. Thus, identification of new antimicrobial
compounds is hampered by their occurrence in low
amounts in complex matrices. Furthermore, they may
not inhibit per se, which hinders the identification by
fractionation techniques. Thus, new approaches are
needed to identify and study these complex relations.
Furthermore, knowledge on the physiology of the spoilage
organisms is required to understand the mechanisms
behind the bioactive compounds. Beside small metabo-
lites, also peptides with antifungal activity have been
reported as resulting from LAB bioactivity. These antifun-
gal peptides are not produced de novo by LABs, however,
they are produced during proteolysis of proteins present in
their surrounding (e.g. wheat or milk proteins). This might
be a co-evolutionary effect as it is beneficial for the mam-
mal, if its milk proteins can be cleaved to bioactive pep-
tides, preventing pathogenic organisms from growing.
More studies should aim at understanding the synergistic
or additive effects and how the consortium of microorgan-
isms as well as host proteins interact with each other.
Conflict of interest statement
Solvej Siedler and Ana Rute Neves are employed at Chr.
Hansen A/S, a company that develops and commercia-
lizes bioprotective cultures.
Acknowledgements
We acknowledge our colleagues in the Discovery function of the R&D
organization as well as the Application Department in Chr. Hansen A/S for
valuable discussions and sharing of results. We acknowledge Eric Johansen
for critical reading of the manuscript.
Current Opinion in Biotechnology 2019, 56:138–146
144 Plant biotechnology
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