antifungal lab2
TRANSCRIPT
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Review
Current perspectives
on antifungal lactic
acid bacteria as
natural
bio-preservatives
Sarah Crowleya,Jennifer Mahonya and
Douwe van Sinderena,b,*aDepartment of Microbiology, University College
Cork, Cork, IrelandbAlimentary Pharmabiotic Centre, University College
Cork, Cork, Ireland (Tel.: D353 21 490 1365;fax: D353 21 490 3101; e-mail: [email protected])
Fungal spoilage of foods represents a major cause of
concern for food manufacturers. The use of lactic acid bacteria
(LAB) to alleviate fungal decay of foods and feeds is a prom-ising solution. The study and application of antifungal LAB
has received a surge of interest in recent years. Significant
progress has been reported on the isolation and characteriza-
tion of antimycotic compounds, which include various organic
acids, cyclic dipeptides and fatty acids, while various food-
based applications of these antifungal LAB have been
described in literature. This review summarizes the current
knowledge on antifungal LAB, their bioactive metabolites, ap-
plications in food systems and interactions with their target
fungi.
Overview of lactic acid bacteriaLactic acid bacteria (LAB) encompass a heterogeneousgroup of Gram-positive, non-sporeforming, non-motile,
aerotolerant, rod and coccus-shaped organisms, which pro-
duce lactic acid as a major end product during carbohydrate
fermentation. Early taxonomy defined four main core
genera involved in food fermentations, namely
Lactobacillus,Leuconostoc,Pediococcusand Streptococcus
(Wessels et al., 2004). However, reclassifications have
amended this original grouping and the LAB group is
currently comprised of the following genera: Aerococcus,
Alloiococcus, Carnobacterium, Dolosigranulum, Entero-
coccus, Globicatella, Lactobacillus, Lactococcus, Lactos-
phaera, Leuconostoc, Mlissococcus, Oenococcus,
Pediococcus, Streptococcus, Tetragenococcus, Vagococcus
and Weisella (Ruas-Madiedo, Sanchez, Hidalgo-
Cantabrana, Margolles, & Laws, 2012). For centuries,
LAB have been exploited as biopreservative microorgan-isms, and as such they perform a critical role in a diversity
of food fermentations involving milk, meats, vegetables and
sourdoughs by inducing rapid acidification of the raw ma-
terial. With increasing pressure from consumers towards
more natural food preservatives, LAB represent ideal can-
didates for commercial exploitation due to their GRAS
(Generally Regarded As Safe) status and their Qualified
Presumption of Safety (QPS) status in the EU, and conse-
quently the scientific exploration of their potential as
biocontrol agents has enjoyed consistent and growing inter-
est. Aside from their preserving qualities, certain LAB are
also associated with health-promoting/probiotic properties.
Members of theLactobacillusand Enterococcusgenera arecommonly exploited for their probiotic potential (Saito,
2004). Proposed mechanisms of action of probiotic LAB
include modulation of the immune response and the pro-
duction of antimicrobial compounds to exclude pathogens,
among others (Dicks & Botes, 2010).
Antifungal metabolites of LABOrganic acids
LAB produce organic acids such as lactic, acetic and
propionic acid as fermentation end products of carbohy-
drate metabolism. The production of these weak organic
acids results in an acidic environment which generally re-
stricts growth of both bacteria and fungi, including many
pathogenic and spoilage microbes (Ross, Morgan, & Hill,2002). The antimicrobial effects of these acids are attrib-
uted to the reduction of pH to a level below the range of
growth and metabolic inhibition by non-dissociated organic
acid molecules (Batish, Roy, Lal, & Grover, 1997). The
mechanisms by which organic acids inhibit fungal growth
are still not fully understood. Acetic acid is believed to
have a synergistic effect with lactic acid in preventing
fungal growth, however, acetic acid is described as more* Corresponding author.
0924-2244/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.tifs.2013.07.004
Trends in Food Science & Technology xx (2013) 1e17
Please cite this article in press as: Crowley, S., et al., Current perspectives on antifungal lactic acid bacteria as natural bio-preservatives, Trends in Food
Science & Technology (2013), http://dx.doi.org/10.1016/j.tifs.2013.07.004
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potent due to its higher pKa value causing it to have a
higher level of dissociation inside the cell (Batish et al.,
1997; Dang, Vermeulen, Ragaert,and Devlieghere, 2009).
Propionic acid also exerts anti-mould and anti-yeast activ-
ities and displays a pKa value of 4.87, which is higher
than that of acetic acid (pKa 4.76) (Lind, Jonsson, &
Schnurer, 2005). Various organic acids produced by LABhave been implemented as fungal inhibitors, where syner-
gistic effects are believed to be involved. For example, a
mixture of acetic, formic, propionic, butyric, caproic and
n-valeric acid, was held responsible for the broad spectrum
anti-mould activity by Lactobacillus sanfranciscensis CB1
(Corsetti, Gobbetti, Rossi, & Damiani, 1998). However,
the short chain fatty acid caproic acid was shown to
contribute the most towards the inhibition ofFusarium gra-
minearum. In a recent report, lactic and acetic acid were the
main antifungal substances produced by Leuconostoc cit-
reum and Weisella confusa isolates (Baek, Kim, Choi,
Yoon, & Kim, 2012), and at concentrations higher than
17.5 mM, these organic acids were shown to be responsible
for retarding growth ofCladosporium sp. YS1 andPenicil-
lium crustosum YS2.
Other carboxylic acids are also receiving attention as
antifungal agents derived from LAB. Nine carboxylic acids
including three cinnamic acid derivatives,D-glucuronic acid
and salicylic acid were all isolated as antifungal compounds
from Lactobacillus amylovorus DSM 19280 (Ryan et al.,
2011). An array of carboxylic acids were detected in silos
inoculated with Lactobacillus plantarum MiLAB 14 and
MiLAB 393 (Table 1) (Broberg, Jacobsson, Strom, &
Schnurer, 2007). Benzoic, vanillic, azealic, hydrocinnamic,
and hydroxybenzoic acids, in conjunction with a number of
other carboxylic acids, were isolated from Weisella cibaria
PS2 and three Lactobacillus species by Brosnan, Coffey,Arendt, and Furey (2012). Furthermore, some of the car-
boxylic acids identified by Broberg et al. (2007) and
Brosnanet al. (2012), i.e. hydrocinnamic, azealic, vanillic,
p-couramic, and 4-hydroxybenzoic acid, were also shown
to be produced by Lactobacillus reuteri eep1 (Guo et al.,
2012).
Phenyllactic acid (PLA)PLA has been widely reported as an antimicrobial com-
pound, which possesses broad spectrum antibacterial and
antifungal action, and which is perhaps one of the most
extensively studied antifungal organic acids from LAB.
Bactericidal activities have been observed against both
Gram-positive and negative bacteria, such as Listeria mono-cytogenes, Staphylococcus aureus and Escherichia coli
(Dieuleveux, Lemarinier, & Gueguen, 1998). PLA has
recurrently been isolated as the causative agent of fungal
inhibition in a number of studies over the last decade and
usually plays a synergistic role with other metabolites
(Dal Bello et al., 2007; Rizzello, Cassone, Coda, &
Gobbetti, 2011; Ryan et al., 2011; Strom, Sjogren,
Broberg, & Schnurer, 2002). The lack of toxicity to animal
and human cell lines and absence of an apparent odour
makes PLA a potential candidate for the control of food
spoilage, possibly in concert with complementary treat-
ments (Lavermicocca, Valerio, & Visconti, 2003). The pro-
duction of phenyllactic acid by LAB was first described by
Lavermicocca et al. (2000), who isolated this compound
from the cell free supernatant of Lb. plantarum strain21B together with its corresponding 4-hydroxy derivative.
Sourdough fermentations started with Lb. plantarum 21B
prevented spoilage by the fungal strain Aspergillus niger
FTDC3227 for at least seven days, as compared to the con-
trol (containing the non-antifungal producer Lactobacillus
brevis1D), which allowed growth of this spoilage strain af-
ter just two days. PLA was also the subject of a study inves-
tigating bakery moulds performed by Lavermicocca et al.
(2003), where it was shown to delay growth of mycotoxi-
genic strains ofPenicillium verrucosumand Penicillium cit-
rinum. Compared to the findings of these authors lower
MIC values, between 6.5 and 12 mg ml1
, were reported
for PLA produced by a Lb. plantarum strain against fungal
spoilers such asAspergillus fumigatusandPenicillium cam-
emberti (Prema, Smila, Palavesam, & Immanuel, 2010). A
variety of Lactobacillus species, such as Lb. plantarum,
Lactobacillus coryniformis, Lb. reuteri, Lactobacillus ros-
siae, Lactobacillus alimentarius, Lactobacillus rhamnosus
and Lactobacillus fermentum have been shown to produce
PLA as an antifungal compound, though production levels
vary from isolate to isolate (Table 1).
Valerio, Lavermicocca, Pascale, and Visconti (2004)
screened a collection of diverse LAB associated with
food preservation, for PLA and 4-hydroxyphenyllactic
acid (OH-PLA) production. Interestingly, each of the 29 as-
sayed strains produced PLA and/or OH-PLA at different
levels, with Leuconostoc mesenteroides subsp. mesenter-oides ITMY30 producing the highest quantity of PLA
(0.57 0.04 mM). Further studies revealed that the pres-
ence of increased levels of the amino acid phenylalanine
(Phe) resulted in increased levels of PLA. In 2007 Li and
colleagues described the conversion of Phe to PLA as a
rate-limiting step and demonstrated that production of
PLA was increased 14-fold through addition of the precur-
sor phenylpyruvic acid (PPA) to the growth medium (Li,
Jiang, & Pan, 2007). Subsequent studies in 2008 reported
the purification and partial characterization of lactate dehy-
drogenase (LDH) from Lactobacillus species SK007 as the
enzyme responsible for conversion of PPA to PLA. Since
LDH catalyzes the reduction of pyruvate to lactate, it was
deduced that the production of PLA by LAB strains maybe due to the conversion of PPA to PLA (Li, Jiang, Pan,
Mu, & Zhang, 2008). Optimization of the growth medium
ofLactobacillus sp. SK007 led to an improved PLA yield
of 2.30 g L1
(Mu, Chen, Li, Zhang, & Jiang, 2009). The
improved medium utilizes corn steep liquor as a replace-
ment to peptone (in MRS agar) as the sole nitrogen source
and may be useful for improving PLA production by
currently used antifungal LAB strains.
2 S. Crowley et al. / Trends in Food Science & Technology xx (2013) 1e17
Please cite this article in press as: Crowley, S., et al., Current perspectives on antifungal lactic acid bacteria as natural bio-preservatives, Trends in Food
Science & Technology (2013), http://dx.doi.org/10.1016/j.tifs.2013.07.004
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Table 1. Isolated and chemically characterized antifungal compounds produced by LAB.
LAB isolate Source Antifungal compound(s) isolated & identified Reference(s)
Lb. sanfranciscensisCB1 Sourdough Acetic, caproic, formic, propionic, butyric andn-valeric acids
Corsettiet al., 1998
Lactobacillus pentosusTV35 Vagina Pentocin TV35b Okkers et al., 1999Lb. plantarumVTT E-78076 Beer Benzoic acid, mevalonolactone,
methylhydantoin and cyclo(glycl-L-leucyl)
Niku-Paavolaet al., 1999
Lb. plantarum21B Sourdough PLA and 4-hydroxyphenyllactic acid Lavermicoccaet al., 2000Lb. plantarumMiLAB 393 Grass silage Cyclo(l-Phe-l-Pro), Cyclo(l-Phe-trans-4-OH-l-Pro)
and 3-PLAStromet al., 2002
Lb. coryniformisSi3 Grass Cyclo(Phe-Pro), cyclo(Phe-4-OH-Pro), PLA,reuterin
Magnussonet al., 2003
Lb. plantarumMiLAB 14 Lilac flowers 3-(R)-hydroxydecanoic acid, 3-hydroxy-5-cis-dodecanoic acid, 3-(R)-hydroxydodecanoicacid and 3-(R)-hydroxytetradecanoic acid
Sjogrenet al., 2003
Lb. plantarumMiLAB 14,Lb. plantarumMiLAB 393
Lilac flowersGrass silage
3-hydroxydecanoic acid, 2-hydroxy-5methylpentanoic acid, benzoic acid,catechol, hydrocinnamic acid, salicylic acid,3-PLA, 4-hydroxybenzoic acid, (trans, trans)-3,4-dihydroxycyclohexane-1-carboxylic acid,p-hydrocouramic acid, vanillic acid, azealicacid, hydroferulic acid, p-coumaric acid,hydrocaffeic acid, ferulic acid and caffeic acid
Broberget al., 2007
Lb. plantarumFST 1.7 Malted barley Lactic acid, PLA, cyclo(L-Leu-L-Pro) andcyclo(L-Phe-L-Pro)
Dal Belloet al., 2007
Lactobacillus paracaseisubsp.paracaseiSM20,P. jenseniiSM11
Raw milk Propionic acid, acetic acid, lactic acid,succinic acid, 2-pyrrolidone-5-carboxylic acid,3-phenyllactic acid and hydroxyphenyllactic acid
Schwenninger et al., 2008
Lb. plantarumstrain Grass silage 3-PLA Premaet al., 2010Lb. plantarumAF1 Kimchi Cyclo(LeueLeu),d-dodecalactone Yang & Chang, 2010;
Yang et al., 2011Lb. plantarumLB1, Lb.rossiaeLB5
Raw wheat germ Lactic acid, PLA and formic acid Rizzelloet al., 2011
Lb. amylovorusDSM 19280
Cereal environment Lactic acid, acetic acid, salicylic acid,D-glucuronic acid, cytidine, 20-deoxycytidine,sodium decanoate, p-coumaric acid,3-phenylpropanoic acid, (E)-2-methylcinnamicacid, 3-PLA, 3-(4 hydroxyphenyl)lactic acid,cyclo(L-Pro-L-Pro), cyclo(L-Leu-L-Pro),
cyclo(L-Try-L-Pro), cyclo(L-Met-L-Pro)and cyclo(L-His-L-Pro)
Ryanet al., 2011
Lb. plantarumVE56,W. paramesenteroidesLC11
Fermented cassava 2-hydroxy-4 methylpentanoic acid Ndaganoet al., 2011
Lb. plantarumIMAU10014 Koumiss 3-PLA; benzeneacetic acid and 2 propenylester
Wang, Shen, et al., 2012
Lb. caseiAST18 Unknown Cyclo-(Leu-Pro), 2,6-diphenyl-piperidine,5,10-diethoxy-2,3,7,8-tetrahydro-1H and6Hdipyrrolo[1,2-a;10,20-d]pyrazine
Liet al., 2012
Lb. amylovorusFST2.1,LactobacillusarizonensisR13,Lb. plantarumFST 1.7,Lb. reuteriR2,W. cibariaPS2
Cereal environment,cheese, maltedbarley, a, a (respectively)
DL-r-hydroxyphenyllactic acid,1,2-dihydroxybenzene, 4-hydroxybenzoicacid, vanillic acid, (S)-()-2-hydroxyisocaproicacid, 3-(4-hydroxy-3-methoxy-3-methoxyphenyl)propanoic acid,p-coumaric acid, azelaic acid, PLA,benzoic acid, hydrocinnamic acid,
3-hydroxydecanoic acid, DL-b-hydroxylauricacid, decanoic acid, 2-hydroxydodecanoicacid, DL-b-hydroxymyrstric acid, salicylic acid,hydrocinnamic acid D9, 1,2 e dihydroxybenzeneand 3-(4-hydroxy-3-methoxyphenyl)propanoic acid
Brosnanet al., 2012
Lb. reuteriee1p Porcine (S)-(-)-2-hydroxyisocapric acid, hydrocinnamic acid,phenyllactic acid, decanoic acid, azealic acid,4-hydroxybenzoic acid, p-coumaric acid,vanillic acid, DL-b-hydroxyphenyllactic acidand 3-hydroxydecanoic acid
Guoet al., 2012
Lb. hammesiiDSM 16381 French wheat sourdough Mono-hydroxy C18:1 fatty acid Blacket al., 2013
a Not specified.
3S. Crowley et al. / Trends in Food Science & Technology xx (2013) 1e17
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ReuterinReuterin is a broad spectrum antimicrobial substance
produced by Lb. reuteri (Axelsson, Chung, Dobrogosz, &
Lindgren, 1989). This low molecular weight compound
was found to exhibit antimicrobial activity against a range
of Gram-positive and Gram-negative bacteria, such as Sal-
monella typhimurium and E. coli K12, while it was alsodemonstrated to be capable of inhibiting growth of a range
of moulds and yeasts, including Candida albicans and
Aspergillus flavus (Axelsson et al., 1989). An increased
antifungal effect was observed when Lb. coryniformis
strains produced 3-HPA from glycerol (Nakanishi et al.,
2002). This activity was further corroborated by evidence
provided by Magnusson, Strom, Roos, Sjogren, and
Schnurer (2003) when the addition of glycerol to the
growth medium of variousLb. coryniformisstrains resulted
in a marked increase in antifungal activity towards a
collection of food-spoilage fungi. Glycerol/diol dehydra-
tase enzymes catalyze the conversion of glycerol to 3-
HPA. The presence of a glycerol/diol dehydratase operon
(pdu operon) in Lb. coryniformis Si3 was confirmed by
PCR amplification of the pdu genes suggesting that the
observed increase in inhibition was attributed to the pro-
duction of reuterin with the breakdown products of glyc-
erol degradation; 1,3-propanediol and 3-HPA, detected in
the culture supernatant of the cells. Production of 3-HPA
by a Lb. coryniformis strain and its associated antifungal
activity against Pichia sp. Y1 was also demonstrated in
silage (Tanaka et al., 2009). The antimicrobial mechanism
of reuterin towards E. coli was recently discerned
(Schaefer et al., 2010). Microarray analysis of E. coli
exposed to reuterin revealed increased expression of genes
under the control of OxyR, a transcriptional regulator
which induces upregulation of genes in response to periodsof oxidative stress. It was determined that the aldehyde
group of reuterin (which is highly reactive) interacts with
thiol groups of small molecules and proteins causing
oxidative stress to the cell, which may then lead to growth
inhibition.
Cyclic dipeptidesCyclic dipeptides, also known as 2,5 dioxopiperazines,
are among the most common peptide derivatives found in
nature. Various bioactive properties are associated with
these dipeptides, including antimicrobial and antitumoral
activities, while they may also be involved in quorum
sensing processes (Rhee, 2004). The property of cyclic di-
peptides produced by LAB to act as antifungal agents hasbeen demonstrated in several studies as described below.
The cyclic dipeptide cyclo(glycyl-L-leucyl) was isolated
from the culture filtrate of Lb. plantarum VTT E-78076
as a compound that retards growth of the Gram-negative
bacterium Pantoea agglomerans as well as the cereal
mould Fusarium avenaceum (Niku-Paavola, Laitila,
Mattila-Sandholm, & Haikara, 1999). Strom et al. (2002)
investigated the antifungal compounds produced by Lb.
plantarum MiLAB 393, a grass silage isolate, which was
shown to exert inhibitory effects towards several moulds
and yeasts, including Fusarium porotrichioides and Kluy-
veromyces marxianus. Two cyclic dipeptides, cyclo(L-
Phe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro), were
shown to be responsible for the observed inhibitory activ-
ities. An MIC value of 20 mg ml1
was determined forcyclo(L-Phe-L-Pro) against A. fumigatus and Penicillium
roqueforti. Weak synergistic effects were demonstrated
against both of these fungi when cyclo(L-Phe-L-Pro) and
PLA were used in combination, resulting in the MIC of
cyclo(L-Phe-L-Pro) being reduced to 10 mg ml1. It is
noteworthy that the MICs of cyclic dipeptides are relatively
high compared to other antimicrobial peptides. Further ev-
idence of antimycotic cyclic dipeptides was presented by
Dal Bello et al. (2007) as cyclo(L-Leu-L-Pro) and
cyclo(L-Phe-L-Pro) were detected in the supernatant of
Lb. plantarumFST 1.7. The presence of cyclic dipeptides
in wheat bread and sourdough started by Lb. plantarum
FST 1.7 was investigated by Ryan, Dal Bello, Arendt,
and Koehler (2009). The latter work showed that acidifica-
tion and temperature play an important role in the produc-
tion of cyclic dipeptides, although their concentrations were
lower than the required MIC for spoilage fungi. Therefore
these authors concluded that the cyclic dipeptides play a
minimal role in bread preservation, yet may impact on sen-
sory attributes. Despite the fact that they are produced by a
variety of lactobacilli, the modus operandiand biochemical
pathways of cyclic dipeptides as antifungal inhibitors has
not yet been defined.
Fatty acidsFatty acids possess both antibacterial and antifungal abil-
ities (Bergsson, Arnfinnsson, Steingrimsson, & Thormar,2001). The chain length of the fatty acid appears to play an
important role in antimicrobial action with longer chain
lengths deemed optimal for inhibition. Previous studies
have shown that lauric (C12) and capric (C10) acids were
the most potent fatty acids against C. albicans (Bergsson
et al., 2001). However, short chain fatty acids with antifungal
activity have also been described. The fungicidal character-
istics of fatty acids and their hydroxy derivatives produced
by LAB have been described in a number of studies.
Sjogren, Magnusson, Broberg, Schnurer, and Kenne (2003)
identified, using a combination of Nuclear Magnetic Reso-
nance (NMR), electrospray ionization mass spectrometry
(ESI-MS) and gas chromatographyemass spectrometry
(GCeMS), four antifungal hydroxylated fatty acidsproduced byLb. plantarumMiLAB 14 as 3-(R)-hydroxyde-
canoic acid, 3-hydroxy-5-cis-dodecenoic acid, 3-(R)-hy-
droxydodecanoic acid and 3-(R)-hydroxytetradecanoic acid
(Table 1). Pronounced antifungal activity was directed to-
wards several moulds and yeasts, however, yeasts were found
to be more sensitive to such hydroxylated fatty acids with re-
ported MICs between 10 and 100 mg ml1. Elevated levels
of two hydroxyl fatty acids, 3-hydroxydecanoic acid and
4 S. Crowley et al. / Trends in Food Science & Technology xx (2013) 1e17
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2-hydroxy-4-methylpentanoic acid, in combination with
other antifungal compounds were detected in silage inocu-
lated withLb. plantarumstrains MiLAB 393 or MiLAB 14
(Broberg et al., 2007). 2-hydroxy-4-methylpentanoic acid
was also retrieved from the concentrated cell-free superna-
tant (cCFS) ofLb. plantarum VE56 and Weisella paramesen-
teroides LC11. This fatty acid is thought to act in synergywith other inhibitory metabolites and was shown to be
responsible for growth arrest ofAspergillusand Penicillium
species (Ndagano, Lamoureux, Dortu, Vandermoten, &
Thonart, 2011). In a recent study (Brosnanet al., 2012), six
fatty acids including 3-hydroxydecanoic acid and DL-b-hy-
droxymyristic acid were detected in the supernatant of
certain antifungal LAB (Table 1). Similarly, three fatty
acids (hydroxyisocapric acid, decanoic acid and
3-hydroxydecanoic acid) isolated from Lb. reuteri ee1p
were found to target dermatophytes (Guo et al., 2012).
LAB are furthermore documented to produce hydroxyl fatty
acids from linoleic acid (Kishimoto et al., 2003). Black,
Zannini, Curtis, and Ganzle (2013)described the conversion
of linoleic acid to a mono-hydroxyoctadecanoic fatty acid by
Lactobacillus hammesi DSM 16381, which displayed anti-
fungal characteristics and a MIC of 0.7 g L1 against
A. niger. The fatty acid was treated to isolate coriolic
(13-hydroxy-9,11-octadecadienoic) acid and ricinoleic
(12-hydroxy-9-octadecenoic) acid, which exhibited MICs
ofupt o2.4gL1
. It wasobservedthat thefatty acid structure
is an important factor in antifungal activity with a require-
ment of at least one hydroxyl group and one double bond
along the carbon backbone. To date there is limited informa-
tion available discerning the mode of action of fatty acids,
however, one such mechanism has been proposed based on
a study of cis-9-heptadecenoic acid, a fatty acid produced
by the filamentous yeast Pseudozyma flocculosa exhibitinginhibitory activities towards several plant pathogenic fungi
(Avis & Belanger, 2001). Antifungal fatty acids are believed
to partition the lipid bilayers of fungal membranes resulting
in loss of membrane integrity. Increased fluidity causes
membrane permeability resulting in uncontrolled release
of intracellular electrolytes and proteins, ultimately leading
to cytoplasmic disintegration of fungal cells (Avis &
Belanger, 2001).
Proteinaceous compoundsStudies concerning antibacterial proteinaceous com-
pounds, e.g. bacteriocins, are extensive in comparison to
proteins with antifungal properties, although during the
last decade various LAB-derived proteinaceous compoundswith anti-yeast and anti-mould abilities have been identi-
fied (Codaet al., 2008; Rizzelloet al., 2011). Initial studies
documented the loss of antifungal activity following treat-
ment with proteolytic enzymes, while subsequent investi-
gations have provided further characterization of such
antifungal proteins. Studies have reported the production
of antifungal proteinaceous compounds from species of
Lactococcus, Streptococcus, Lactobacillus and
Pediococcus with activity against a broad spectrum of
food-associated fungi (Table 2). It is noteworthy that the
Lactobacillus species are the most predominant isolates
associated with such proteinaceous antifungal compounds
(Table 2).
Recent studies on sourdough lactobacilli have provided
further evidence of bioactive antimycotic peptides. Fiveantifungal peptides were identified in water-soluble extracts
of sourdough fermented with Lb. brevisAM7. Activity was
observed towards P. roqueforti DPPMAF1 with MICs
ranging between 3.5 and 8.2 mg ml1
. An even lower
MIC of 0.95 mg ml1 was obtained when two of the pep-
tides were used in combination. One peptide was shown to
be similar to the defensin-like protein found in pear. Further-
more, two tripeptides were shown to correspond to anti-
hypersensitive and antimicrobial peptides contained in ca-
seins (Coda et al., 2008). An in-depth investigation of the
water/salt soluble extracts from sourdough fermented with
Lb. plantarum1A7 revealed the action of nine novel anti-
fungal peptides having MICs between 2.5 and 10 mg ml1
(Codaet al., 2011). One of these peptides showed homology
to the lantibiotic lacticin 3147.Rizzelloet al. (2011)tested
the antagonistic effects of methanol and water/salt soluble
extracts from wheat germ sourdough, towards a variety of
bakery moulds. The water/salt-soluble extracts contained
four antifungal peptides with MICs between 2.5 and
15.2 mg ml1
, and sequence homology to antimicrobial
and antifungal peptides. Finally, peptides targeting Asper-
gillus japonicuswere found in extracts from sourdough fer-
mented with Lb. rossiae LD108 and Lactobacillus
paralimenariusPB127 (Garofaloet al., 2012). The LD108
sourdough peptides were shown to correspond to proteolytic
fragments from wheat a-gliadin.
A further investigation into these antifungal peptides iscritical as their mode of action in fungal inhibition has
yet to be elucidated.
Miscellaneous antifungal compoundsRyan etal. (2011) reportedthe isolation of two nucleosides
with antifungal activity from the culture filtrate ofLb. amylo-
vorus DSM19280. Cytidine and2 0-deoxycytidine were iden-
tified from a cocktail of 17 antifungal compoundsand possess
MIC values > 200 mg ml1 againstA. fumigatusJ9.
Lactones, produced by two Lb. plantarum isolates from
beer and kimchi, have previously been demonstrated to
elicit antibacterial and antiviral activities (Kishimoto,
Sugihara, Mochida, & Fujita, 2005; Miyazawa et al.,
2000), while they also exhibit antifungal activity. Anti-fungal lactones from LAB were first reported by Niku-
Paavola et al. (1999) when mevanolactone showed to be
produced by Lb. plantarum VTT E-78076. Yang, Kim,
and Chang (2011)reported the purification ofd-dodecalac-
tone produced by Lb. plantarum AF1 with associated MIC
values that ranged from 350 to 6250mg ml1 against mem-
bers of theAspergillusgenus as well asP. roqueforti. d-do-
decalactone is associated with fruity aromas and may
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impart desirable organoleptic characteristics making it a
preferred choice for food applications.
Isolation, purification and identification of antifungalmetabolites
Antifungal compounds of LAB have previously been
described as complex in nature and indeed several studies
have reported the difficulties encountered during the
isolation process (Li, Liu, Zhang, Cui, & Lv, 2012;
Magnusson & Schnurer, 2001; Niku-Paavol et al., 1999;
Yang & Chang, 2010). For this reason, many studies merely
report the antifungal activity and therefore the availability
of data relating to the isolation of such compounds is
limited. Another limitation of this work is that the com-
pounds produced under laboratory conditions may differ
from those produced in food matrices and, therefore, the
Table 2. Proteinaceous antifungal compounds produced by LAB.
LAB isolate(s) Protein responsible Activity spectrum Reference(s)
Lc. lactissubsp. diacetylactisDRC1
Peptide esensitive to pronase E andtrypsin
A. flavus Batish, Grover, & Lal, 1989
Lb. caseisubsp.pseudoplantarum
(commercial silageinoculant)
Peptide with antimycotoxigenicproperties esensitive to trypsin
and a-chymotrypsin
A. flavus Gourama & Bullerman, 1995
Lb. caseiDSM 20312,Lb. caseiCCM1825
Anitmycotoxigenic peptidessensitive to trypsin and pepsin
P. citrinum, Penicilliumexpansum
Gourama & Bullerman, 1997
Lc. lactissubsp. lactisCHD-28.3
Peptide esensitive to chymotrypsin,trypsin and pronase E
A. flavusIARI, A. flavusNCIM555, Aspergillus parasiticusNCIM 898 and Fusariumspp.
Roy, Batish, Grover, &Neelakantan, 1996
Lb. pentosusTV35b Bacteriocin-like peptide pentocinTV35b, 3.9 kDa
C. albicans Okkerset al., 1999
Lb. coryniformisspp.coryniformisstrain Si3
3 kDa, heat stable, active betweenpH 3.0-4.5
Broad spectrum Magnusson & Schnurer, 2001
Lb. paracaseisubsp.paracaseistrain M3
43 kDa, hydrophobic bacteriocin C. albicansNBIMCC 72,Candida blankiiNBIMCC 85,Candida pseudointermediaNBIMCC 1532 strain SU
Atanassova et al., 2003
Lb. plantarumVLT01 Peptide esensitive to proteinase K,
trypsin and protease
Broad spectrum Colorettiet al., 2007
Lb. plantarumCM8,W. confusaI5,Pediococcus pentosaceousR47,W. cibariaR16
CFS sensitive to proteinase K Broad spectrum Rouseet al., 2008
Lb. brevisAM7 Five antifungal peptides P. roquefortiDPPMAF1 Codaet al., 2008FiveLactobacillusstrains Peptide esensitive to pepsin,
trypsin,a-chymotrypsin, andproteinase K
PenicilliumM1 Voulgari et al., 2010
Lb. brevisNCDC 02 Hydrophobic peptide between1 and 5 kDa in size
Broad spectrum Falguni, Shilpa, & Mann, 2010
Lb. brevisPS1 Peptide esensitive to proteinaseK and pronase E
Fusariumspecies Mauchet al., 2010
Lb. fermentumTe007,Ped. pentosaceousTe010
Peptide esensitive to proteinase K A. niger Muhialdini et al., 2011
Lb. plantarumNB and SDR Peptideesensitive to proteinase K Penicilliumsp. Zhao, 2011Lb. plantarum1A7 Nine sourdough peptides P. roquefortiDPPMAF1 Codaet al., 2011Lb. plantarumLB1 andLb. rossiaeLB5
Four antifungal sourdough peptides P. roquefortiDPPMAF1 Rizzelloet al., 2011
Lb. plantarumIMAU10014 Peptide esensitive to proteinase Kand trypsin
P. roqueforti, A niger Wang et al., 2012
Lb. sakeiKTU05-06,Ped. acidilacticiKTU05-7,Ped. pentosaceusKTU05-8,KTU05-9 and KTU05-10
Bacteriocin-like inhibitorysubstances esakacin KTU05-6,pediocin KTU05-8 KTU05-9,KTU05-10 and AcKTU05-67
Broad spectrum Digaitiene, Hansen, Juodeikiene,Eidukonyte, & Josephsen, 2012
Lb. rossiaeLD108,Lb. paralimentariusPB127
Sourdough peptides A. japonicus Garofaloet al., 2012
Lactobacillus fermentumCRL 251
Peptides esensitive to trypsin,
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isolation of antifungal molecules should ideally be per-
formed using the food matrix itself rather than from exper-
imental media where possible. Advanced methodologies
for the improved isolation and identification of antifungal
compounds have resulted in an increase in the number of
novel compounds identified over the last few years. The
majority of extraction procedures enlist either liquideliquidextraction (LLE) or solid phase extraction (SPE), whereby
the compounds of interest are retained in the organic frac-
tion or sorbent of the column, respectively. Separation of
the compounds is largely achieved using reverse phase
HPLC (RP-HPLC) systems equipped with C18 columns to
separate the components, while the final identification of
the compounds usually employs NMR and MS.
Lavermicocca et al. (2000) reported one of the first
extraction procedures for antifungal metabolites derived
from LAB. The inhibitory compounds from Lb. plantarum
21B were isolated through a series of extraction steps. The
CFS of the bacterium was firstly subjected to multiple LLE
steps using ethyl acetate followed by thin layer chromatog-
raphy (TLC) which was used for partial purification. The
active fractions were subsequently identified through com-
parison of standard sample spectra using GC/MS. LLE-
based procedures have also been used as the first extraction
step by a number of other groups working on the purifica-
tion of antifungal compounds (Brosnanet al., 2012; Wang,
Shen, Xiao, Zhou, & Dai, 2012; Wang, Yan, Wang, Zhang,
& Qi, 2012) (Fig. 1).
SPE combined with hydrophobic C18 column chroma-
tography has been successfully used and widely applied
for the isolation of antifungal compounds (Strom et al.,
2002). A bioassay-guided isolation procedure was devised
employing a microtitre well spore germination test for A.
fumigatus J9. Sample preparation, separation and structureelucidation were all essential parameters considered in the
aforementioned assay (Sjogren, 2005). Sample preparation
involved the separation of the CFS of Lb. plantarum Mi-
LAB 393 into hydrophilic and hydrophobic fractions on a
SPE column. The pooled active hydrophobic fractions
were then separated by RP-HPLC using a C18 column
and an elution gradient of 5e100% acetonitrile, after which
fractions were collected and bioassayed against the target
organism. Active fractions were further fractionated using
a Hypercarb porous graphitic column coupled to the
bioassay, after which compound identification was per-
formed through a combination of NMR, MS and GC.
This extraction procedure has been used as the basis for a
multitude of subsequent studies covering the isolation andidentification of anti-yeast and mould compounds from
LAB with some variations including the introduction of re-
cycling preparative HPLC to re-separate the fractions until
a single peak is obtained (Fig. 2) (Dal Bello et al., 2007;
Magnusson et al., 2003; Ryan et al., 2011; Schwenninger
et al., 2008; Sjogren et al., 2003; Yang & Chang, 2010;
Yanget al., 2011). An optimized method for the determina-
tion of PLA in MRS broth has been devised (Armaforte,
Carri, Ferri, & Caboni, 2006), based on a previously
described method (Strom et al., 2002), which generated
inconsistent yields caused by interactions between bacterial
metabolites and the stationary phase of column resulting in
unwanted retention of PLA on the column. The bacterial
supernatant obtained by centrifugation was microfiltered
and directly assessed by HPLC with a RP C18 column.All interfering components eluted at the beginning of a
chromatographic run and PLA was then clearly separated,
with high reproducibility and recovery rates reported
(Armaforte et al., 2006).
Antifungal peptides have recurrently been the subject
of antifungal LAB reports and can be purified by a num-
ber of methods. Okkers, Dicks, Silvester, Joubert, and
Odendaal (1999) reported on the purification of a
3.9 kDa antifungal peptide using ammonium sulphate pre-
cipitation followed by cation-exchange chromatography
using an Sulphopropyl (SP)-Sepharose column to obtain
purified fractions. Concentrated culture broth from Lb.
coryniformisSi3 was used as the starting material for pep-
tide purification (Magnusson & Schnurer, 2001). The first
step involved ion-exchange chromatography after which
the active fractions were subjected to ammonium sulphate
precipitation. Dissolved pellets were then applied to a gel
filtration column to reveal the estimated size of the anti-
fungal peptide. Anion exchange chromatography was
used for the isolation of the proteinaceous antifungal
compounds derived from Lactobacillus paracasei subsp.
paracasei strain M3, where active fractions were then
applied on a RP C4 column and further purified on a
C18 HPLC system, followed by ESI-MS analysis
(Atanassova et al., 2003). An alternative method was pre-
sented by Coda et al. (2008) for the extraction of
sourdough-derived peptides. Water soluble extracts werefirstly fractioned by ultrafiltration to separate the active
fractions into various sizes according to the membrane
cut-off. The active fractions were applied to reversed-
phase fast-performance liquid chromatography (RP-
FPLC) and fractions with antifungal activity were then
separated by SDS-PAGE and identified by nano-LC-ESI-
MS/MS. The identified peptides were synthesized and
further investigated. This separation procedure was also
used to isolate antifungal sourdough peptides in subse-
quent studies (Coda et al., 2011; Rizzello et al., 2011).
Most recently a rapid method for the detection of anti-
fungal compounds from LAB was developed by Brosnan
et al. (2012). Extracellular metabolites produced by anti-
fungal LAB isolates were screened for the presence of anti-fungal compounds, and compared to known antifungal
standards, by LC coupled with MS. Five isolates displaying
strong inhibitory activities were thus screened and the ob-
tained mass spec profiles were then compared to that of a
panel of twenty five known antifungal metabolites,
including PLA, vanillic acid and cytidine. Minimal prepa-
ration was required as the samples were either filtered
and directly injected into the system, or extracted using
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ethyl acetate prior to injection. Separation of the individual
compounds was achieved through a Gemini C18 column,
while identification was performed using the linear ion
trap quadrupole (LTQ) Orbitrap hybrid Fourier transform
mass spectrometer (FTMS). The developed method boastsa short analysis time of just 23 min, while it also eliminates
the need for additional analytical methods, such as
GCeMS and NMR, as the whole process can be performed
in a single run. This innovative technique may assist food
manufacturers in the rapid selection of antifungal LAB
for application in various food fermentations, such as
sourdough production, on the basis of specific antifungal
compounds produced. Moreover this technique was sub-
stantiated byGuo et al. (2011) to identify ten metabolites
from the culture broth of Lb. reuteri ee1p targeting human
pathogenic fungi such as Epidermophyton floccosum.
Further promise has been afforded by Watrous et al.
(2012), who developed a novel method enabling metabolic
profiling of live colonies straight from a petri dish. Theantifungal effects ofPseudomonas sp. SH-C52 were deter-
mined by applying this new approach combining nanospray
desorption ESI-MS and alignment of MS data and molecu-
lar networking. Thanamycin, the mediator of antifungal ac-
tivity in Pseudomonas sp. SH-C52, was detected by this
methodology where it had previously remained unidentified
by other approaches. This molecule is produced transiently
in small quantities emphasizing the sensitivity of this
technique. Such a strategy may allow for the antifungal me-
tabolites of LAB colonies to be discerned in a similar
manner and represents a highly sensitive, real time, cost-
effective identification method. Although the number of
techniques has increased, consolidated methods need tobe established to improve the ease of purifying these
compounds.
Application of antifungal LAB as bio-control agents infood and feed systems
An overview of the various food and feed applications of
antifungal LAB is presented inTable 3. The global food in-
dustrysectoris under constant pressure from both consumers
and regulatory bodies to provide high quality fresh food with
minimal processing. Consequently, research in recent years
has significantly focused on the discovery of alternative stra-
tegies to prevent food spoilage. Despite the physical and
chemical barriers currently implemented to prevent food
decay, the consumers preference for safe preservative-freeproducts, are increasing. The use of antifungal LAB to
circumvent fungal spoilage has been studied in a multitude
of food and feed settings encompassing fresh fruits and veg-
etables, bakery, dairy products and silages. In situtesting is
essential to substantiate the potential application of these
generally regarded as safe (GRAS) organisms as bio-
protectants against fungalrot andspoilage, as well as sensory
and safety involvements. Indeed, various studies have
Fig. 1. Chemical structures of various antifungal compounds produced by LAB.
8 S. Crowley et al. / Trends in Food Science & Technology xx (2013) 1e17
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demonstrated the successful application of LAB to alleviate
fungal spoilage in various foods renderingthem feasible sub-
stitutes or complements to chemical preservatives.
Fruits & vegetablesFresh fruits and vegetables provide an opportune niche
for many undesirable fungi due to high water availability
and long term storage during transport, with Fusarium,
Penicillium, Alternaria and Botrytis species, amongst
others, identified as major fungal spoilers. Sathe, Nawani,
Dhakephalkar, and Kapadnis (2007)demonstrated the abil-ity of Lb. plantarum CUK501 to inhibit growth of four
different fungi on cucumbers for up to eight days compared
to an untreated control. Penicillium spoilage was delayed
on apples, pears, plums and grapes through the use ofPed-
iococcus and Weisella isolates (Crowley, Mahony, & van
Sinderen, 2012b; Lan, Chen, Wu, & Yanagida, 2012;
Rouse, Harnett, Vaughan, & van Sinderen, 2008). The cul-
ture filtrate of Lb. plantarum IMAU10014 was found to
reduce Botrytis cinerea growth on tomato leaves (Wang,
Shen, et al., 2012; Wang, Yan, et al., 2012). The most
recent fruit application involved a mutant strain ofLb. plan-
tarum IMAU10014 (Wang et al., 2013). An enhanced
antifungal-producing strain (F3C2) was generated through
genome shuffling and eliminated growth ofPenicillium dig-
itatum KM08 on the surface of kumquats compared to the
wild type (Table 3). The above reports support the use of
antifungal LAB and/or their metabolites for the delay of
fungal growth during transport and storage of fresh fruits
and vegetables.
Dairy productsDairy products, including cheeses and yoghurt, are also
susceptible to fungal attack. LAB are routinely used as
starter cultures in fermented dairy products and their ability
to reduce fungal contamination has been demonstrated. Yo-
ghurts have been primarily targeted as they are liable to
yeast growth due to their low pH, storage at refrigerationtemperatures and presence of fruit in certain products. A
co-culture ofLb. paracasei subsp.paracasei and Propioni-
bacterium jensenii was found to retard growth of various
Candida species in an in situ yoghurt model as well as on
cheese surface (Schwenninger & Meile, 2004). Another
study demonstrated that a selection of antifungal adjuncts
such as Lactobacillus harbinensis K.V9.3.1Np and Lb.
rhamnosus K.C8.3.1I exhibited protective properties
against a number of fungi including Debaryomyces hanse-
nii and Rhizopus mucilaginosa in yoghurts, while they did
not alter the growth or acidification rates of the yoghurt
starters, nor did they affect the pH, lactic or acetic acid
levels (Delavenne, Ismail, Pawtowski, Mounier, &
Barbier, 2012). Cheeses are also susceptible to spoilageby psychrotolerant moulds capable of withstanding low ox-
ygen environments such as P. roqueforti. Three antifungal
Lb. plantarum isolates demonstrated anti-mould capabil-
ities when used as adjuncts during cheddar cheese produc-
tion (Zhao, 2011). Furthermore, processed cheese slices
and cheese shelf-life were improved after treatment with
antifungal LAB (Garcha & Natt, 2011; Muhialdini,
Hassan, Sadon, Zulkifli, & Azfari, 2011). Use of the
Fig. 2. Flow diagram detailing the isolation and identification of anti-fungal compounds from LAB. 1. Antifungal metabolites derived fromculture supernatant can be separated using either LiquideLiquidExtraction or Solid Phase Extraction where the organic phase containshydrophobic compounds while the supernatant contains hydrophiliccompounds.2. Fractions are assessed by bioassay against a fungal in-
dicator. 3. Active fractions are subsequently separated using HPLCwith column of choice and this process may be repeated several timesto further purify active fractions. 4. Eluted fractions are tested for anti-fungal activity again following chromatographic separation. 5. Thestructural details of the compound(s) that produce positive fractions
are then identified through MS, NMR and/or GC.
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Table 3. Application of antifungal LAB as protectants in foods and feed.
Food(s) examined Antifungal LAB Activity spectrum Reference
Fruits & vegetablesCucumber Lb. plantarumCUK501 A. flavus, F. graminearum,
Rhizopus stolonifer, Bt. cinereaSatheet al., 2007
Corn Lb. plantarumPTCC 1058 A. flavus Khanafari, Soudi, &
Miraboulfathi, 2007Apple Ped. pentosaceousR47 P. expansum Rouseet al., 2008Soybean Lb. plantarumAF1 A. flavus Yang & Chang, 2010Fresh mango Lb. acidilophidusNCDC 291 A. alternata Garcha & Natt, 2011Tomato leaves Lb. plantarumIMAU10014 Bt. cinerea Wang, Yan, et al., 2012Grape W. cibaria861006 Penicillium oxalicum Lanet al., 2012Pear, plum, grape Ped. pentosaceous54 P. expansum Crowleyet al., 2012bKumquat Lb. plantarumIMAU10014 strain
F3A3 (mutant)P. digitatumKM08 Wanget al., 2013
Dairy productsYoghurt, cheese Lb. paracaseisubsp. paracasei Candidaspecies Schwenninger & Meile, 2004Indian cheese Lb. acidilophidusNCDC 291 A. alternata Garcha & Natt, 2011Cheddar cheese Lb. plantarumNB, Lb. plantarum
SDR andLb. plantarumDC2Penicilliumsp. Zhao, 2011
Cheese slices Lb. fermentumTe007, Ped.pentosaceousTe010
A. oryzae, A. niger Muhialdinet al., 2011
Yoghurt Lb. harbinensisK.V9.3.1Np, Lb.rhamnosusK.C8.3.1I andLb.paracaseiK.C8.3.1Hc1
D. hansenii, R. mucilaginosa, K.marxianus, K. lactis, Yarrowialipolytica, Penicilliumbrevicompactum
Delavenne et al., 2012a
Yoghurt Lb. plantarum16 (NCIMB41875)andLb. plantarum62(NCIMB41876)
R. mucilaginosa Crowleyet al., 2012a
BreadsSourdough Lb. plantarum21B A. nigerFTDC3227 Lavermicoccaet al., 2000Sourdough Lb. plantarum, Lb. caseiand Lb.
fermentum
a Fazeli, Shahverdi, Sedaghat,Jamalifar, & Samadi, 2004
Gluten free bread,wheat bread
Lb. plantarumFST 1.7 Fusariaspecies Dal Belloet al., 2007, Moore,Dal Bello, & Arendt, 2008
Sourdough Lb. plantarumFST 1.7 & 1.9 A. niger, F. culmorum, P. expansum,P. roqueforti
Ryan, Dal Bello, & Arendt, 2008
Bread Lb. brevisAM7 P. roquefortiDPPMAF1 Codaet al., 2008Sourdough Lb. buchneriFUA 3525, and Lb.
diolovoransDSM 14421A. clavatus, Cladisporiumspp.,Mortierellaspp., S. cervisiae, P.roqueforti
Zhanget al., 2010
Bread Lb. plantarumCRL 778 Penicilliumsp. Gerezet al., 2010Bread Lb. amylovorusDSM 19280 F. culmorumFST 4.05, A. niger
FST4.21,P. expansumFST 4.22,P.roquefortiFST 4.11, bakery fungalflora
Ryan et al., 2011
Bread Lb. plantarum1A7 Penicillium, AspergillusandEurotiumspecies
Codaet al., 2011
Bread, panettone Lb. rossiaeLD108; Lb.paralimentariusPB127
A. japoniucs Garofaloet al., 2012
Wheat sourdough L. citreumH012 andW.koreensisH020
P. roqueforti, A. niger Choi, Kim, Hwang, Kim, &Yoon, 2012
Sangak (Traditionalflat bread)
Lb. plantarumssp. plantarum,strain ATCC 20179, Lb.
acidipholus,strain ATCC 20079andL. mesenteroidesssp.mesenteroides,strain 1591
Moulds Najafi, Rezaei, Safari, &Razavi, 2012
Bread Ped. acidilacticiKTU05-7, Ped.pentosaceousKTU05-8 andPed.pentosaceousKTU05-8
Moulds Cizeikieneet al., 2013
Sourdough Lb. hammesiiDSM 16381 A. niger, P. roqueforti,environmental contaminants
Blacket al., 2013
SilageBarley silage Lb. buchneri Yeasts Kung & Ranjit, 2001
10 S. Crowley et al. / Trends in Food Science & Technology xx (2013) 1e17
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aforementioned isolates provides manufacturers with a nat-
ural option to the use of preservatives such as sodium ben-
zoate, sorbic acids and natamycin in yoghurt and cheese
production.
Bakery productsPoor bread quality attributable to fungal growth has
proven problematic for the food industry in terms of both
economic and health costs for numerous years. An in-
depth investigation of antifungal LAB sourdough starters
has been performed with the majority of reports harnessing
the antifungal properties of lactobacilli, in particular Lb.plantarum isolates, to enhance shelf-life and quality of
the products (Table 3). The earliest documentation of the
application of an antifungal LAB sourdough starter was
the use of sourdough isolate Lb. plantarum 21B in a co-
fermentation with Saccharomyces cerevisiae to retard the
growth of A. niger FTDC3227 over a seven day storage
period (Lavermicocca et al., 2000). However, no sensory
analysis of the final product was conducted to assess the
impact ofLb. plantarum21B on the organoleptic properties
of the sourdough. Other Lb. plantarum isolates have all
shown antifungal potential in bread fermentations (See
Table 3). Additional Lactobacillus species have come to
the fore as fungal inhibitors in bread production. The
shelf-life of wheat bread containing Lb. amylovorus DSM
19280 was improved, with inhibition observed against
Aspergillus, Fusarium and Penicillium moulds. Recently
Lb. rossiae LD108 and Lb. paralimentarius PB127 were
used in the production of bread and panettone, and found
to prevent growth ofA. japonicus with shelf lives ranging
from 11 to 32 days as compared to bread prepared withbakers yeast dough (Garofaloet al., 2012). Antifungal ped-
iococci have also proved successful in the control of mould
growth in bread (Cizeikiene, Juodeikiene, Paskevicius, &
Bartkiene, 2013). Pediococcus acidilactici KTU05-7, and
Pediococcus pentosaceous KTU05-8 and KTU05-10 strains
provided protection against mould development when
sprayed on the surface of bread, a treatment that proved
effective against a number of food related fungi such as
Table 3 (continued)
Food(s) examined Antifungal LAB Activity spectrum Reference
Grass silage Lb. buchneri, Lb. plantarumandPed. pentosaceous
Yeasts & moulds Driehuis et al., 2002
Wheat silage,corn silage
Lb. buchneriand Lb. plantarum Yeasts & moulds Weinberget al., 2002
Corn silage Lb. buchneri40788 Yeasts Taylor & Kung, 2002Maize silage Lb. buchneri40788 Yeasts Ranjit, Taylor, & Kung, 2002Crimped wheatgrains
Lb. buchneri Yeasts Adesogan, Salawu, Ross, Davies,and Brooks (2003)
Corn silage Lb. buchneri Yeasts Nishinoet al., 2004Corn silage Lb. buchneri40788 andPed.
pentosaceousR1094Yeasts Kleinschmit, Schmidt, &
Kung, Jr., 2005Maize silage Lb. buchneri Yeasts Filya, Sucu, & Karabulut, 2006Grass silage Lb. plantarumMiLAB 393 and 14 Pichia anomala Broberg et al., 2007Corn silage Lb. buchneri40788 Moulds Kung, Schmidt, Ebling, &
Hu, 2007Alfalfa silage Lb. buchneriand Lb. plantarum Yeasts Zhanget al., 2009Alfalfa silage Lb. bucnheriand Ped.
pentosaceousYeasts & moulds Schmidt, Hu, Mills, &
Kung, 2009Corn silage Lb. buchneri40788, Lb.
plantarumand Ped. acidilacitiYeasts Reich & Kung, 2010
Corn silage Lb. buchneriand Ped.
pentosaceous
Yeasts Schmidt & Kung, 2010
Corn silage Lb. buchneriLN4637 andLb.buchneriLN40177
Yeasts Tabacco, Piano, Revello-Chion,& Borreani, 2011
Miscellaneous foodsFermented seaweedbeverage
Lb. plantarumDW1 Unidentified yeasts Prachyakij et al., 2008
Raw smoked sausage Lc. lactisssp. lactisK-205 and194
Eurotium repens Stoyanova et al., 2010
Raw poultry meat Lb. acidophilusNCDC 291 A. alternata Garcha & Natt, 2011Orange juice Lb. plantarum16 (NCIMB41875)
and 62 (NCIMB41876)R. mucilaginosa Crowleyet al., 2012a
Rice cakes Leuc. citreumC5, W. confusaHO24 and W. confusaD2-96
Cadisporiumsp. YS1,Penicillium crustosumYS2,Neurosorasp. YS3
Baeket al., 2012
a Not specified.
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Fusarium culmorum Al-2 and Candida parapsilosis C.7.2.
Co-fermentation of sourdough with Lactobacillus buchneri
FUA 3525 and Lactobacillus diolovorans DSM 14421 de-
ferred growth of a number of bread-spoiling fungi,
including Aspergillus clavatus and Cladosporium spp.,
through the accumulation of acetate and propionate
(Zhang, Brandtb, Schwaba, & Ganzlea, 2010).
Animal feedAnimal feed is also under threat of fungal decay during
storage and feeding. Silage is the product of anaerobic
fermentation of water soluble carbohydrates (WSC) to
organic acids in forage crops, of which LAB play a domi-
nating role (Schmidt & Kung, 2010). Oxygen may acciden-
tally be introduced into silage during ensiling, storage and
feeding, encouraging troublesome aerobic spoilers such as
yeasts and moulds to proliferate, resulting in spoilage and
decreased nutritive value, especially in hot climates
(Kung, Taylor, Lynch, & Neylon, 2003; Taylor & Kung,
2002). A plethora of investigations on the potentials of
LAB as silage additives to produce high quality feeds
have been performed with the majority of reports domi-
nated by the application of the hetero-fermentative Lb.
buchneri. The production of acetic acid and 1,2-
propanediol during anaerobic degradation of lactic acid is
an important factor in the preserving attributes ofLb. buch-
neri (Oude Elferinket al., 2001). The aerobic stability of
whole crop maize, maize, corn and barley silages has
been improved with Lb. buchneri as a silage inoculant
(See Table 3). The use of homo-fermentative LAB is
important in the ensiling process as rapid lactic acid pro-
duction from fermentation of WSC decreases pH, thereby
improving forage preservation. However, preservation can
be compromised as lactic acid can be oxidized by aerobicmicroorganisms and there is a reduced production in anti-
fungal volatile fatty acids to prevent the growth of aerobic
moulds and yeasts with this additive choice (Nishino,
Wada, Yoshida, & Shiota, 2004; Reich & Kung, 2010;
Weinberg et al., 2002). This drawback may be overcome
by combining homo-fermentative LAB with Lb. buchneri
and several reports have compared the use of Lb. buchneri
alone and in combination with other LAB to improve silage
quality, although conflicting results were documented. A
combination approach was favoured by some authors
(Driehuis, Oude Elferink, & Van Wikselaar, 2002; Reich
& Kung, 2010; Zhang et al., 2009), while others preferred
the sole use ofLb. buchnerito improve forage stability (Hu,
Schmidt, McDonell, Klingerman, & Kung, 2009; Weinberget al., 2002). Lb. plantarum strains MiLAB 393 and Mi-
LAB 14 were previously shown to have inhibitory activities
towards a spectrum of fungi. Antifungal metabolites pro-
duced by these isolates in cultured broth, such as 3-PLA
and 3-hydroxydecanoic acid, were also identified in silage
when used as inoculants. Furthermore additional antifungal
components such as azealic acid were detected in silage in-
oculants highlighting the potential for these strains in silage
preservation (Broberget al., 2007). Lb. plantarum MiLAB
393 has since been patented and used as a commercial
silage inoculant known as Feedtech
Silage F3000.
Miscellaneous foodsAntifungal LAB have further promoted increased quality
andshelf-life of a miscellany of other foods. Muhialdini etal.(2011)demonstrated the antagonistic effects of four LAB
isolates againstA. nigerandAspergillus oryzae in tomato pu-
ree. Beverages have also benefited from the application of
antifungal LAB. The shelf-life of orange juice spiked with
R. mucilaginosawas improved by the addition of the anti-
fungalLb. plantarum16 (NCIMB41875) steep water isolate
(Crowley, Mahony, & van Sinderen, 2012a), while a fer-
mented seaweed beverage was found to contain a reduced
yeast count after introduction of Lb. plantarum DW1
(Prachyakij, Charernjiratrakul, & Kantachote, 2008). More
recently Baek et al. (2012) demonstrated the potential of
Leuc.citreumC5, W. confusa HO24 andW. confusa D2-96
as antifungal rice cake starters. Limited applications of anti-
fungal LAB in the preservation of meats exist. Interestingly,
Lactobacillus acidilophidus NCDC 291 exerted a 0.4 log
reduction in viable numbers ofAspergillus alternata when
inoculated into raw poultry meat (Garcha & Natt, 2011).
Additionally the shelf-life of raw smoked sausages was
extended after application of twoLactococcus lactisssp. lac-
tis strains K-205 and194 (Stoyanova, Ustyugova, Sultimova,
& Bilanenko, 2010).
Antifungal LABefungal interactionsWhile all the above-mentioned studies endorse the appli-
cation of antifungal LAB, little information is available
about the interactions of these antifungal metabolites and
their target fungal species. Antifungal metabolite targetsites and modes of action are as of yet a poorly explored
territory. In a bid to address this knowledge caveat, studies
examining fungal protein expression as well as the physical
effects of the antifungal metabolites on fungal development
by microscopy represent the first attempt to gain an insight
into these elusive interactions.
One of the first studies to investigate antifungal LAB-
efungal interactions was reported by Strom, Schnurer,
and Melin (2005). A co-cultivation assay was devised using
Lb. plantarumMiLAB 393 and its target Aspergillus nidu-
lans. Physical changes during growth were examined
microscopically, while changes in protein expression using
2-D gels were also investigated. Reported morphological
changes upon co-cultivation included interrupted mycelialbranching in addition to swollen hyphal tips. Three proteins
were found to be differentially upregulated (designated Px,
P1 and P11) and one protein, P2/K3, was thought to be
shifted to an alternative location following exposure to
the antifungal substances.
Aside from proteomics, microscopy has also been
exploited to study LABefungal interactions more recently.
A macroconidia germination assay was monitored
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Please cite this article in press as: Crowley, S., et al., Current perspectives on antifungal lactic acid bacteria as natural bio-preservatives, Trends in Food
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microscopically in order to determine what effects cCFS
from a Lb. brevis PS1 culture had on F. culmorum growth
(Mauch, Dal Bello, Coffey, & Arendt, 2010). It was noted
that germ tube outgrowth was slightly delayed compared to
a control upon treatment of conidia with 5% cCFS. Further-
more germ tube formation was completely restricted after
treatment with 10% cCFS. Similar findings were reportedby Guo et al. (2011), where conidia germination tests
were also used to evaluate the impact of Lb. reuteri R2
CFS on the dermatophyte Trichophytan tonsurans. The sus-
pected mode of action of brevicin SG1 on C. albicans and
P. citrinum fungal cells was also investigated (Adebayo &
Aderiye, 2011). The effects of this bacteriocin on these two
target organisms were examined by Transmitted Scanning
Electron Microscopy (TSEM). Treatment of yeast cells re-
sulted in reduced hyphal branching and irregular shaped
cells. A dose-dependent response was observed whereby
at lower concentrations (500 AU ml1) initiation of new
hyphae failed to develop, while at 1000 AU ml1
hyphal
development was completely arrested withC. albicanscells
exhibiting growth that was reminiscent of that of a budding
yeast. The suspected mode of action on yeast cells was
thought to be antibiosis and targeting the cell wall-
synthesizing enzymes. SG1 induced morphological
changes and decreased total biomass of P. citrinum.
TSEM revealed swelling, lysis, damage to hyphae and total
disruption of the cell wall. The mode of action was deemed
to be both cytolytic and fungiolytic with the fungal wall
presumed to be the primary target. In a recent paper Scan-
ning Electron Microscopy (SEM) has revealed reduction in
conidial size and undulation of the mycelial surface of
Aspergillus parasiticus MTCC 2796 after exposure to the
antifungal compound of Ped. acidilactici LAB 5 (Mandal,
Sen, & Mandal, 2013). From the limited studies that haveattempted to elucidate how antifungal LAB impact on their
sensitive fungi it appears that the primary target site of the
antifungal compounds is the fungal cell wall, which is
different from the previously held notion that the LAB-
produced short chain fatty acids caused interference with
membrane potential and leakage of membrane contents.
While both mechanisms may be responsible for the anti-
fungal effect, current data have not allowed a firm conclu-
sion as regards to the reasons for strain/species-specific
antifungal action of LAB and further studies may well
reveal additional modes of action.
A relatively unexplored approach to investigate the mo-
lecular targets of the antifungal LAB-derived metabolites is
by means of transcriptome analysis. Microarrays have beenemployed to study the transcriptional responses of a variety
of fungi, such as Candida and Aspergillus species, to anti-
fungal drugs (De Backer et al., 2001; Gautamet al., 2008).
The genes most often affected appear to be those involved
in ergosterol biosynthesis, the major sterol component in
fungal plasma membranes. Azoles target the 14-a-deme-
thylase enzyme, product of the CYP51, thus interfering
with ergosterol biosynthesis (Ferreira et al., 2005).
Transcriptional profiling of C. albicans in a co-culture
with the probiotic strainsLb. rhamnosus GR-1 and Lb. reu-
teri RC-14 was determined by Kohler, Assefa, and Reid
(2012) in order to elucidate the molecular targets involved
in probiotic interference. Upregulation of genes including
those involved in lactic acid utilization, stress response
and signalling was reported, while downregulation of,amongst others, genes associated with filamentous growth,
cell wall organization and ergosterol biosynthesis provides
an insight into the transcriptional response of this fungal
pathogen. These strategies may also be applied to the un-
derstanding of antifungal LABefungal interactions. Micro-
array technology may thus provide an opportunity to
elucidate which genes and associated metabolic or physio-
logical functions of a given fungal spoiler are targeted by
antifungal compounds, such as PLA and d-dodecalacetone.
The so far published work performed on revealing such in-
teractions between antifungal drugs and fungal pathogens
provide an excellent basis for future work.
Conclusions & future perspectivesVery significant advances in the field of antifungal LAB
have been achieved during the last decade. However,
certain limitations and knowledge gaps still need to be ad-
dressed. Whilst there have been many publications on anti-
fungal applications in recent years, just a small number of
such studies have investigated final product quality,
including sensory analysis. It is also interesting that very
few commercial cultures are available, possibly due to the
fact that the anti-fungal activity of any given strain is
dependent on many physico-chemical parameters, the
food production process and the ability of the strains to pro-
duce the compounds in situ in the food product. The latter
will be a prerequisite for a full assessment of antifungalLAB application in foods, as the inhibitory metabolites or
their producing LAB may alter the visual and/or organo-
leptic properties of the produced food. Safety concerns
such as health effects are also important considerations
which so far have not been addressed for all antifungal
strains. Safety assessments should be included as a standard
practice when characterizing an antifungal strain, as was
done in the case of the antifungal strain Lb. plantarum
DW3, for which an acute oral toxicity test was performed
on mice, indicating that the isolate is safe for human con-
sumption (Kantachote et al., 2010). Such assessments
should include analysis of acquired antibiotic resistance
and potential biogenic amine production in compliance
with the EU qualified presumption of safety evaluation.Although in most instances sensory and safety assessments
remain incomplete for a given antifungal strain, high-
lighting the need for additional evidence to ensure the
safety of implementing these compounds in food matrices,
the mentioned antifungal LAB have become highly adapted
to a range of environments as highlighted by their diverse
in vivo and in vitro food applications. The development
of more ready-to-use antifungal combinations such as the
13S. Crowley et al. / Trends in Food Science & Technology xx (2013) 1e17
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antifungal slurry formulated by Gerez, Torino, Obregozo,
and Font de Valdez (2010) would prove far more advanta-
geous for the food manufacturer and provides an alternative
approach to meeting consumer demands.
Standardization of isolation and purification processes is
required with procedures needing to be rapid, sensitive,
reproducible, and cost effective (Fig. 3). The development
of sensitive and rapid isolation procedures may ultimatelylead to the discovery of additional antifungal compounds.
In time antifungal LAB may even replace chemical preser-
vatives as bio-protectants in foods. As more genome se-
quences become available transcriptomic approaches
represent an amenable method to determine the molecular
targets of antifungal metabolites derived from LAB. As
of yet these targets are unknown and forthcoming studies
should invest in microarray or other omics technologies
to determine the effects of various LAB-produced anti-
fungal compounds on fungi. Future efforts should also be
oriented towards expanding our knowledge regarding the
genetic mechanisms and metabolic pathways behind anti-
fungal production (Fig. 3). Moreover, if the genetic machin-
ery responsible for antifungal production is discerned thismay lead to the ability to transfer antifungal properties to
starter cultures already routinely in use. Ultimately, the
antifungal substances produced by LAB will need to be
characterized to the same detailed extent as their antibacte-
rial equivalents.
AcknowledgementsS. Crowley is the recipient of a Lauritzson Foundation
scholarship. D. van Sinderen is a recipient of a Science
Foundation Ireland (SFI) Principal Investigator award
(Ref. No. 08/IN.1/B1909).
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