Antifungal LAB2

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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




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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.

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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





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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





8/17

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.





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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





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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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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





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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).

References

Adebayo, C. O., & Aderiye, B. I. (2011). Suspected mode ofantimycotic action of brevicin SG1 against Candida albicansandPenicillium citrinum. Food Control, 22, 1814e1820.

Adesogan, A. T., Salawu, M. B., Ross, A. B., Davies, D. R., &Brooks, A. E. (2003). Effect ofLactobacillus buchneri,Lactobacillus fermentum, Leuconostoc mesenteroidesinoculants,or a chemical additive on the fermentation, aerobic stability, andnutritive value of crimped wheat grains. Journal of Dairy Science,86, 1789e1796.

Armaforte, E., Carri, S., Ferri, G., & Caboni, M. F. (2006). High-performance liquid chromatography determination of phenyllacticacid in MRS broth. Journal of Chromatography, 1131, 281e284.

Atanassova, M., Choiset, Y., Dalgalarrondo, M., Chobert, J. M.,Dousset, X., Ivanova, I., et al. (2003). Isolation and partialbiochemical characterization of a proteinaceous anti-bacteria andanti-yeast compound produced by Lactobacillus paracaseisubsp.

paracaseistrain M3. International Journal of Food Microbiology,87, 63e73.Avis, T. J., & Belanger, R. R. (2001). Specificity and mode of action of

the antifungal fatty acid cis-9-heptadecenoic acid produced byPseudozyma flocculosa. Applied and EnvironmentalMicrobiology, 67, 956e960.

Axelsson, L. T., Chung, T. C., Dobrogosz, W. J., & Lindgren, S. E.(1989). Production of a broad spectrum antimicrobial substancebyLactobacillus reuteri.Microbial Ecology in Health and Disease,2, 131e136.

Baek, E., Kim, H., Choi, H., Yoon, S., & Kim, J. (2012). Antifungalactivity ofLeuconostoc citreumand Weissella confusa in ricecakes.Journal of Microbiology, 50, 842e848.

Batish, V. K., Grover, S., & Lal, R. (1989). Screening lactic startercultures for antifungal activity. Cultured Dairy Products Journal,24, 23e25.

Batish, V. K., Roy, U., Lal, R., & Grover, S. (1997). Antifungal attributes

of lactic acid bacteria ea review. Critical Reviews inBiotechnology, 17, 209e225.

Bergsson, G., Arnfinnsson, J., Steingrimsson, O., & Thormar, H.(2001).In vitrokilling ofCandida albicansby fatty acids andmonoglycerides.Antimicrobial Agents and Chemotherapy, 45,3209e3212.

Black, B. A., Zannini, E., Curtis, J. M., & Ganzle, M. G. (2013).Antifungal hydroxy-fatty acids produced during sourdoughfermentation: microbial and enzymatic pathways, and antifungalactivity in bread.Applied and Environmental Microbiology, 79(6),1866e1873.

Broberg, A., Jacobsson, K., Strom, K., & Schnurer, J. (2007).Metabolite profiles of lactic acid bacteria in grass silage. Appliedand Environmental Microbiology, 73, 5547e5552.

Brosnan, B., Coffey, A., Arendt, E. K., & Furey, A. (2012). Rapididentification, by use of the LTQ Orbitrap hybrid FT mass

spectrometer, of antifungal compounds produced by lactic acidbacteria.Analytical and Bioanalytical Chemistry, 403,2983e2995.

Choi, H., Kim, Y. W., Hwang, I., Kim, J., & Yoon, S. (2012). EvaluationofLeuconostoc citreumHO12 and Weissella koreensisHO20isolated from kimchi as a starter culture for whole wheatsourdough.Food Chemistry, 134, 2208e2216.

Cizeikiene, D., Juodeikiene, G., Paskevicius, A., & Bartkiene, E.(2013). Antimicrobial activity of lactic acid bacteria againstpathogenic and spoilage microorganism isolated from food andtheir control in wheat bread. Food Control, 31, 539e545.

Coda, R., Cassone, A., Rizzello, C. G., Nionelli, L., Cardinali, G., &Gobbetti, M. (2011). Antifungal activity ofWickerhamomycesanomalusand Lactobacillus plantarumduring sourdoughfermentation: identification of novel compounds and long-termeffect during storage of wheat bread.Applied and Environmental

Microbiology, 77, 3484e

3492.Coda, R., Rizzello, C. G., Nigro, F., De Angelis, M., Arnault, P., &Gobbetti, M. (2008). Long-term fungal inhibitory activity of water-soluble extracts ofPhaseolus vulgariscv. Pinto and sourdoughlactic acid bacteria during bread storage. Applied andEnvironmental Microbiology, 74, 7391e7398.

Coloretti, F., Carri, S., Armaforte, E., Chiavari, C., Grazia, L., &Zambonelli, C. (2007). Antifungal activity of lactobacilli isolatedfrom salami. FEMS Microbiology Letters, 271, 245e250.

Corsetti, A., Gobbetti, M., Rossi, J., & Damiani, P. (1998). Antimouldactivity of sourdough lactic acid bacteria: identification of a

Standardized isolationmethod-rapid, easy &reproducible

Novel compounds

Isolation &Identification

Improved sensoryanalysis

In situ testing

Safety assessment

Applications

Elucidation of inhibitorymechanism

Target sites

Transcriptomic approach

Mode ofaction

Fig. 3. Future directions in the field.





15/17

mixture of organic acids produced byLactobacillus sanfranciscoCB1.Applied Microbiology and Biotechnology, 50, 253e256.

Crowley, S., Mahony, J., & van Sinderen, D. (2012a). Broad-spectrumantifungal-producing lactic acid bacteria and their application infruit models. Folia Microbiologica (Praha), http://dx.doi.org/10.1007/s12223-012-0209-3.

Crowley, S., Mahony, J., & van Sinderen, D. (2012b). Comparative

analysis of two antifungal Lactobacillus plantarumisolates andtheir application as bioprotectants in refrigerated foods. Journal ofApplied Microbiology, 113, 1417e1427.

Dal Bello, F., Clarke, C. I., Ryan, L. A. M., Ulmer, H., Schober, T. J.,Strom, K., et al. (2007). Improvement of the quality and shelf-life ofwheat breadby fermentationwith theantifungalstrain LactobacillusplantarumFST 1.7.Journal of Cereal Science, 45, 309e318.

Dang, T. D. T., Vermeulen, A., Ragaert, P., & Devlieghere, F. (2009). Apeculiar stimulatory effect of acetic and lactic acid on growth andfermentative metabolism ofZygosaccharomyces bailii. FoodMicrobiology, 26, 320e327.

De Backer, M. D., Ilyina, T., Ma, X., Vandoninc, S.,Luyten, W. H. M. L., & Bossche, H. V. (2001). Genomic profiling ofthe response ofCandida albicansto itraconazole treatment using aDNA microarray. Antimicrobial Agents and Chemotherapy, 45,1660e1670.

Delavenne, E., Ismail, R., Pawtowski, A., Mounier, J., & Barbier, B.

(2012). Assessment of lactobacilli strains as yoghurt bioprotectivecultures.Food Control, 30, 206e213.

Dicks, L. M., & Botes, M. (2010). Probiotic lactic acid bacteria in thegastro-intestinal tract: health benefits, safety and mode of action.Beneficial Microbes, 1, 11e29.

Dieuleveux, V., Lemarinier, S., & Gueguen, M. (1998). Antimicrobialspectrum and target site of D-3-phenyllactic acid. InternationalJournal of Food Microbiology, 40, 177e183.

Digaitiene, A., Hansen, A. S., Juodeikiene, G., Eidukonyte, D., &Josephsen, J. (2012). Lactic acid bacteria isolated from ryesourdoughs produce bacteriocin-like inhibitory substances activeagainstBacillus subtilisand fungi. Journal of AppliedMicrobiology, 112, 732e742.

Driehuis, F., Oude Elferink, S. J. W. H., & Van Wikselaar, P. G. (2002).Fermentation characteristics and aerobic stability of grass silageinoculated withLactobacillus buchneri, with or without

homofermentative lactic acid bacteria. Grass Forage Science, 56,330e343.

Falguni, P., Shilpa, V., & Mann, B. (2010). Production of proteinaceousantifungal substances from Lactobacillus brevisNCDC 02.International Journal of Dairy Technology, 63, 70e76.

Fazeli, M. R., Shahverdi, A. R., Sedaghat, B., Jamalifar, H., &Samadi, N. (2004). Sourdough-isolatedLactobacillus fermentumas a potent anti-mould preservative of a traditional Iranian bread.European Food Research and Technology, 218(6), 554e556.

Ferreira, M. E., Colombo, A. L., Paulsen, I., Ren, Q., Wortman, J.,Huang, J., et al. (2005). The ergosterol biosynthesis pathway,transporter genes, and azole resistance inAspergillus fumigatus.Medical Mycology, 43(Suppl. 1), S313eS319.

Filya, I., Sucu, E., & Karabulut, A. (2006). The effect ofLactobacillusbuchnerion the fermentation, aerobic stability and ruminaldegradability of maize silage. Journal of Applied Microbiology,

101, 1216e

1223.Garcha, S., & Natt, N. K. (2011). In situcontrol of food spoilagefungus usingLactobacillus acidophilusNCDC 291. Journal ofFood Science and Technology, 49, 643e648.

Garofalo, C., Zannini, E., Aquilanti, L., Silvestri, G., Fierro, O.,Picariello, G., et al. (2012). Selection of sourdough lactobacilliwith antifungal activity for use as biopreservatives in bakeryproducts.Journal of Agricultural and Food Chemistry, 60,7719e7728.

Gautam, P., Shankar, J., Madan, T., Sirdeshmukh, R., Sundaram, C. S.,Gade, W. N., et al. (2008). Proteomic and transcriptomic analysis

ofAspergillus fumigatuson exposure to amphotericin B.Antimicrobial Agents and Chemotherapy, 52, 4220e4227.

Gerez, C. L., Torino, M. I., Obregozo, M. D., & Font de Valdez, G.(2010). A ready-to-use antifungal starter culture improves the shelflife of packaged bread. Journal of Food Protection, 73, 758e762.

Gerez, C. L., Torres, M. J., de Valdez, G. F., & Rollan, G. (2013).Control of spoilage fungi by lactic acid bacteria. Biological

Control, 64, 231e

237.Guo, J., Brosnan, B., Furey, A., Arendt, E., Murphy, P., & Coffey, A.(2012). Antifungal activity ofLactobacillusagainst Microsporumcanis, Microsporum gypseumand Epidermophyton floccosum.Bioengineered Bugs, 3, 104e113.

Guo,J., Mauch, A.,Galle, S.,Murphy, P., Arendt, E. K.,& Coffey,A. (2011).Inhibition of growth ofTrichophyton tonsuransbyLactobacillusreuteri.Journal of Applied Microbiology, 111, 474e483.

Gourama, H., & Bullerman, L. B. (1995). Inhibition of growth andaflatoxin production ofAspergillus flavusbyLactobacillusspecies.Journal of Food Protection, 58, 1249e1256.

Gourama, H., & Bullerman, L. B. (1997). Anti-aflatoxigenic activity ofLactobacillus casei pseudoplantarum. International Journal ofFood Microbiology, 34, 131e143.

Hu, W., Schmidt, R. J., McDonell, E. E., Klingerman, C. M., &Kung, L., Jr. (2009). The effect ofLactobacillus buchneri40788 orLactobacillus plantarumMTD-1 on the fermentation and aerobic

stability of corn silages ensiled at two dry matter contents. Journalof Dairy Science, 92, 3907e3914.

Kantachote, D., Prachyakij, P., Charernjiratrakul, W., Ongsakul, M.,Duangjitcharoen, Y., Chaiyasut, C., et al. (2010).Characterization of the antiyeast compound and probioticproperties of a starter Lactobacillus plantarumDW3 for possibleuse in fermented plant beverages. Electronic Journal ofBiotechnology, 13, 1e15.

Khanafari, A., Soudi, H., & Miraboulfathi, M. (2007). Biocontrol ofAspergillus flavusand aflatoxin B1 production in corn. IranianJournal of Environmental Health Science and Engineering, 4,163e168.

Kishimoto, N., Sugihara, S., Mochida, K., & Fujita, T. (2005). In vitroantifungal and antiviral activities of gamma- and delta-lactoneanalogs utilized as food flavoring.Biocontrol Science, 10, 31e36.

Kishimoto, N., Yamamoto, I., Toraishi, K., Yoshioka, S., Saito, K.,

Masuda, H., et al. (2003). Two distinct pathways for the formationof hydroxy FA from linoleic acid by lactic acid bacteria.Lipids, 38,1269e1274.

Kleinschmit, D. H., Schmidt, R. J., & Kung, L., Jr. (2005). The effects ofvarious antifungal additives on the fermentation and aerobicstability of corn silage. Journal of Dairy Science, 88, 2130e2139.

Kohler, G. A., Assefa, S., & Reid, G. (2012). Probiotic interference ofLactobacillus rhamnosusGR-1 andLactobacillus reuteriRC-14with the opportunistic fungal pathogen Candida albicans.Infectious Diseases in Obstetrics and Gynecology, 1e14.

Kung, L., Jr., Schmidt, R. J., Ebling, T. E., & Hu, W. (2007). The effect ofLactobacillus buchneri40788 on the fermentation and aerobicstability of ground and whole high-moisture corn.Journal of DairyScience, 90, 2309e2314.

Kung, L., Jr., Taylor, C. C., Lynch, M. P., & Neylon, J. M. (2003). Theeffect of treating alfalfa with Lactobacillus buchneri40788 on

silage fermentation, aerobic stability, and nutritive value forlactating dairy cows. Journal of Dairy Science, 86, 336e343.Kung, L., Jr., & Ranjit, N. K. (2001). The effect ofLactobacillus

buchneriand other additives on the fermentation and aerobicstability of barley silage. Journal of Dairy Science, 84,1149e1155.

Lan, W. T., Chen, Y. S., Wu, H. C., & Yanagida, F. (2012). Bio-protective potential of lactic acid bacteria isolated from fermentedwax gourd. Folia Microbiologica, 57, 99e105.

Lavermicocca, P., Valerio, F., Evidente, A., Lazzaroni, S., Corsetti, A.,& Gobbetti, M. (2000). Purification and characterization of novel





16/17

antifungal compounds from the sourdough Lactobacillusplantarumstrain 21B. Applied and Environmental Microbiology,66, 4084e4090.

Lavermicocca, P., Valerio, F., & Visconti, A. (2003). Antifungalactivity of phenyllactic acid against molds isolated from bakeryproducts.Applied and Environmental Microbiology, 69,634e640.

Li, H., Liu, L., Zhang, S., Cui, W., & Lv, J. (2012). Identification ofantifungal compounds produced byLactobacillus caseiAST18.Current Microbiology, 65, 156e161.

Li, X., Jiang, B., & Pan, B. (2007). Biotransformation of phenylpyruvicacid to phenyllactic acid by growing and resting cells of aLactobacillussp. Biotechnology Letters, 29, 593e597.

Li, X., Jiang, B., Pan, B., Mu, W., & Zhang, T. (2008). Purification andpartial characterization ofLactobacillus speciesSK007 lactatedehydrogenase (LDH) catalyzing phenylpyruvic acid (PPA)conversion into phenyllactic acid (PLA). Journal of Agriculturaland Food Chemistry, 56, 2392e2399.

Lind, H., Jonsson, H., & Schnurer, J. (2005). Antifungal effect of dairypropionibacteria

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