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Trends in Food Science & Technology xx (2013) 1e17


Current perspectives on antifungal lactic acid bacteria as natural bio-preservatives

Sarah Crowleya, Jennifer Mahonya and Douwe van Sinderena,b,*
Department of Microbiology, University College Cork, Cork, Ireland b Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland (Tel.: D353 21 490 1365; fax: D353 21 490 3101; e-mail: d.vansinderen@ucc.ie)
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 promising solution. The study and application of antifungal LAB has received a surge of interest in recent years. Signicant progress has been reported on the isolation and characterization of antimycotic compounds, which include various organic acids, cyclic dipeptides and fatty acids, while various foodbased applications of these antifungal LAB have been described in literature. This review summarizes the current knowledge on antifungal LAB, their bioactive metabolites, applications in food systems and interactions with their target fungi.

Lactobacillus, Leuconostoc, Pediococcus and Streptococcus (Wessels et al., 2004). However, reclassications have amended this original grouping and the LAB group is currently comprised of the following genera: Aerococcus, Alloiococcus, Carnobacterium, Dolosigranulum, Enterococcus, Globicatella, Lactobacillus, Lactococcus, Lactosphaera, Leuconostoc, Mlissococcus, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weisella (Ruas-Madiedo, Sanchez, HidalgoCantabrana, Margolles, & Laws, 2012). For centuries, LAB have been exploited as biopreservative microorganisms, and as such they perform a critical role in a diversity of food fermentations involving milk, meats, vegetables and sourdoughs by inducing rapid acidication of the raw material. With increasing pressure from consumers towards more natural food preservatives, LAB represent ideal candidates for commercial exploitation due to their GRAS (Generally Regarded As Safe) status and their Qualied Presumption of Safety (QPS) status in the EU, and consequently the scientic exploration of their potential as biocontrol agents has enjoyed consistent and growing interest. Aside from their preserving qualities, certain LAB are also associated with health-promoting/probiotic properties. Members of the Lactobacillus and Enterococcus genera are commonly exploited for their probiotic potential (Saito, 2004). Proposed mechanisms of action of probiotic LAB include modulation of the immune response and the production of antimicrobial compounds to exclude pathogens, among others (Dicks & Botes, 2010). Antifungal metabolites of LAB Organic acids LAB produce organic acids such as lactic, acetic and propionic acid as fermentation end products of carbohydrate metabolism. The production of these weak organic acids results in an acidic environment which generally restricts growth of both bacteria and fungi, including many pathogenic and spoilage microbes (Ross, Morgan, & Hill, 2002). The antimicrobial effects of these acids are attributed 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

Overview of lactic acid bacteria Lactic acid bacteria (LAB) encompass a heterogeneous group of Gram-positive, non-sporeforming, non-motile, aerotolerant, rod and coccus-shaped organisms, which produce lactic acid as a major end product during carbohydrate fermentation. Early taxonomy dened four main core genera involved in food fermentations, namely
* Corresponding author.
0924-2244/$ - see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tifs.2013.07.004

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

S. Crowley et al. / Trends in Food Science & Technology xx (2013) 1e17

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 activities 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 LAB have been implemented as fungal inhibitors, where synergistic 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 of Fusarium graminearum. In a recent report, lactic and acetic acid were the main antifungal substances produced by Leuconostoc citreum 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 of Cladosporium sp. YS1 and Penicillium 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 carboxylic acids identied by Broberg et al. (2007) and Brosnan et 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 compound, 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 monocytogenes, 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 treatments (Lavermicocca, Valerio, & Visconti, 2003). The production of phenyllactic acid by LAB was rst described by Lavermicocca et al. (2000), who isolated this compound from the cell free supernatant of Lb. plantarum strain 21B 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 control (containing the non-antifungal producer Lactobacillus brevis 1D), which allowed growth of this spoilage strain after just two days. PLA was also the subject of a study investigating bakery moulds performed by Lavermicocca et al. (2003), where it was shown to delay growth of mycotoxigenic strains of Penicillium verrucosum and Penicillium citrinum. Compared to the ndings 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 as Aspergillus fumigatus and Penicillium camemberti (Prema, Smila, Palavesam, & Immanuel, 2010). A variety of Lactobacillus species, such as Lb. plantarum, Lactobacillus coryniformis, Lb. reuteri, Lactobacillus rossiae, 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 assayed strains produced PLA and/or OH-PLA at different levels, with Leuconostoc mesenteroides subsp. mesenteroides ITMY30 producing the highest quantity of PLA (0.57 0.04 mM). Further studies revealed that the presence 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 precursor phenylpyruvic acid (PPA) to the growth medium (Li, Jiang, & Pan, 2007). Subsequent studies in 2008 reported the purication and partial characterization of lactate dehydrogenase (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 may be due to the conversion of PPA to PLA (Li, Jiang, Pan, Mu, & Zhang, 2008). Optimization of the growth medium of Lactobacillus 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 replacement to peptone (in MRS agar) as the sole nitrogen source and may be useful for improving PLA production by currently used antifungal LAB strains.

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

S. Crowley et al. / Trends in Food Science & Technology xx (2013) 1e17 Table 1. Isolated and chemically characterized antifungal compounds produced by LAB. LAB isolate Lb. sanfranciscensis CB1 Lactobacillus pentosus TV35 Lb. plantarum VTT E-78076 Lb. plantarum 21B Lb. plantarum MiLAB 393 Lb. coryniformis Si3 Lb. plantarum MiLAB 14 Source Sourdough Vagina Beer Sourdough Grass silage Grass Lilac owers Antifungal compound(s) isolated & identied Acetic, caproic, formic, propionic, butyric and n-valeric acids Pentocin TV35b Benzoic acid, mevalonolactone, methylhydantoin and cyclo(glycl-L-leucyl) PLA and 4-hydroxyphenyllactic acid Cyclo(l-Phe-l-Pro), Cyclo(l-Phe-trans-4-OH-l-Pro) and 3-PLA Cyclo(Phe-Pro), cyclo(Phe-4-OH-Pro), PLA, reuterin 3-(R)-hydroxydecanoic acid, 3-hydroxy-5-cisdodecanoic acid, 3-(R)-hydroxydodecanoic acid and 3-(R)-hydroxytetradecanoic acid 3-hydroxydecanoic acid, 2-hydroxy5methylpentanoic 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, azealic acid, hydroferulic acid, p-coumaric acid, hydrocaffeic acid, ferulic acid and caffeic acid Lactic acid, PLA, cyclo(L-Leu-L-Pro) and cyclo(L-Phe-L-Pro) Propionic acid, acetic acid, lactic acid, succinic acid, 2-pyrrolidone-5-carboxylic acid, 3-phenyllactic acid and hydroxyphenyllactic acid 3-PLA Cyclo(LeueLeu), d-dodecalactone Lactic acid, PLA and formic acid Lactic acid, acetic acid, salicylic acid, 0 D-glucuronic acid, cytidine, 2 -deoxycytidine, sodium decanoate, p-coumaric acid, 3-phenylpropanoic acid, (E)-2-methylcinnamic acid, 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) 2-hydroxy-4 methylpentanoic acid 3-PLA; benzeneacetic acid and 2 propenyl ester Cyclo-(Leu-Pro), 2,6-diphenyl-piperidine, 5,10-diethoxy-2,3,7,8-tetrahydro-1H and 6Hdipyrrolo[1,2-a;10,20-d]pyrazine DL-r-hydroxyphenyllactic acid, 1,2-dihydroxybenzene, 4-hydroxybenzoic acid, vanillic acid, (S)-()-2-hydroxyisocaproic acid, 3-(4-hydroxy-3-methoxy-3methoxyphenyl)propanoic acid, p-coumaric acid, azelaic acid, PLA, benzoic acid, hydrocinnamic acid, 3-hydroxydecanoic acid, DL-b-hydroxylauric acid, decanoic acid, 2-hydroxydodecanoic acid, DL-b-hydroxymyrstric acid, salicylic acid, hydrocinnamic acid D9, 1,2 e dihydroxybenzene and 3-(4-hydroxy-3-methoxyphenyl)propanoic acid (S)-(-)-2-hydroxyisocapric acid, hydrocinnamic acid, phenyllactic acid, decanoic acid, azealic acid, 4-hydroxybenzoic acid, p-coumaric acid, vanillic acid, DL-b-hydroxyphenyllactic acid and 3-hydroxydecanoic acid Mono-hydroxy C18:1 fatty acid Reference(s) Corsetti et al., 1998 Okkers et al., 1999 Niku-Paavola et al., 1999 Lavermicocca et al., 2000 Strom et al., 2002 Magnusson et al., 2003 Sjogren et al., 2003

Lb. plantarum MiLAB 14, Lb. plantarum MiLAB 393

Lilac owers Grass silage

Broberg et al., 2007

Lb. plantarum FST 1.7 Lactobacillus paracasei subsp. paracasei SM20, P. jensenii SM11 Lb. plantarum strain Lb. plantarum AF1 Lb. plantarum LB1, Lb. rossiae LB5 Lb. amylovorus DSM 19280

Malted barley Raw milk

Dal Bello et al., 2007 Schwenninger et al., 2008

Grass silage Kimchi Raw wheat germ Cereal environment

Prema et al., 2010 Yang & Chang, 2010; Yang et al., 2011 Rizzello et al., 2011 Ryan et al., 2011

Lb. plantarum VE56, W. paramesenteroides LC11 Lb. plantarum IMAU10014 Lb. casei AST18

Fermented cassava Koumiss Unknown

Ndagano et al., 2011 Wang, Shen, et al., 2012 Li et al., 2012

Lb. amylovorus FST2.1, Lactobacillus arizonensis R13, Lb. plantarum FST 1.7, Lb. reuteri R2, W. cibaria PS2

Cereal environment, cheese, malted barley, a, a (respectively)

Brosnan et al., 2012

Lb. reuteri ee1p


Guo et al., 2012

Lb. hammesii DSM 16381


French wheat sourdough

Black et al., 2013

Not specied.

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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Reuterin Reuterin 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 Salmonella typhimurium and E. coli K12, while it was also demonstrated to be capable of inhibiting growth of a range of moulds and yeasts, including Candida albicans and Aspergillus avus (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 various Lb. coryniformis strains resulted in a marked increase in antifungal activity towards a collection of food-spoilage fungi. Glycerol/diol dehydratase enzymes catalyze the conversion of glycerol to 3HPA. The presence of a glycerol/diol dehydratase operon ( pdu operon) in Lb. coryniformis Si3 was conrmed by PCR amplication of the pdu genes suggesting that the observed increase in inhibition was attributed to the production of reuterin with the breakdown products of glycerol 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 periods of 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 dipeptides Cyclic 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 dipeptides produced by LAB to act as antifungal agents has been demonstrated in several studies as described below. The cyclic dipeptide cyclo(glycyl-L-leucyl) was isolated from the culture ltrate 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 Kluyveromyces marxianus. Two cyclic dipeptides, cyclo(LPhe-L-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro), were shown to be responsible for the observed inhibitory activities. An MIC value of 20 mg ml1 was determined for cyclo(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 evidence 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. plantarum FST 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 acidication and temperature play an important role in the production 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 sensory attributes. Despite the fact that they are produced by a variety of lactobacilli, the modus operandi and biochemical pathways of cyclic dipeptides as antifungal inhibitors has not yet been dened. Fatty acids Fatty acids possess both antibacterial and antifungal abilities (Bergsson, Arnnnsson, 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 characteristics 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) identied, using a combination of Nuclear Magnetic Resonance (NMR), electrospray ionization mass spectrometry (ESI-MS) and gas chromatographyemass spectrometry (GCeMS), four antifungal hydroxylated fatty acids produced by Lb. plantarum MiLAB 14 as 3-(R)-hydroxydecanoic acid, 3-hydroxy-5-cis-dodecenoic acid, 3-(R)-hydroxydodecanoic acid and 3-(R)-hydroxytetradecanoic acid (Table 1). Pronounced antifungal activity was directed towards several moulds and yeasts, however, yeasts were found to be more sensitive to such hydroxylated fatty acids with reported MICs between 10 and 100 mg ml1. Elevated levels of two hydroxyl fatty acids, 3-hydroxydecanoic acid and

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

S. Crowley et al. / Trends in Food Science & Technology xx (2013) 1e17

2-hydroxy-4-methylpentanoic acid, in combination with other antifungal compounds were detected in silage inoculated with Lb. plantarum strains MiLAB 393 or MiLAB 14 (Broberg et al., 2007). 2-hydroxy-4-methylpentanoic acid was also retrieved from the concentrated cell-free supernatant (cCFS) of Lb. plantarum VE56 and Weisella paramesenteroides LC11. This fatty acid is thought to act in synergy with other inhibitory metabolites and was shown to be responsible for growth arrest of Aspergillus and Penicillium species (Ndagano, Lamoureux, Dortu, Vandermoten, & Thonart, 2011). In a recent study (Brosnan et al., 2012), six fatty acids including 3-hydroxydecanoic acid and DL-b-hydroxymyristic 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-hydroxy octadecanoic fatty acid by Lactobacillus hammesi DSM 16381, which displayed antifungal 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 of up to 2.4 g L1. It was observed that the fatty acid structure is an important factor in antifungal activity with a requirement of at least one hydroxyl group and one double bond along the carbon backbone. To date there is limited information 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 lamentous yeast Pseudozyma occulosa exhibiting inhibitory 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 uidity causes membrane permeability resulting in uncontrolled release of intracellular electrolytes and proteins, ultimately leading to cytoplasmic disintegration of fungal cells (Avis & Belanger, 2001). Proteinaceous compounds Studies concerning antibacterial proteinaceous compounds, e.g. bacteriocins, are extensive in comparison to proteins with antifungal properties, although during the last decade various LAB-derived proteinaceous compounds with anti-yeast and anti-mould abilities have been identied (Coda et al., 2008; Rizzello et al., 2011). Initial studies documented the loss of antifungal activity following treatment with proteolytic enzymes, while subsequent investigations 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. Five antifungal peptides were identied in water-soluble extracts of sourdough fermented with Lb. brevis AM7. 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 peptides were used in combination. One peptide was shown to be similar to the defensin-like protein found in pear. Furthermore, two tripeptides were shown to correspond to antihypersensitive and antimicrobial peptides contained in caseins (Coda et al., 2008). An in-depth investigation of the water/salt soluble extracts from sourdough fermented with Lb. plantarum 1A7 revealed the action of nine novel antifungal peptides having MICs between 2.5 and 10 mg ml1 (Coda et al., 2011). One of these peptides showed homology to the lantibiotic lacticin 3147. Rizzello et 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 Aspergillus japonicus were found in extracts from sourdough fermented with Lb. rossiae LD108 and Lactobacillus paralimenarius PB127 (Garofalo et al., 2012). The LD108 sourdough peptides were shown to correspond to proteolytic fragments from wheat a-gliadin. A further investigation into these antifungal peptides is critical as their mode of action in fungal inhibition has yet to be elucidated. Miscellaneous antifungal compounds Ryan et al. (2011) reported the isolation of two nucleosides with antifungal activity from the culture ltrate of Lb. amylovorus DSM 19280. Cytidine and 20 -deoxycytidine were identied from a cocktail of 17 antifungal compounds and possess MIC values > 200 mg ml1 against A. fumigatus J9. 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. Antifungal lactones from LAB were rst reported by NikuPaavola et al. (1999) when mevanolactone showed to be produced by Lb. plantarum VTT E-78076. Yang, Kim, and Chang (2011) reported the purication of d-dodecalactone produced by Lb. plantarum AF1 with associated MIC values that ranged from 350 to 6250 mg ml1 against members of the Aspergillus genus as well as P. roqueforti. d-dodecalactone is associated with fruity aromas and may

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

S. Crowley et al. / Trends in Food Science & Technology xx (2013) 1e17 Table 2. Proteinaceous antifungal compounds produced by LAB. LAB isolate(s) Lc. lactis subsp. diacetylactis DRC1 Lb. casei subsp. pseudoplantarum (commercial silage inoculant) Lb. casei DSM 20312, Lb. casei CCM1825 Lc. lactis subsp. lactis CHD-28.3 Lb. pentosus TV35b Lb. coryniformis spp. coryniformis strain Si3 Lb. paracasei subsp. paracasei strain M3 Protein responsible Peptide e sensitive to pronase E and trypsin Peptide with antimycotoxigenic properties e sensitive to trypsin and a-chymotrypsin Anitmycotoxigenic peptides sensitive to trypsin and pepsin Peptide e sensitive to chymotrypsin, trypsin and pronase E Bacteriocin-like peptide pentocin TV35b, 3.9 kDa 3 kDa, heat stable, active between pH 3.0-4.5 43 kDa, hydrophobic bacteriocin Activity spectrum A. avus A. avus Reference(s) Batish, Grover, & Lal, 1989 Gourama & Bullerman, 1995

P. citrinum, Penicillium expansum A. avus IARI, A. avus NCIM 555, Aspergillus parasiticus NCIM 898 and Fusarium spp. C. albicans Broad spectrum C. albicans NBIMCC 72, Candida blankii NBIMCC 85, Candida pseudointermedia NBIMCC 1532 strain SU Broad spectrum Broad spectrum

Gourama & Bullerman, 1997 Roy, Batish, Grover, & Neelakantan, 1996 Okkers et al., 1999 Magnusson & Schnurer, 2001 Atanassova et al., 2003

Lb. plantarum VLT01 Lb. plantarum CM8, W. confusa I5, Pediococcus pentosaceous R47, W. cibaria R16 Lb. brevis AM7 Five Lactobacillus strains

Peptide e sensitive to proteinase K, trypsin and protease CFS sensitive to proteinase K

Coloretti et al., 2007 Rouse et al., 2008

Lb. brevis NCDC 02 Lb. brevis PS1 Lb. fermentum Te007, Ped. pentosaceous Te010 Lb. plantarum NB and SDR Lb. plantarum 1A7 Lb. plantarum LB1 and Lb. rossiae LB5 Lb. plantarum IMAU10014 Lb. sakei KTU05-06, Ped. acidilactici KTU05-7, Ped. pentosaceus KTU05-8, KTU05-9 and KTU05-10 Lb. rossiae LD108, Lb. paralimentarius PB127 Lactobacillus fermentum CRL 251

Five antifungal peptides Peptide e sensitive to pepsin, trypsin, a-chymotrypsin, and proteinase K Hydrophobic peptide between 1 and 5 kDa in size Peptide e sensitive to proteinase K and pronase E Peptide e sensitive to proteinase K Peptide e sensitive to proteinase K Nine sourdough peptides Four antifungal sourdough peptides Peptide e sensitive to proteinase K and trypsin Bacteriocin-like inhibitory substances e sakacin KTU05-6, pediocin KTU05-8 KTU05-9, KTU05-10 and AcKTU05-67 Sourdough peptides Peptides e sensitive to trypsin, <10 kDa, thermostable and active in the pH range 4e7

P. roqueforti DPPMAF1 Penicillium M1

Coda et al., 2008 Voulgari et al., 2010

Broad spectrum Fusarium species A. niger Penicillium sp. P. roqueforti DPPMAF1 P. roqueforti DPPMAF1 P. roqueforti, A niger Broad spectrum

Falguni, Shilpa, & Mann, 2010 Mauch et al., 2010 Muhialdini et al., 2011 Zhao, 2011 Coda et al., 2011 Rizzello et al., 2011 Wang et al., 2012 Digaitiene, Hansen, Juodeikiene, Eidukonyte, & Josephsen, 2012

A. japonicus A. niger CH101, Penicillium sp. CH 102, F. graminearum CH 103, Geotrichium citri-aurantii INTA1 and Penicillium digitatum INTA2

Garofalo et al., 2012 Gerez, Torres, de Valdez, & Rollan, 2013

impart desirable organoleptic characteristics making it a preferred choice for food applications. Isolation, purication and identication of antifungal metabolites Antifungal compounds of LAB have previously been described as complex in nature and indeed several studies have reported the difculties 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 compounds produced under laboratory conditions may differ from those produced in food matrices and, therefore, the

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isolation of antifungal molecules should ideally be performed using the food matrix itself rather than from experimental media where possible. Advanced methodologies for the improved isolation and identication of antifungal compounds have resulted in an increase in the number of novel compounds identied over the last few years. The majority of extraction procedures enlist either liquideliquid extraction (LLE) or solid phase extraction (SPE), whereby the compounds of interest are retained in the organic fraction 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 nal identication of the compounds usually employs NMR and MS. Lavermicocca et al. (2000) reported one of the rst 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 rstly subjected to multiple LLE steps using ethyl acetate followed by thin layer chromatography (TLC) which was used for partial purication. The active fractions were subsequently identied through comparison of standard sample spectra using GC/MS. LLEbased procedures have also been used as the rst extraction step by a number of other groups working on the purication of antifungal compounds (Brosnan et al., 2012; Wang, Shen, Xiao, Zhou, & Dai, 2012; Wang, Yan, Wang, Zhang, & Qi, 2012) (Fig. 1). SPE combined with hydrophobic C18 column chromatography 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 structure elucidation were all essential parameters considered in the aforementioned assay (Sjogren, 2005). Sample preparation involved the separation of the CFS of Lb. plantarum MiLAB 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 identication was performed 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 and identication of anti-yeast and mould compounds from LAB with some variations including the introduction of recycling 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; Yang et al., 2011). An optimized method for the determination 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 microltered 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 puried by a number of methods. Okkers, Dicks, Silvester, Joubert, and Odendaal (1999) reported on the purication of a 3.9 kDa antifungal peptide using ammonium sulphate precipitation followed by cation-exchange chromatography using an Sulphopropyl (SP)-Sepharose column to obtain puried fractions. Concentrated culture broth from Lb. coryniformis Si3 was used as the starting material for peptide purication (Magnusson & Schnurer, 2001). The rst step involved ion-exchange chromatography after which the active fractions were subjected to ammonium sulphate precipitation. Dissolved pellets were then applied to a gel ltration column to reveal the estimated size of the antifungal 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 puried on a C18 HPLC system, followed by ESI-MS analysis (Atanassova et al., 2003). An alternative method was presented by Coda et al. (2008) for the extraction of sourdough-derived peptides. Water soluble extracts were rstly fractioned by ultraltration to separate the active fractions into various sizes according to the membrane cut-off. The active fractions were applied to reversedphase fast-performance liquid chromatography (RPFPLC) and fractions with antifungal activity were then separated by SDS-PAGE and identied by nano-LC-ESIMS/MS. The identied peptides were synthesized and further investigated. This separation procedure was also used to isolate antifungal sourdough peptides in subsequent studies (Coda et al., 2011; Rizzello et al., 2011). Most recently a rapid method for the detection of antifungal compounds from LAB was developed by Brosnan et al. (2012). Extracellular metabolites produced by antifungal LAB isolates were screened for the presence of antifungal compounds, and compared to known antifungal standards, by LC coupled with MS. Five isolates displaying strong inhibitory activities were thus screened and the obtained mass spec proles were then compared to that of a panel of twenty ve known antifungal metabolites, including PLA, vanillic acid and cytidine. Minimal preparation was required as the samples were either ltered and directly injected into the system, or extracted using

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Fig. 1. Chemical structures of various antifungal compounds produced by LAB.

ethyl acetate prior to injection. Separation of the individual compounds was achieved through a Gemini C18 column, while identication was performed using the linear ion trap quadrupole (LTQ) Orbitrap hybrid Fourier transform mass spectrometer (FTMS). The developed method boasts a 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 specic antifungal compounds produced. Moreover this technique was substantiated by Guo et al. (2011) to identify ten metabolites from the culture broth of Lb. reuteri ee1p targeting human pathogenic fungi such as Epidermophyton occosum. Further promise has been afforded by Watrous et al. (2012), who developed a novel method enabling metabolic proling of live colonies straight from a petri dish. The antifungal effects of Pseudomonas sp. SH-C52 were determined by applying this new approach combining nanospray desorption ESI-MS and alignment of MS data and molecular networking. Thanamycin, the mediator of antifungal activity in Pseudomonas sp. SH-C52, was detected by this methodology where it had previously remained unidentied 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 metabolites of LAB colonies to be discerned in a similar manner and represents a highly sensitive, real time, costeffective identication method. Although the number of techniques has increased, consolidated methods need to be established to improve the ease of purifying these compounds. Application of antifungal LAB as bio-control agents in food and feed systems An overview of the various food and feed applications of antifungal LAB is presented in Table 3. The global food industry sector is under constant pressure from both consumers and regulatory bodies to provide high quality fresh food with minimal processing. Consequently, research in recent years has signicantly focused on the discovery of alternative strategies to prevent food spoilage. Despite the physical and chemical barriers currently implemented to prevent food decay, the consumers preference for safe preservative-free products, 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 vegetables, bakery, dairy products and silages. In situ testing is essential to substantiate the potential application of these generally regarded as safe (GRAS) organisms as bioprotectants against fungal rot and spoilage, as well as sensory and safety involvements. Indeed, various studies have

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Fruits & vegetables Fresh 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, identied as major fungal spoilers. Sathe, Nawani, Dhakephalkar, and Kapadnis (2007) demonstrated the ability 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 of Pediococcus and Weisella isolates (Crowley, Mahony, & van Sinderen, 2012b; Lan, Chen, Wu, & Yanagida, 2012; Rouse, Harnett, Vaughan, & van Sinderen, 2008). The culture ltrate 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 of Lb. plantarum IMAU10014 (Wang et al., 2013). An enhanced antifungal-producing strain (F3C2) was generated through genome shufing and eliminated growth of Penicillium digitatum 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 products Dairy 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. Yoghurts have been primarily targeted as they are liable to yeast growth due to their low pH, storage at refrigeration temperatures and presence of fruit in certain products. A co-culture of Lb. paracasei subsp. paracasei and Propionibacterium 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 hansenii and Rhizopus mucilaginosa in yoghurts, while they did not alter the growth or acidication 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 spoilage by psychrotolerant moulds capable of withstanding low oxygen environments such as P. roqueforti. Three antifungal Lb. plantarum isolates demonstrated anti-mould capabilities when used as adjuncts during cheddar cheese production (Zhao, 2011). Furthermore, processed cheese slices and cheese shelf-life were improved after treatment with antifungal LAB (Garcha & Natt, 2011; Muhialdini, Hassan, Sadon, Zulkii, & Azfari, 2011). Use of the

Fig. 2. Flow diagram detailing the isolation and identication of antifungal compounds from LAB. 1. Antifungal metabolites derived from culture supernatant can be separated using either LiquideLiquid Extraction or Solid Phase Extraction where the organic phase contains hydrophobic compounds while the supernatant contains hydrophilic compounds. 2. Fractions are assessed by bioassay against a fungal indicator. 3. Active fractions are subsequently separated using HPLC with column of choice and this process may be repeated several times to further purify active fractions. 4. Eluted fractions are tested for antifungal activity again following chromatographic separation. 5. The structural details of the compound(s) that produce positive fractions are then identied through MS, NMR and/or GC.

demonstrated the successful application of LAB to alleviate fungal spoilage in various foods rendering them feasible substitutes or complements to chemical preservatives.

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Table 3. Application of antifungal LAB as protectants in foods and feed. Food(s) examined Fruits & vegetables Cucumber Corn Apple Soybean Fresh mango Tomato leaves Grape Pear, plum, grape Kumquat Dairy products Yoghurt, cheese Indian cheese Cheddar cheese Cheese slices Yoghurt Antifungal LAB Lb. plantarum CUK501 Lb. plantarum PTCC 1058 Ped. pentosaceous R47 Lb. plantarum AF1 Lb. acidilophidus NCDC 291 Lb. plantarum IMAU10014 W. cibaria 861006 Ped. pentosaceous 54 Lb. plantarum IMAU10014 strain F3A3 (mutant) Lb. paracasei subsp. paracasei Lb. acidilophidus NCDC 291 Lb. plantarum NB, Lb. plantarum SDR and Lb. plantarum DC2 Lb. fermentum Te007, Ped. pentosaceous Te010 Lb. harbinensis K.V9.3.1Np, Lb. rhamnosus K.C8.3.1I and Lb. paracasei K.C8.3.1Hc1 Lb. plantarum 16 (NCIMB41875) and Lb. plantarum 62 (NCIMB41876) Lb. plantarum 21B Lb. plantarum, Lb. casei and Lb. fermentum Lb. plantarum FST 1.7 Lb. plantarum FST 1.7 & 1.9 Lb. brevis AM7 Lb. buchneri FUA 3525, and Lb. diolovorans DSM 14421 Lb. plantarum CRL 778 Lb. amylovorus DSM 19280 Activity spectrum A. avus, F. graminearum, Rhizopus stolonifer, Bt. cinerea A. avus P. expansum A. avus A. alternata Bt. cinerea Penicillium oxalicum P. expansum P. digitatum KM08 Reference Sathe et al., 2007 Khanafari, Soudi, & Miraboulfathi, 2007 Rouse et al., 2008 Yang & Chang, 2010 Garcha & Natt, 2011 Wang, Yan, et al., 2012 Lan et al., 2012 Crowley et al., 2012b Wang et al., 2013

Candida species A. alternata Penicillium sp. A. oryzae, A. niger D. hansenii, R. mucilaginosa, K. marxianus, K. lactis, Yarrowia lipolytica, Penicillium brevicompactum R. mucilaginosa

Schwenninger & Meile, 2004 Garcha & Natt, 2011 Zhao, 2011 Muhialdin et al., 2011 Delavenne et al., 2012a


Crowley et al., 2012a

Breads Sourdough Sourdough Gluten free bread, wheat bread Sourdough Bread Sourdough

A. niger FTDC3227

Fusaria species A. niger, F. culmorum, P. expansum, P. roqueforti P. roqueforti DPPMAF1 A. clavatus, Cladisporium spp., Mortierella spp., S. cervisiae, P. roqueforti Penicillium sp. F. culmorum FST 4.05, A. niger FST4.21, P. expansum FST 4.22, P. roqueforti FST 4.11, bakery fungal ora Penicillium, Aspergillus and Eurotium species A. japoniucs P. roqueforti, A. niger Moulds

Lavermicocca et al., 2000 Fazeli, Shahverdi, Sedaghat, Jamalifar, & Samadi, 2004 Dal Bello et al., 2007, Moore, Dal Bello, & Arendt, 2008 Ryan, Dal Bello, & Arendt, 2008 Coda et al., 2008 Zhang et al., 2010

Bread Bread

Gerez et al., 2010 Ryan et al., 2011

Bread Bread, panettone Wheat sourdough Sangak (Traditional at bread)

Lb. plantarum 1A7 Lb. rossiae LD108; Lb. paralimentarius PB127 L. citreum H012 and W. koreensis H020 Lb. plantarum ssp. plantarum, strain ATCC 20179, Lb. acidipholus, strain ATCC 20079 and L. mesenteroides ssp. mesenteroides, strain 1591 Ped. acidilactici KTU05-7, Ped. pentosaceous KTU05-8 and Ped. pentosaceous KTU05-8 Lb. hammesii DSM 16381

Coda et al., 2011 Garofalo et al., 2012 Choi, Kim, Hwang, Kim, & Yoon, 2012 Naja, Rezaei, Safari, & Razavi, 2012



Cizeikiene et al., 2013

Sourdough Silage Barley silage

A. niger, P. roqueforti, environmental contaminants Yeasts

Black et al., 2013

Lb. buchneri

Kung & Ranjit, 2001

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S. Crowley et al. / Trends in Food Science & Technology xx (2013) 1e17 Table 3 (continued ) Food(s) examined Grass silage Wheat silage, corn silage Corn silage Maize silage Crimped wheat grains Corn silage Corn silage Maize silage Grass silage Corn silage Alfalfa silage Alfalfa silage Corn silage Corn silage Corn silage Miscellaneous foods Fermented seaweed beverage Raw smoked sausage Raw poultry meat Orange juice Rice cakes Antifungal LAB Lb. buchneri, Lb. plantarum and Ped. pentosaceous Lb. buchneri and Lb. plantarum Lb. buchneri 40788 Lb. buchneri 40788 Lb. buchneri Lb. buchneri Lb. buchneri 40788 and Ped. pentosaceous R1094 Lb. buchneri Lb. plantarum MiLAB 393 and 14 Lb. buchneri 40788 Lb. buchneri and Lb. plantarum Lb. bucnheri and Ped. pentosaceous Lb. buchneri 40788, Lb. plantarum and Ped. acidilaciti Lb. buchneri and Ped. pentosaceous Lb. buchneri LN4637 and Lb. buchneri LN40177 Lb. plantarum DW1 Lc. lactis ssp. lactis K-205 and 194 Lb. acidophilus NCDC 291 Lb. plantarum 16 (NCIMB41875) and 62 (NCIMB41876) Leuc. citreum C5, W. confusa HO24 and W. confusa D2-96 Activity spectrum Yeasts & moulds Yeasts & moulds Yeasts Yeasts Yeasts Yeasts Yeasts Yeasts Pichia anomala Moulds Yeasts Yeasts & moulds Yeasts Yeasts Yeasts Reference Driehuis et al., 2002 Weinberg et al., 2002


Taylor & Kung, 2002 Ranjit, Taylor, & Kung, 2002 Adesogan, Salawu, Ross, Davies, and Brooks (2003) Nishino et al., 2004 Kleinschmit, Schmidt, & Kung, Jr., 2005 Filya, Sucu, & Karabulut, 2006 Broberg et al., 2007 Kung, Schmidt, Ebling, & Hu, 2007 Zhang et al., 2009 Schmidt, Hu, Mills, & Kung, 2009 Reich & Kung, 2010 Schmidt & Kung, 2010 Tabacco, Piano, Revello-Chion, & Borreani, 2011 Prachyakij et al., 2008 Stoyanova et al., 2010 Garcha & Natt, 2011 Crowley et al., 2012a Baek et al., 2012

Unidentied yeasts Eurotium repens A. alternata R. mucilaginosa Cadisporium sp. YS1, Penicillium crustosum YS2, Neurosora sp. YS3

Not specied.

aforementioned isolates provides manufacturers with a natural option to the use of preservatives such as sodium benzoate, sorbic acids and natamycin in yoghurt and cheese production. Bakery products Poor 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 indepth 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 cofermentation 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 nal product was conducted to assess the

impact of Lb. plantarum 21B 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 of A. japonicus with shelf lives ranging from 11 to 32 days as compared to bread prepared with bakers yeast dough (Garofalo et al., 2012). Antifungal pediococci 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

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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 deferred growth of a number of bread-spoiling fungi, including Aspergillus clavatus and Cladosporium spp., through the accumulation of acetate and propionate (Zhang, Brandtb, Schwaba, & G anzlea, 2010). Animal feed Animal 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 dominating role (Schmidt & Kung, 2010). Oxygen may accidentally 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 dominated by the application of the hetero-fermentative Lb. buchneri. The production of acetic acid and 1,2propanediol during anaerobic degradation of lactic acid is an important factor in the preserving attributes of Lb. buchneri (Oude Elferink et 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 production from fermentation of WSC decreases pH, thereby improving forage preservation. However, preservation can be compromised as lactic acid can be oxidized by aerobic microorganisms and there is a reduced production in antifungal 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 conicting 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 of Lb. buchneri to improve forage stability (Hu, Schmidt, McDonell, Klingerman, & Kung, 2009; Weinberg et al., 2002). Lb. plantarum strains MiLAB 393 and MiLAB 14 were previously shown to have inhibitory activities towards a spectrum of fungi. Antifungal metabolites produced by these isolates in cultured broth, such as 3-PLA and 3-hydroxydecanoic acid, were also identied in silage when used as inoculants. Furthermore additional antifungal components such as azealic acid were detected in silage inoculants highlighting the potential for these strains in silage

preservation (Broberg et al., 2007). Lb. plantarum MiLAB 393 has since been patented and used as a commercial silage inoculant known as Feedtech Silage F3000. Miscellaneous foods Antifungal LAB have further promoted increased quality and shelf-life of a miscellany of other foods. Muhialdini et al. (2011) demonstrated the antagonistic effects of four LAB isolates against A. niger and Aspergillus oryzae in tomato puree. Beverages have also beneted from the application of antifungal LAB. The shelf-life of orange juice spiked with R. mucilaginosa was improved by the addition of the antifungal Lb. plantarum 16 (NCIMB41875) steep water isolate (Crowley, Mahony, & van Sinderen, 2012a), while a fermented 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. citreum C5, W. confusa HO24 and W. confusa D2-96 as antifungal rice cake starters. Limited applications of antifungal LAB in the preservation of meats exist. Interestingly, Lactobacillus acidilophidus NCDC 291 exerted a 0.4 log reduction in viable numbers of Aspergillus alternata when inoculated into raw poultry meat (Garcha & Natt, 2011). Additionally the shelf-life of raw smoked sausages was extended after application of two Lactococcus lactis ssp. lactis strains K-205 and 194 (Stoyanova, Ustyugova, Sultimova, & Bilanenko, 2010). Antifungal LABefungal interactions While all the above-mentioned studies endorse the application of antifungal LAB, little information is available about the interactions of these antifungal metabolites and their target fungal species. Antifungal metabolite target sites 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 rst attempt to gain an insight into these elusive interactions. One of the rst studies to investigate antifungal LABefungal interactions was reported by Strom, Schnurer, and Melin (2005). A co-cultivation assay was devised using Lb. plantarum MiLAB 393 and its target Aspergillus nidulans. 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 mycelial branching 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. Furthermore germ tube formation was completely restricted after treatment with 10% cCFS. Similar ndings were reported by 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 suspected 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 resulted 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 with C. albicans cells 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 wallsynthesizing 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 Scanning 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 have attempted 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 LABproduced short chain fatty acids caused interference with membrane potential and leakage of membrane contents. While both mechanisms may be responsible for the antifungal effect, current data have not allowed a rm conclusion as regards to the reasons for strain/species-specic antifungal action of LAB and further studies may well reveal additional modes of action. A relatively unexplored approach to investigate the molecular targets of the antifungal LAB-derived metabolites is by means of transcriptome analysis. Microarrays have been employed to study the transcriptional responses of a variety of fungi, such as Candida and Aspergillus species, to antifungal drugs (De Backer et al., 2001; Gautam et 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-demethylase enzyme, product of the CYP51, thus interfering with ergosterol biosynthesis (Ferreira et al., 2005).

Transcriptional proling of C. albicans in a co-culture with the probiotic strains Lb. rhamnosus GR-1 and Lb. reuteri 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 lamentous 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 understanding of antifungal LABefungal interactions. Microarray technology may thus provide an opportunity to elucidate which genes and associated metabolic or physiological 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 interactions between antifungal drugs and fungal pathogens provide an excellent basis for future work. Conclusions & future perspectives Very signicant advances in the eld of antifungal LAB have been achieved during the last decade. However, certain limitations and knowledge gaps still need to be addressed. Whilst there have been many publications on antifungal applications in recent years, just a small number of such studies have investigated nal 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 produce the compounds in situ in the food product. The latter will be a prerequisite for a full assessment of antifungal LAB application in foods, as the inhibitory metabolites or their producing LAB may alter the visual and/or organoleptic 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 consumption (Kantachote et al., 2010). Such assessments should include analysis of acquired antibiotic resistance and potential biogenic amine production in compliance with the EU qualied presumption of safety evaluation. Although in most instances sensory and safety assessments remain incomplete for a given antifungal strain, highlighting 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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S. Crowley et al. / Trends in Food Science & Technology xx (2013) 1e17 Armaforte, E., Carri, S., Ferri, G., & Caboni, M. F. (2006). Highperformance liquid chromatography determination of phenyllactic acid 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 partial biochemical characterization of a proteinaceous anti-bacteria and anti-yeast compound produced by Lactobacillus paracasei subsp. paracasei strain M3. International Journal of Food Microbiology, 87, 63e73. Avis, T. J., & Belanger, R. R. (2001). Specicity and mode of action of the antifungal fatty acid cis-9-heptadecenoic acid produced by Pseudozyma occulosa. Applied and Environmental Microbiology, 67, 956e960. Axelsson, L. T., Chung, T. C., Dobrogosz, W. J., & Lindgren, S. E. (1989). Production of a broad spectrum antimicrobial substance by Lactobacillus reuteri. Microbial Ecology in Health and Disease, 2, 131e136. Baek, E., Kim, H., Choi, H., Yoon, S., & Kim, J. (2012). Antifungal activity of Leuconostoc citreum and Weissella confusa in rice cakes. Journal of Microbiology, 50, 842e848. Batish, V. K., Grover, S., & Lal, R. (1989). Screening lactic starter cultures 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 e a review. Critical Reviews in Biotechnology, 17, 209e225. Bergsson, G., Arnnnsson, J., Steingrimsson, O., & Thormar, H. (2001). In vitro killing of Candida albicans by fatty acids and monoglycerides. Antimicrobial Agents and Chemotherapy, 45, 3209e3212. Black, B. A., Zannini, E., Curtis, J. M., & Ganzle, M. G. (2013). Antifungal hydroxy-fatty acids produced during sourdough fermentation: microbial and enzymatic pathways, and antifungal activity in bread. Applied and Environmental Microbiology, 79(6), 1866e1873. Broberg, A., Jacobsson, K., Strom, K., & Schnurer, J. (2007). Metabolite proles of lactic acid bacteria in grass silage. Applied and Environmental Microbiology, 73, 5547e5552. Brosnan, B., Coffey, A., Arendt, E. K., & Furey, A. (2012). Rapid identication, by use of the LTQ Orbitrap hybrid FT mass spectrometer, of antifungal compounds produced by lactic acid bacteria. Analytical and Bioanalytical Chemistry, 403, 2983e2995. Choi, H., Kim, Y. W., Hwang, I., Kim, J., & Yoon, S. (2012). Evaluation of Leuconostoc citreum HO12 and Weissella koreensis HO20 isolated from kimchi as a starter culture for whole wheat sourdough. Food Chemistry, 134, 2208e2216. Cizeikiene, D., Juodeikiene, G., Paskevicius, A., & Bartkiene, E. (2013). Antimicrobial activity of lactic acid bacteria against pathogenic and spoilage microorganism isolated from food and their control in wheat bread. Food Control, 31, 539e545. Coda, R., Cassone, A., Rizzello, C. G., Nionelli, L., Cardinali, G., & Gobbetti, M. (2011). Antifungal activity of Wickerhamomyces anomalus and Lactobacillus plantarum during sourdough fermentation: identication of novel compounds and long-term effect during storage of wheat bread. Applied and Environmental Microbiology, 77, 3484e3492. Coda, R., Rizzello, C. G., Nigro, F., De Angelis, M., Arnault, P., & Gobbetti, M. (2008). Long-term fungal inhibitory activity of watersoluble extracts of Phaseolus vulgaris cv. Pinto and sourdough lactic acid bacteria during bread storage. Applied and Environmental Microbiology, 74, 7391e7398. Coloretti, F., Carri, S., Armaforte, E., Chiavari, C., Grazia, L., & Zambonelli, C. (2007). Antifungal activity of lactobacilli isolated from salami. FEMS Microbiology Letters, 271, 245e250. Corsetti, A., Gobbetti, M., Rossi, J., & Damiani, P. (1998). Antimould activity of sourdough lactic acid bacteria: identication of a

Standardized isolation method-rapid, easy & reproducible Novel compounds Improved sensory analysis In situ testing Safety assessment Elucidation of inhibitory mechanism Target sites Transcriptomic approach

Isolation & Identification

Mode of action

Fig. 3. Future directions in the eld.

antifungal slurry formulated by Gerez, Torino, Obregozo, and Font de Valdez (2010) would prove far more advantageous for the food manufacturer and provides an alternative approach to meeting consumer demands. Standardization of isolation and purication 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 ultimately lead to the discovery of additional antifungal compounds. In time antifungal LAB may even replace chemical preservatives as bio-protectants in foods. As more genome sequences 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 antifungal compounds on fungi. Future efforts should also be oriented towards expanding our knowledge regarding the genetic mechanisms and metabolic pathways behind antifungal production (Fig. 3). Moreover, if the genetic machinery responsible for antifungal production is discerned this may 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 antibacterial equivalents. Acknowledgements S. 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
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