International Journal of Food Microbiology 239 (2016) 79–85

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International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Identification and quantification of antifungal compounds produced by

lactic acid bacteria and propionibacteria
Céline Le Lay a, Emmanuel Coton a, Gwenaëlle Le Blay a,1, Jean-Marc Chobert b, Thomas Haertlé b, Yvan Choiset b,
Nicolas Nguyen Van Long a, Laurence Meslet-Cladière a, Jérôme Mounier a,⁎
a
Université de Brest, EA 3882, Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, ESIAB, Technopôle Brest-Iroise, 29280 Plouzané, France
b
Institut National de la Recherche Agronomique, UR 1268 Biopolymères Interactions Assemblages, équipe Fonctions et Interactions des Protéines, B.P. 71627, 44316 Nantes Cedex 3, France

a r t i c l e i n f o a b s t r a c t

Article history: Fungal growth in bakery products represents the most frequent cause of spoilage and leads to economic losses for
Received 1 February 2016 industrials and consumers. Bacteria, such as lactic acid bacteria and propionibacteria, are commonly known to
Received in revised form 31 May 2016 play an active role in preservation of fermented food, producing a large range of antifungal metabolites. In a pre-
Accepted 19 June 2016
vious study (Le Lay et al., 2016), an extensive screening performed both in vitro and in situ allowed for the selec-
Available online 20 June 2016
tion of bacteria exhibiting an antifungal activity. In the present study, active supernatants against Penicillium
Keywords:
corylophilum and Aspergillus niger were analyzed to identify and quantify the antifungal compounds associated
Moulds with the observed activity. Supernatant treatments (pH neutralization, heating and addition of proteinase K) sug-
Antifungal gested that organic acids played the most important role in the antifungal activity of each tested supernatant. Dif-
Lactic acid bacteria ferent methods (HPLC, mass spectrometry, colorimetric and enzymatic assays) were then applied to analyze the
Germination supernatants and it was shown that the main antifungal compounds corresponded to lactic, acetic and propionic
Growth acids, ethanol and hydrogen peroxide, as well as other compounds present at low levels such as phenyllactic,
hydroxyphenyllactic, azelaic and caproic acids. Based on these results, various combinations of the identified
compounds were used to evaluate their effect on conidial germination and fungal growth of P. corylophilum
and Eurotium repens. Some combinations presented the same activity than the bacterial culture supernatant
thus confirming the involvement of the identified molecules in the antifungal activity. The obtained results sug-
gested that acetic acid was mainly responsible for the antifungal activity against P. corylophilum and played an
important role in E. repens inhibition.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction As stated above, fungal contamination is suppressed through baking,

In the bakery product context, moulds are responsible for the spoil-
age of a variety of products otherwise poorly susceptible to spoilage or
pathogen microorganism contaminations due to their preparation (bak-
ing treatment) and intrinsic characteristics (low water activity) (Le Lay
et al., 2016). These fungal contaminations are responsible for substantial
economic losses and may also impact the brand image (Pitt and
Hocking, 2009). The most common fungi isolated from bakery products
belong to the Penicillium, Aspergillus, Eurotium but also Mucor,
Cladosporium and Wallemia genera (Legan, 1993; Le Lay et al., 2016;
Poute and Tsen, 1987).
⁎ Corresponding author at: Laboratoire Universitaire de Biodiversité et Ecologie
Microbienne, Parvis Blaise-Pascal, Technopôle Brest-Iroise, 29280 Plouzané, France.
E-mail address: jerome.mounier@univ-brest.fr (J. Mounier).
1
Present address: Laboratoire de Microbiologie des Environnements Extrêmes (LMEE),
Université de Bretagne Occidentale, Institut Universitaire Européen de la Mer (IUEM),
UMR 6197 (UBO, CNRS, IFREMER), Place Nicolas Copernic, Technopôle Brest-Iroise,
29280 Plouzané, France.
but bakery products can be contaminated after this step via spores pres-
ent in the factory air, during cooling, slicing or storage step (Legan,
1993). Therefore, the control of these contaminants is of great impor-
tance. Up to now, different methods or combinations of methods (hur-
dle technologies) have been used to protect food products and extend
their shelf-life. These methods include air treatment, modified atmo-
sphere packaging and addition of chemical preservatives (propionate,
sorbate, ethanol) (Schnürer and Magnusson, 2005). Regarding the latter
compounds, consumers are more and more requesting preservative-
free products and therefore natural preservation alternatives, biopres-
ervation being one that was particularly explored as recently reviewed
by Axel et al. (2016b).
Lactic acid bacteria (LAB) and propionibacteria are good candidates
for biopreservation as they have a long history of safe use. LAB are
used in various fermented food like dairy (yogurt, kefir or cheese) and
bakery products (sourdough) but also in non-fermented products
such as meat, vegetables and seafood (Buckenhüskes et al., 1997;
Leistner, 1995; Saithong et al., 2010). Propionibacteria are used as
probiotics (Mantere-Alhonen and Makinen, 1987), as well as ripening

http://dx.doi.org/10.1016/j.ijfoodmicro.2016.06.020
0168-1605/© 2016 Elsevier B.V. All rights reserved.
80 C. Le Lay et al. / International Journal of Food Microbiology 239 (2016) 79–85
cultures in the production of Swiss type cheese (Biede and Hammond,
1979). During their fermentation, bacteria produce a large spectrum of
compounds in food matrices; these metabolites play an important role
in the product organoleptic properties but also in preservation
(Caplice and Fitzgerald, 1999). Bacterial fermentation generally induces
a rapid matrix acidification, due to the production of organic acids, such
as lactic, acetic or propionic acids, which play a role in the biopreserva-
tion of fermented foods (Batish et al., 1997; Leroy and De Vuyst, 2004).
Regarding the antifungal activity of bioprotective cultures, it often re-
sults from the synergistic effect of different molecules, organic acids
not being the only known active molecules. Indeed, other molecules
such as fatty acids (Bergsson et al., 2001, Sjogren et al., 2003), reuterin
(Axelsson et al., 1989; Magnusson et al., 2003), cyclic dipeptides
(Niku-Paavola, 1999; Strom et al., 2002), and proteinaceous compounds
(Coda et al., 2008; Rizzello et al., 2011) may be involved in antifungal
activity.
In this study, we characterized the nature of antifungal compounds
present in culture supernatants that previously showed an efficient in-
hibition, both in vitro and in situ, of bakery fungal contaminants (Le
Lay et al., 2016). In parallel, different biochemical methods were used
to look for known antifungal metabolites present in active supernatants.
Finally, combinations of the identified antifungal metabolites were eval-
uated to verify if these molecules were indeed responsible for this
activity.
2. Materials and methods
2.1. Bacterial strains and culture conditions
In this study, 6 bacteria were selected based on a previous study (Le
Lay et al., 2016). Four LAB (Leuconostoc citreum L123, Lactobacillus brevis
Lu35, Lactobacillus reuteri 5529 and Lactobacillus spicheri O15) and one
propionibacteria (Propionibacterium freudenreichii LSaci68), all
exhibiting antifungal activities in vitro and in situ, were studied. A nega-
tive control, corresponding to a LAB without antifungal activity (Lacto-
bacillus casei Lu53), was tested in parallel. Bacteria were grown in
wheat flour hydrolysate (WFH) broth as previously described (Le Lay
et al., 2016). After incubation at 30 °C for 48 h or 72 h for LAB and
propionibacteria, respectively, supernatants were obtained after centri-
fugation for 10 min at 8422 g and filtration on 0.2 μm pore size PTFE fil-
ter (VWR, France).
2.2. Nature of antifungal compounds
Antifungal activity of supernatant was confirmed by pouring in
Petri dishes a mix of supernatants obtained after centrifugation and
filtration with a 4 × concentrated agar solution (75%/25%, v/v).
After polymerization, a top layer of PDA medium (Potato Dextrose
Broth supplemented with 0.7% agar) was added. Then, 50 conidia
of Penicillium corylophilum UBOCC-A-112081 or Aspergillus niger
UBOCC-A-112064 (UBO Culture Collection, Plouzané, France), both
isolated from spoiled bakery products (Le Lay et al., 2016), were in-
oculated in the middle of the Petri dishes and incubated at 25 °C for
7 days. Radial fungal growth was measured every day, with 2 per-
pendicular measures.
Preliminary characterization of the compounds harboring anti-
fungal activities was performed using a double layer activity test as
described above by using supernatants that endured the following
treatments: i) neutralization (at pH 7 using 2 M NaOH), ii) heat
treatment (90 °C or 120 °C for 20 min) and iii) proteinase K treat-
ment. For the last treatment, supernatants were neutralized at pH 7
followed by digestion with 10 mM of proteinase K for 2 h at 37 °C;
at the end of reaction, pH was readjusted to its initial value by
using 5 M HCl.
2.3. Quantification of lactic, acetic and propionic acids
Supernatants were centrifuged for 15 min at 7500 g at 4 °C and half
diluted with sulfuric acid (0.02 N) then frozen at −20 °C during at least
2 h for protein precipitation. Samples were centrifuged to eliminate the
precipitate and were filtered on PVFD 0.45 μm filter. Acetic, lactic
and propionic acids were analyzed after injection of 10 μL of the
obtained solution in a HPLC Dionex system with an Aminex-A column
(Bio-Rad). Organic acid detection was performed at 210 nm using a
UV/Visible and a refractometer detectors as described by Thierry et al.
(2002).
2.4. Ethanol quantification
Ethanol was quantified with gas chromatography using the internal
standard method. Supernatants were first directly injected into the Gas
Chromatograph (GC 3900 Varian) equipped with a Chrompack Capillary
Column (CP-Sil 8 CB LB/MS CP 8752, 30 m × 0.32 mm, 0.25 μm) for
ethanol concentration estimation.
For quantification, 0.02%, 0.05% and 0.2% ethanol standards were
prepared and mixed with isopropanol at the same concentration. The
same protocol was applied to the tested samples: 500 μL of sample
were mixed (v/v) with isopropanol at the same concentrations. Analy-
ses were performed on 1 μL of each mix. Three biological replicates
and 3 technical replicates per biological replicate were performed.
2.5. Hydrogen peroxide quantification
These analyses were performed using the OxiSelect Hydrogen Per-
oxide Assay Kit (Cell Biolabs, Inc., San Diego, USA) following the
manufacturer's instructions. A solution at 8.8 mM was first prepared
using a stock solution of hydrogen peroxide diluted at 1:1000 in WFH
broth. Then, H2O2 standard solutions with concentrations ranging
from 0 μM to 50 μM were prepared by dilution in WFH medium. Plates
were read at 595 nm using a Multiskan FC Microplate Photometer
(Thermo Scientific). Sample absorbances were compared to a standard
curve and H2O2 concentrations were determined.
2.6. Reuterin quantification
Reuterin was measured using the colorimetric method of Circle et al.
(1945). Acrolein (Aldrich) standard solutions ranging from 0.05 to
6 mM were prepared in WFH broth medium. To 1 mL of each sample
and standard solution, 0.75 mL of a 10 mM tryptophan solution (dis-
solved in 0.05 M HCl) was added. After homogenization, the mix was in-
cubated at 37 °C for 20 min. The absorbance was recorded at 560 nm
using a Genesys 10S UV–Vis spectrophotometer (Thermo scientific).
Sample concentrations were determined using linear response of
acrolein.
2.7. Antifungal compounds identification by LC-QToF
2.7.1. Standard preparation
Twenty-six compounds (noted A to Z) described in the literature for
their antifungal properties (Brosnan et al., 2012) were used (Table 1).
Stock solutions at 2 mg/mL were prepared and stored at − 20 °C. In
parallel, a stock solution corresponding to a mix of the 26 compounds
(at 100 μg/mL each) was prepared by diluting the stock solutions with
H2O/acetonitrile (ACN) (90/10, v/v) into amber vials. Standard mixes
were prepared at different concentrations, i.e., 1, 5, 10, 30 and 50 ppm,
by dilutions of the standard stock solution with H2O/ACN (90/10, v/v).
2.7.2. Sample preparation
Bacterial supernatants were extracted using the QuEChERS disper-
sive SPE (dSPE) (Agilent Technologies) as described by Brosnan et al.
(2014). Briefly, for each sample, 10 mL of supernatant were mixed in
 C. Le Lay et al. / International Journal of Food Microbiology 239 (2016) 79–85 81

Table 1 solvent gradient (A: H2O + 10 mM ammonium formate + 0.1% formic

Name, chemical formula, theoretical mass and detected mass for each tested antifungal acid, B: ACN + 0.1% formic acid) as described in Table S1.
standard.

Chemical [M-H]− [M-H]− 2.8. Antifungal activity of natural and artificial supernatants on conidial
Antifungal compound formula theoretical m/z found m/z germination and radial growth
A D-Glucuronic acid C6H10O7 193.03468 193.0356
B Cytidine C9H13N3O5 242.07726 242.0786 To better identify the compounds supporting antifungal activity, a
C 2-Deoxycitidine C9H13N3O4 226.08263 226.9658 highly antifungal supernatant obtained after 48 h growth of L. citreum
D Succinic acid C4H6O4 117.09 117.0901
L123 in WFH medium was selected. In parallel, 4 different “artificial su-
E DL-ρ-Hydroxyphenyllactic acid C9H10O4 181.04997 181.059
pernatants” were created by mixing, at the same concentrations than
F 1,2-Dihydroxybenzene C6H6O2 109.02908 109.0294
G 4-Hydroxybenzoic acid C7H6O3 137.0238 137.0245 those observed in the supernatant, the different potential antifungal
H 3,4-Dihydroxyhydrocinnamic acid C9H10O4 181.05 181.0509 molecules identified in L. citreum L123 supernatant. The first mix
I Vanillic acid C8H8O4 167.03443 167.035 contained lactic and acetic acids, ethanol, H2O2 and azelaic, phenyllactic,
J Caffeic acid C9H8O4 179.03436 179.0348
hydroxyphenyllactic and hydroxyisocaproic acids at 2.94 g/L, 2.35 g/L,
K (S)-(−)-Hydroxyisocaproic acid C6H12O3 131.07086 131.0712
L 3-(4-Hydroxyphenyl)propionic acid C9H10O3 165.05513 165.056
0.017%, 0.5799 μM, 1.88 μg/mL, 6.70 μg/mL, 1.03 μg/mL and 7.26 μg/mL,
M Phenyllactic acid C10H12O3 165.05517 165.0556 respectively. The second mix contained the first 4 molecules. The third
N ρ-Coumaric acid C9H8O3 163.03952 163.04 mix only contained the last four molecules, while the last mix contained
O 3-(4-Hydroxy-3-methoxyphenyl) C10H12O4 195.06554 195.066 all molecules except acetic acid. Physiological water was used as control.
propanoic acid
The different mixes were prepared and the pH adjusted to 3.5 with
P Salicylic acid C7H6O3 137.02387 137.0243
Q Ferulic acid C10H10O4 193.05008 193.0509 1 M HCl (the same pH than in the L. citreum L123 supernatant after
R Methylcinnamic acid C10H10O2 161.06025 161.0453 48 h incubation at 25 °C in WFH medium) and solutions were sterilized
S Benzoic acid C7H6O2 121.02895 121.0296 on 0.2 μm filters.
T Azelaic acid C9H16O4 187.09703 187.0975
To evaluate the effect on conidial germination, the artificial superna-
U Hydrocinnamic acid C9H10O2 149.06025 149.0609
V 3-Hydroxydecanoic acid C10H20O3 187.13342 187.1339
tants (29.1 mL) were mixed with 4 × concentrated agar (9.4 mL) and
W DL-β-Hydroxylauric acid C12H24O3 215.16472 215.1654 poured into Petri dishes. After polymerization, a second layer of
X 2-Hydroxydodecanoic acid C12H24O3 215.16472 215.1653 12.5 mL PDA medium (PDB + 0.7% agar) was added. The Petri dishes
Y Decanoic acid C10H20O2 171.1385 171.1391 were then inoculated uniformly with 100 μL of a conidia suspension at
Z DL-β-Hydroxymyristic acid C14H28O3 243.19602 243.1965
105 conidia/mL of either P. corylophilum UBOCC-A-112081or E. repens
UBOCC-A-112,075. The same experiment was performed with L. citreum
L123 supernatant (positive control) and with physiological water (neg-
50 mL Falcon tube with 10 mL of ethyl acetate and 1% formic acid ative control).
(Sigma Aldrich), 1 g of NaCl and 4 g of MgSO4 (Carlo Erba). The mixture Petri dishes were incubated at 25 °C and germinated and non-germi-
was shaken for 1 min and centrifuged for 10 min at 9000 rpm The top nated conidia were counted by phase-contrast microscopy (at 40 ×
organic phase was recovered and added to the dSPE (150 mg C18, magnitude) every 3 h using a constant reading frame of 1 mm by
900 mg MgSO4) (10 mL) and shacked for 1 min. After centrifugation 25 mm and without opening the Petri dish (Nguyen Van Long, personal
for 10 min at 5000 rpm, the liquid phase was transferred into a glass communication). Conidia were considered germinated when the length
tube containing 100 μL of DMSO and dried under N2. of a germinating tube was superior to the diameter of the conidia. Three
Samples were finally reconstituted with 900 μL of H2O/ACN (90/10, counts per Petri dishes and 2 replicates per sample were performed.
v/v) and filtered on 0.2 μm pore size PTFE filter (Whatman) into 2 mL Germination time (τ) and Pmax, which respectively represents the
amber vials. time (expressed in h) at which 50% of conidia have germinated and
the maximum percentage of germinated conidia were calculated as pre-
2.7.3. Method validation viously described (Dantigny et al., 2011).
To validate the method, 3 calibration tests were performed in order To study the effects of supernatant on fungal growth, a
to highlight the medium effect and to determine the extraction recov- similar experimental design was used except that 50 conidia of either
ery. Two types of calibration curves were performed to test the medium P. corylophilum UBOCC-A-112081 or E. repens UBOCC-A-112075 were
effect. The first one corresponded to a mixture of antifungal compounds inoculated in a central spot. Fungal radial growth at 25 °C was measured
at different concentrations (1, 5, 10, 30 and 50 ppm, all compounds being every day, with 2 measures per sample, for 7 days and the growth rate
at the same concentration in each mix) in H2O/ACN (90/10, v/v). The sec- (expressed in mm/day) was determined.
ond one corresponded to the same standard mixes diluted in WFH medi-
um with pH adjusted to 3.5 with lactic acid (Fluka). For recovery 3. Results and discussion
determination, calibration curves were obtained using the same standard
mixture of 26 antifungal compounds in WFH medium, using ethyl 3.1. Antifungal activity in supernatant
acetate and the QuEChERS dSPE kit as used for the supernatant. A
control for WFH medium was obtained by extracting in the same condi- The bacterial strains selected for this study (L. citreum L123, L. brevis
tions non-inoculated WFH medium just after acidification at pH 3.5 Lu35, L. reuteri 5529, L. spicheri O15 and Propionibacterium freudenreichii
with lactic acid. LSaci68) were those exhibiting antifungal activities of interest against
different bakery product fungal contaminants after a sequential screen-
2.7.4. LC-QToF analysis ing process (Le Lay et al., 2016). The screening corresponded to an in
Detection and quantification of antifungal compounds were per- vitro screening of the antifungal activity of 270 lactic acid bacteria and
formed on a 1260 Infinity binary HPLC combined with a 6530 Accurate 50 propionibacteria followed by in situ tests corresponding to the appli-
Mass LC-QToF LC/MS (Agilent Technologies). Ten microliters of sample cation of the most effective bacteria, after optimization, either by milk
were injected into a Zorbax Extend-C18 column (201 × 150 mm, 5 μ) bread roll and pound cake surface spraying or incorporation in milk
equipped with a Zorbax Extend-C18 pre-column (2.1 × 12.5 mm, 5 μ) bread sourdough. These tested fungal contaminants, namely Aspergillus
(Agilent Technologies) at 30 °C with 0.1% formic acid and 10 mM am- niger (UBOCC-A-112064), Cladosporium sphaerospermum (UBOCC-A-
monium formate. It was operated in negative electrospray ionization 112116), Eurotium repens (UBOCC-A-112075), Penicillium corylophilum
mode. Separation of compounds was performed thanks to a binary (UBOCC-A-112081) and Wallemia sebi (UBOCC-A-112090) were
82 C. Le Lay et al. / International Journal of Food Microbiology 239 (2016) 79–85
selected base on their prevalence and occurrence on milk bread rolls (a
bakery product produced using a sourdough) and pound cakes (Le Lay
et al., 2016). However, the antifungal activities were observed for the
entire bacterial culture, which means in presence of the bacterial cells.
The first part of this study then consisted in evaluating whether the cor-
responding supernatants (culture depleted of the bacterial cells) still
harbored antifungal activities. The evaluation was performed on P.
corylophilum UBOCC-A-112081 and A. niger UBOCC-A-112064 as the
previous study by Le Lay et al. (2016) showed that very few isolates
were able to inhibit these strains and therefore that they were among
the most difficult to inhibit in the considered context. The performed
experiments showed that supernatants still exhibited antifungal activi-
ties against both fungi in comparison to the 2 negative controls, non-in-
oculated WFH medium, and L. casei Lu53 (with no antifungal activity)
supernatant. However, in comparison to the entire cultures, lower anti-
fungal activities were observed. Indeed, while complete inhibitions
were observed after 7 days in the previous study (Le Lay et al., 2016),
a growth delay of about 3 days according to the considered species
was observed in the present study. The most active supernatants against
the two fungal targets were those of L. brevis Lu35 and L. citreum L123.
Regarding the radial growth of fungal target in presence of superna-
tant, the less active were those of P. freudenreichii LSaci68 and L. reuteri
5529 (data not shown). The difference in antifungal activities between
the cultures and the supernatants may be explained by i) the fact that
part of the antifungal activity could be associated with the cells or ii)
the ¼ dilution factor due to the addition of concentrated agar. Neverthe-
less, the obtained results indicated that bacterial molecules present in
the supernatants were supporting the observed antifungal activities.
These results are corroborated by Corsetti et al. (1998) and Ryan et al.
(2011), who showed that the respective antifungal activities of Lactoba-
cillus sanfranciscensis CB1 and Lactobacillus amylovorus DSM 19280
were due to organic acids produced in the medium and that these activ-
ities were still present in cell-free supernatants.
3.2. Nature of active molecules
According to the literature, antifungal activity can result from a large
panel of active molecules of various chemical families (e.g. organic acids
including carboxylic acids, proteinaceous compounds, fatty acids, etc.)
(Crowley et al., 2013). In order to characterize the nature of the mole-
cules harboring the antifungal activity in the studied supernatants,
different treatments were performed. Proteinase K treatment is classi-
cally used to determine if an antimicrobial compounds is of a proteina-
ceous nature. Heat treatment is also used to evaluate the heat stability of
the molecules. pH neutralization is used to evaluate if the activity is as-
sociated with a pH or organic acid effect.
The antifungal activity of each bacterial supernatant treated or not
with proteinase K was the same, suggesting that proteins were not in-
volved in the antifungal activity. As for heat treatments, all supernatant
antifungal activities were affected by heating (90 °C, 20 min), except for
L. citreum L123 activity against P. corylophilum UBOCC-A-112081 that
was conserved; nevertheless, this activity was lost after heating at
120 °C for 20 min. This suggested that the antifungal activities were sup-
ported at least partly by heat sensitive molecules. Finally, after pH neu-
tralization, all treated supernatants were inactive against the 2 fungal
targets as the observed fungal diameters were similar to those of the
negative control. Overall, the obtained results suggested that organic
acids supported the majority of the observed antifungal activity against
P. corylophilum UBOCC-A-112081 and A. niger UBOCC-A-112064. In
order, to evaluate which molecules with antifungal potential were in-
deed present in the supernatant, a number of compounds were targeted
for detection and quantification analyses. Organic acids include a large
spectrum of compounds, and several of them are known to be efficient
antifungal compounds and to be produced by LAB and propionibacteria
(Crowley et al., 2013).
3.3. Quantitative analysis of antifungal molecules in supernatants
Beyond organic acids, other molecules exhibiting antifungal activi-
ties and potentially produced by LAB or propionibacteria may explain
the observed results, among those reuterin, H2O2 and ethanol are of
special interest. Concerning reuterin, corresponding to a mixture of
monomeric, hydrated monomeric and cyclic dimeric forms of 3-
hydroxypropionaldehyde (Talarico and Dobrogosz, 1989), this com-
pound has been shown to be a broad-spectrum antimicrobial substance
(Cleusix et al., 2007; Langa et al., 2014). However, in the tested condi-
tions, reuterin was not detected in any of the antifungal supernatants,
including that of L. reuteri 5529. Noteworthy, Guo et al. (2012) showed
that reuterin was undetectable in 4 lactobacilli strains (including a
strain of L. reuteri) exhibiting antifungal activities against Microsporum
canis, Microsporum gypseum and Epidermophyton floccosum.
As for hydrogen peroxide, an oxidative agent also known for its an-
timicrobial activity, which has been shown to affect food-borne patho-
genic bacteria (Ito et al., 2003) and fungi (Lindgren and Dobrogosz,
1990), H2O2 was detected in all samples including in the negative con-
trol. The observed quantities were similar and ranged from 0.5181 to
0.5799 μM, L. citreum L123 being the highest producer (Table 2).
Ethanol, a basal metabolite in strictly heterofermentative LAB, is
commonly used as a preservative in the bakery industry and its antifun-
gal activity has been demonstrated (Dantigny et al., 2005; Dao et al.,
2010). Its presence was observed in each sample with the exception
of the negative control L. casei Lu53 (facultative heterofermentative)
and P. freudenreichii LSaci68. In the positive supernatants, ethanol con-
centrations ranged from 0 to 0.16% (Table 2), the highest concentration
being associated to L. spicheri O15 while L. citreum L123, one of the most
active antifungal strains showed a relatively low production. Although
hydrogen peroxide and ethanol were produced by basically all the test-
ed bacteria, their levels were rather low and therefore suggested a weak
direct involvement in the antifungal activity. However, it should be
noted that synergistic effects may exist between molecules at low
concentrations.
Lactic, acetic and propionic acids correspond to the main organic
acids known to be produced by LAB and/or propionibacteria as part of
their primary metabolism, and to be involved in antifungal activity
(Corsetti et al., 1998; Dal Bello et al., 2007; Schwenninger et al., 2008,
Huh and Hwang, 2016). As expected, lactic acid was found in each test-
ed LAB supernatant. Lactic acid was quantified in a range of 2.47 to
8.51 g/L for the supernatants with antifungal activity. Noteworthy, the
supernatant of the negative control L. casei Lu53 presented a lactic
acid concentration of 5.07 ± 1.62 g/L. The fact that a non-antifungal
strain produced lactic acid at a concentration higher than the best anti-
fungal strain suggests that this molecule is not directly responsible for
the observed antifungal activities. This is in agreement with the results
of Dagnas et al. (2015) who showed that lactic acid alone had a weak in-
hibitory effect against the same fungal strains. Acetic acid was detected
in each bacterial supernatant and its concentration ranged between
0.005 ± 0.005 g/L and 2.35 ± 0.255 g/L for the negative control and
the most active strain (L. citreum L123), respectively. Contrary to lactic
acid, the yield of acetic acid production seemed to be correlated to the
observed antifungal activity. Acetic acid has a higher pKa (pKa =
4.75) than lactic acid (pKa = 3.86) and was therefore at a much higher
concentration in its non-dissociated form; this form is able to cross the
membrane and dissociates into the cell causing an acidic stress (Batish
et al., 1997; Dang et al., 2009). Interestingly, the simultaneous presence
of acetic and lactic acids was shown to have a synergistic effect against P.
corylophilum and E. repens (Dagnas et al., 2015).
As expected, propionic acid was only found in P. freudenreichii
LSaci68 with a concentration of 2.14 g/L that was consistent with its
ability to ferment sugar and lactic acid to produce propionic and acetic
acids. However, in the tested conditions, P. freudenreichii LSaci68 super-
natants possessed a low inhibitory activity. This reduced antifungal ac-
tivity may be related to the low concentrations of acetic and propionic
 C. Le Lay et al. / International Journal of Food Microbiology 239 (2016) 79–85 83

acids. It could be interesting to optimize the medium composition, for

Cytidine
example by addition of lactic acid in order to increase the production

+
+
+
+
of these antifungal metabolites.
In this study, we also searched for 26 antifungal compounds report-
ed in the literature (Brosnan et al., 2012). Standard mixes were pre-

Vanillic
pared in WFH medium, and a HPLC method was validated for their

acid

+
+
+
+
detection (Fig. S1). The QuEChERS purification method used on the an-
DL-ρ-hydroxyphenyl lactic tifungal supernatants (Brosnan et al., 2014) and the rapid method used
for their analysis (LC-QToF) allowed to investigate a large range of
antifungal compounds. Among the 26 targeted compounds, 6 molecules
were detected in the supernatants (azelaic acid, phenyllactic acid, (S)-
1.03 ± 0.32d
(−)-2-hydroxyisocaproic acid, DL-ρ-hydroxyphenyllactic acid, vanillic

6.21 ± 1.05a
3.92 ± 0.32b

0.10 ± 0.03e

0.13 ± 0.01e
1.98 ± 0.19c
acid and cytidine) but only the first 4 were quantified (Table 3).
The highest concentrations of azelaic, phenyllactic and DL-ρ-
mg/L

hydroxyphenyllactic acids were observed in L. spicheri O15 supernatant

acid

(2.71 ± 0.22 μg/mL, 22.04 ± 2.04 μg/mL and 6.21 ± 1.05 μg/mL, respec-
tively). The highest concentration of (S)-(−)-2-hydroxyisocaproic acid
(S)-(−)-2-hydroxyiso-caproic

was detected in L. reuteri 5529 supernatant.

These organic acids have been highlighted in previous studies as an-
tifungal compounds produced by LAB and therefore could be of interest
to extend bakery product shelf-life (Axel et al., 2016a, b). Dal Bello et al.
(2007), Lavermicocca et al. (2000) and Magnusson et al. (2003) report-
10.25 ± 0.40ab

10.92 ± 1.96ab
12.73 ± 3.73a
7.26 ± 4.73bc

5.63 ± 1.91c

ed the production of phenyllactic acid in antifungal lactobacilli isolated

from different sources. This compound was active against several Asper-
mg/ L

gillus, Eurotium and Penicillium species and its minimal inhibitory con-
acid

0d
Detection and quantification of potential antifungal compounds in antifungal supernatants (values in bold correspond to the highest observed values).

centration (MIC) against A. niger at pH 4 was 20 mg/mL (Lavermicocca

et al., 2003) while it was 2.5 mg/mL for Aspergillus fumigatus (Ryan et
⁎ Within a same column, means with different letters are significantly different according to a Fisher's least significant difference test (p b 0.05).
0.34 ± 0.08d

0.37 ± 0.05d
6.70 ± 2.01b

6.15 ± 1.23b
2.71 ± 0.22 a 22.04 ± 2.04a
3.65 ± 0.52c
Phenyllactic

al., 2011). Interestingly, the recent study by Valerio et al. (2016) showed
that the addition of phenylpyruvic acid, a precursor of phenyllactic acid,
mg/L

to the growth medium contributed to improve the antifungal activity of

acid

LAB strains. Phenyllactic acid, azelaic acid, DL-ρ-hydroxyphenyllactic

1.92 ± 0.02 a
0.29 ± 0.27b
2.09 ± 0.56a

1.88 ± 0.33a

2.05 ± 1.31a

acid, (S)-(−)-2-hydroxyisocaproic acid and vanillic acid have previous-

Azelaic acid

ly been identified in L. reuteri supernatant able to inhibit fungal derma-

mg/L

tophytes as well as Aspergillus niger and Aspergillus fumigatus (Guo et al.,

2012). Cytidine was also produced by a Lactobacillus amylovorus anti-
fungal LAB (Ryan et al., 2011). However, cytidine was found to be poorly
effective against A. fumigatus with a MIC at pH 4 N 200 mg/mL (Ryan et
0.53 ± 0.00b
0.58 ± 0.00c
0.52 ± 0.00a

0.52 ± 0.00a
0.52 ± 0.00a
0.52 ± 0.00a

al., 2011). Recently, Honoré et al. (2016) showed that formation of six 2-
Hydrogen
peroxide

hydroxy acids by L. paracasei strains, combined with glucose consump-

tion and acidification by lactic acid, was mainly responsible for minimal
μM

Penicillium spp. growth. It is worth noting that some concentrations re-

0.13 ± 0.02ab

ported in the present study for these compounds were higher in the
0.16 ± 0.09a
0.01 ± 0.02b

negative control than those produced by some active strains, suggesting

0.017 ±
Ethanol

that these compounds played a minor role in the observed antifungal

0.01b
0b

0b
%

activity. However, because these compounds can act synergistically, it

would be interesting to evaluate their MICs in combination with various
2.14 ± 0.06a

concentrations of lactic and acetic acids.

Propionic
acid
g/L

0
0
0

3.4. Antifungal activity of natural and artificial supernatants on conidial

0.99 ± 0.015c
Acetic acid g/L

0.53 ± 0.02d
1.68 ± 0.03b
1.74 ± 0.40b
2.35 ± 0.25a
0.005 ± 0.00e

germination and radial growth

In order to better characterize the involvement of the antifungal

compounds found in the active supernatants, we compared the effect
of the most active supernatant (L. citreum L123) with 4 artificial super-
2.94 ± 0.02bc

3.82 ± 0.17b
5.07 ± 1.62a

2.61 ± 0.01c
2.47 ± 0.29c

natants (consisting of various compounds mixtures at the concentra-

Lactic acid

tions and pH observed in L. citreum L123 supernatant) on conidial

germination and radial growth of P. corylophilum UBOCC-A-112081
g/L⁎

0d

and E. repens UBOCC-A-112075. These fungal strains were selected,

based on the previous study by Le Lay et al. (2016), as they exhibited
(negative control)

contrasted sensibilities during the in vitro antifungal activity screening.

P. freudenreichii
L. citreum L123

As stated previously, P. corylophilum was only inhibited by a small num-

L. spicheri O15
L. reuteri 5529
L. brevis Lu35
L. casei Lu53

ber of strains, while E. repens was one of the most frequently inhibited
Lsaci68

fungi. Moreover, L. citreum L123 culture significantly delayed E. repens

Strain
Table 2

growth but not that of P. corylophilum when applied by spraying on

milk bread roll surface but, when incorporated in the sourdough,
84 C. Le Lay et al. / International Journal of Food Microbiology 239 (2016) 79–85

Table 3
Effect of natural and artificial supernatants on the maximum percentage of germinated conidia (Pmax), germination time (τ), growth rate (μ) and diameter after 7 d incubation in P.
corylophilum and E. repens.

Tested supernatants⁎

1 2 3 4 5 6

Penicillium corylophilum
Pmax (%) 96.5 ± 0.50a 97.8 ± 1.19a 97.6 ± 0.26a 97.6 ± 2.75a 96.5 ± 0.98a 96.7 ± 3.81a
τ (h) 14.9 ± 0.54b 18.1 ± 0.37a 18.2 ± 0.76a 17.4 ± 0.99a 13.4 ± 0.44b 13.7 ± 0.78b
μ (mm/d) 2.20 ± 0.14b 1.75 ± 0.18c 1.35 ± 0.16d 1.625 ± 0.21cd 2.25 ± 0.12b 2.70 ± 0.19a
Diameter after 7 d (cm) 2.18 ± 0.07b 1.80 ± 0.22c 1.45 ± 0.17d 1.70 ± 0.26cd 2.13 ± 0.12b 2.47 ± 0.08a

Eurotium repens
Pmax (%) 100 ± 1 × 10–7a 8.9 ± 2.59c 14.2 ± 6.65c 8.2 ± 1.86c 94.9 ± 8.85a 38.3 ± 5.45b
τ (h) 30.4 ± 1.65a NR NR NR 24.1 ± 6.03a NR
μ (mm/d) 1.0 ± 0.07a NG NG NG 1.1 ± 0.04a NG
Diameter after 7 d (cm) 1.1 ± 0.24a NG NG NG 1 ± 0.06a NG

NR: the population of germinated conidia never reached 50% even after 80 h of incubation.
NG: no growth.
⁎ 1, Physiological water at pH 3.5, 2, Leuconostoc citreum L123 supernatant, 3, lactic and acetic acids, ethanol, H2O2 and azelaic, phenyllactic, hydroxyphenyllactic and hydroxyisocaproic
acids, 4, lactic and acetic acids, ethanol, H2O2, 5, azelaic, phenyllactic, hydroxyphenyllactic and hydroxyisocaproic acids, 6, lactic acid, ethanol, H2O2 and azelaic, phenyllactic,
hydroxyphenyllactic and hydroxyisocaproic acids (see Materials and Methods' section for compound concentrations). Within a same row, means with different letters are significantly
different according to a Fisher's least significant difference test (p b 0.05).

showed delay effect on P. corylophilum after 7 days of incubation at 25 °C
as compared to a control (Le Lay et al., 2016).
Concerning the effect on conidial germination, for P. corylophilum
UBOCC-A-112081, maximum of sporulation was not affected regardless
of the tested supernatant, as ca 100% of conidia germinated (Table 3).
However, germination time (τ) was affected by certain conditions. In-
terestingly, as compared to the τ value observed with L. citreum L123 su-
pernatant (18 h), that of the mix with all molecules was identical, and
that of the mix with only basal metabolism molecules was similar.
Thus, it indicates that the large majority of the antifungal activity was
supported by these molecules. The experiments with the mix without
basal metabolism molecules and the mix without acetate gave a τ
value comparable (about 14 h) to that of the negative control (water).
These results indicated that identification and quantification of antifun-
gal compounds were reliable. Moreover, as the mix with only basal me-
tabolism molecules showed almost the same antifungal activity as the
positive control (L. citreum L123) and the complete mix, the antifungal
activity was principally supported by these 4 molecules However, in ab-
sence of acetic acid, the result was the same than that obtained with
water (negative control). Thus, it can be concluded that the antifungal
activity of L. citreum L123 against P. corylophilum UBOCC-A-112081
was mainly due toacetic acid production.
For E. repens, Pmax for water and mix without basal metabolism
molecules achieved approximately 100%. In contrast, Pmax remained
below 15% for L. citreum L123 supernatant, the mix with all molecules
and the one with basal metabolism molecules but (Table 3). Thus,
these 3 samples prevented conidia germination of E. repens. Moreover,
when acetate was removed from the mix, the Pmax was around 40%
suggesting that if acetate was responsible for an important part of the
observed antifungal activity, other molecules may also play a role. The
germination time was strongly negatively affected by all conditions ex-
cept for the mix without basal metabolism molecules (τ around 25 h).
For the other samples, Pmax never reached 50% and was comprised be-
tween 8 and 15%, preventing the determination of the τ value (Table 3).
Concerning the effect on fungal growth, for P. corylophilum UBOCC-
A-112081 fungal growth was slowed down in the presence of L. citreum
L123 supernatant, mix with all molecules and mix with basal metabo-
lism molecules. The μ value for the mix without acetate was similar to
that of the negative control (water) confirming the hypothesis that
acetic acid supported the large majority of the antifungal activity against
P. corylophilum UBOCC-A-112081.
As for E. repens UBOCC-A-112075, the determined μ value indicated
that growth in presence of the mix without basal metabolism molecules
was similar to the one in water. For the other tested mixes, as stated
above, germination was blocked, so no fungal growth was observed,
even for the mix without acetate (Table 3). In this context it would be
of interest to further investigate the molecules supporting the antifun-
gal activity against E. repens UBOCC-A-112075.
In conclusion, from an applied point of view, the obtained results
confirmed the potential of some lactic acid bacteria cultures, superna-
tants or active metabolite mixtures to be used as antifungal agents
and therefore as natural alternative to preservatives. Interestingly, to
prevent post-treatment aerocontamination by mold spores, various ap-
plication methods could be used according to the considered type of
bakery product. Indeed, to protect bakery product prepared without a
sourdough, surface spraying of any of these solutions could be used,
provided that they do not impact the organoleptic qualities of the final
product. However, the protection would only be on the surface raising
the question of fungal contaminations on sliced products. While for
sourdough products, both surface spraying and incorporation in the
sourdough could be considered. In the latter case, the solutions could
be used as either a sourdough ingredient (no or little bacterial growth)
or even as a sourdough starter (growth). The use as sourdough ingredi-
ent or starter, if efficient, is of interest as it would correspond to a whole
product protection compatible with slicing. From a mechanistic point of
view, the approach used allowed determining the main compounds
supporting the antifungal activity of LAB and propionibacteria. Howev-
er, as a targeted method does not allow for identification of unknown
antifungal compounds, the combination with an untargeted method
would be of interest. The obtained results suggested that for L. citreum
L123, acetate was harboring a predominant part of the antifungal activ-
ity; although species-dependent behaviors were observed. In this con-
text, evaluation of the activity spectrum of this bacterium against
other fungal species and strains would be of interest.
Acknowledgements
This work was funded by the Bretagne, Pays de La Loire and Basse-
Normandie regions in the framework of the ACIPPEV project. We
thank Nathalie Roland from Laboratoires Standa for acetic, lactic and
propionic acid HPLC analyses.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.ijfoodmicro.2016.06.020.
 C. Le Lay et al. / International Journal of Food Microbiology 239 (2016) 79–85
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