Conversion of the mycotoxin patulin to the less toxic desoxypatulinic acid by the biocontrol yeast Rhodosporidium kratochvilovae strain LS11

Article abstract of DOI:10.1021/jf203098v

The infection of stored apples by the fungus Penicillium expansum causes the contamination of fruits and fruit-derived products with the mycotoxin patulin, which is a major issue in food safety. Fungal attack can be prevented by beneficial microorganisms, so-called biocontrol agents. Previous time-course thin layer chromatography analyses showed that the aerobic incubation of patulin with the biocontrol yeast Rhodosporidium kratochvilovae strain LS11 leads to the disappearance of the mycotoxin spot and the parallel emergence of two new spots, one of which disappears over time. In this work, we analyzed the biodegradation of patulin effected by LS11 through HPLC. The more stable of the two compounds was purified and characterized by nuclear magnetic resonance as desoxypatulinic acid, whose formation was also quantitated in patulin degradation experiments. After R. kratochvilovae LS11 had been incubated in the presence of ¹³C-labeled patulin, label was traced to desoxypatulinic acid, thus proving that this compound derives from the metabolization of patulin by the yeast. Desoxypatulinic acid was much less toxic than patulin to human lymphocytes and, in contrast to patulin, did not react in vitro with the thiol-bearing tripeptide glutathione. The lower toxicity of desoxypatulinic acid is proposed to be a consequence of the hydrolysis of the lactone ring and the loss of functional groups that react with thiol groups. The formation of desoxypatulinic acid from patulin represents a novel biodegradation pathway that is also a detoxification process.

Full text of DOI:10.1021/jf203098v

ARTICLE
pubs.acs.org/JAFC
Conversion of the Mycotoxin Patulin to the Less Toxic Desoxypatulinic
Acid by the Biocontrol Yeast Rhodosporidium kratochvilovae
Strain LS11
Raffaello Castoria,*,†,r Luisa Mannina,‡,O Rosa Durꢀan-Patrꢀon,§ Francesca Maffei,|| Anatoly P. Sobolev,^
Dario V. De Felice,† Cristina Pinedo-Rivilla,§ Alberto Ritieni,# Rosalia Ferracane,# and Sandra A. I. Wright†
†Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, and ‡Dipartimento di Scienze e Tecnologie Agro-Alimentari, Ambientali e
Microbiologiche, Universitꢁa degli Studi del Molise, Via F. De Sanctis snc, 86100 Campobasso, Italy
§Departamento de Química Orgꢀanica, Facultad de Ciencias, Universidad de Cꢀadiz, Apdo 40, 11510 Puerto Real, Cꢀadiz, Spain
Dipartimento di Farmacologia, Universitꢁa degli Studi di Bologna, Via Irnerio 48, 40126 Bologna, Italy
^Istituto di Metodologie Chimiche, CNR, Laboratorio di Risonanza Magnetica “Annalaura Segre”, 00015 Monterotondo Stazione
(Rome), Italy
#Dipartimento di Scienza degli Alimenti, Universitꢁa di Napoli “Federico II”, Parco Gussone, 80055 Portici, Italy
S Supporting Information
b
ABSTRACT: The infection of stored apples by the fungus Penicillium expansum causes the contamination of fruits and fruit-derived
products with the mycotoxin patulin, which is a major issue in food safety. Fungal attack can be prevented by beneficial
microorganisms, so-called biocontrol agents. Previous time-course thin layer chromatography analyses showed that the aerobic
incubation of patulin with the biocontrol yeast Rhodosporidium kratochvilovae strain LS11 leads to the disappearance of the
mycotoxin spot and the parallel emergence of two new spots, one of which disappears over time. In this work, we analyzed the
biodegradation of patulin effected by LS11 through HPLC. The more stable of the two compounds was purified and characterized by
nuclear magnetic resonance as desoxypatulinic acid, whose formation was also quantitated in patulin degradation experiments. After
R. kratochvilovae LS11 had been incubated in the presence of 13C-labeled patulin, label was traced to desoxypatulinic acid, thus
proving that this compound derives from the metabolization of patulin by the yeast. Desoxypatulinic acid was much less toxic than
patulin to human lymphocytes and, in contrast to patulin, did not react in vitro with the thiol-bearing tripeptide glutathione. The
lower toxicity of desoxypatulinic acid is proposed to be a consequence of the hydrolysis of the lactone ring and the loss of functional
groups that react with thiol groups. The formation of desoxypatulinic acid from patulin represents a novel biodegradation pathway
that is also a detoxification process.
KEYWORDS: patulin, metabolization, desoxypatulinic acid, detoxification, biocontrol yeast
’ INTRODUCTION
fungi, through the appropriate handling of fruit and the treatment
with fungicides.2 Fungicide treatments cannot completely pre-
vent infection by P. expansum. Furthermore, selecting healthy
fruits on an industrial scale does not ensure the absence of patulin
in the final product.10 Detoxification of mycotoxins (i.e., the
conversion or degradation of mycotoxins to less toxic
compounds) in food for human consumption has recently gained
interest, with particular focus on mycotoxins present in beverages
that derive from crops that are susceptible to the attack of
mycotoxigenic pathogens.11,12
Over the past two decades, research interest on preventive
strategies such as the biocontrol of postharvest pathogenic fungi
by utilizing harmless bacteria and yeasts has steadily increased, as
has public concern regarding fungicide residues in food. Some
biocontrol agents have been developed for commercial use.13,14
Patulin is a mycotoxin that is synthesized by a number of fungi.
It is a polyketide secondary metabolite, which represents a
serious health hazard and is a major issue for food safety.1 Its
major producer, the fungus Penicillium expansum Link., is an
important postharvest pathogen of apples and pears and causes
the accumulation of patulin in infected pome fruits. This leads to
the contamination with patulin in pome fruit-derived juices and
baby foods, consumed by children, who have a low tolerance level
to patulin.2 Patulin has diverse toxic effects such as immunosup-
pression, embryo and maternal toxicity in mice, and genotoxicity
to cultured human cells.3ꢀ5 The mechanism of patulin toxicity has
been proposed to be due to its reactivity with thiol-bearing mol-
ecules such as the important cellular antioxidant glutathione.6,7
Acute symptoms caused by patulin in humans are gastritis
and nausea.2 Therefore, many countries, including the U.S. and
those of the European Union, have established maximum toler-
able levels of patulin contamination for fruit-derived products.8,9
The most effective way of avoiding mycotoxin contamination
is to stop the fruit from being attacked by mycotoxin-producing
Received: March 25, 2011
Accepted: September 19, 2011
Revised:
September 17, 2011
Published: September 19, 2011
r
2011 American Chemical Society
11571
dx.doi.org/10.1021/jf203098v J. Agric. Food Chem. 2011, 59, 11571–11578
|

Journal of Agricultural and Food Chemistry
ARTICLE
The mechanisms of action of biocontrol agents have been
examined, with the final goal to understand how biocontrol
agents operate and to improve their efficacy.13,15ꢀ17 Preharvest
application of biocontrol agents for control of postharvest
diseases has been proposed and tested.18 Interestingly, some
biocontrol agents appear to have both the ability to prevent
fungal infections on fruit and the potential for degrading the
mycotoxins that are produced by these same fungi. Strains of
Aureobasidium pullulans effectively protected wine-grapes from
infection by Aspergillus carbonarius in the vineyard and prevented
their subsequent contamination with ochratoxin A. They were
also able to degrade the mycotoxin to the less toxic compound
ochratoxin α in synthetic medium and fresh grape must.19
Another biocontrol yeast, strain LS11 of the basidiomycete
Rhodosporidium kratochvilovae [a strain that was formerly desig-
nated Rhodotorula glutinis, but was renamed R. kratochvilovae
following the analysis of the sequence data of the internal
transcribed spacer (ITS) region (unpublished data)], can pre-
vent apples from the attack of postharvest rot fungi20 and lower
patulin contamination in stored fruit.21 Less patulin accumulated
in apples that had been pretreated with the yeast and infected by
P. expansum than in non-yeast-treated, infected fruits.21 Further-
more, strain LS11 can resist very high concentrations of patulin
(500 μg/mL) and is able to metabolize this mycotoxin under
aerobic conditions, leading to the formation of two products with
Rf (retention factor) 0.46 and Rf 0.38, as detected by TLC.21 The
compound(s) with Rf 0.46 appears to be more stable and is most
likely the major final product(s) of the degradation process,
because it is present at the end of the degradation process, when
the other product is no longer detectable (unpublished data).
Preliminary observations indicated that the two biodegradation
products were less toxic than patulin to Escherichia coli
(unpublished data). However, their toxicity to eukaryotic cells,
such as human cells, is unknown.
Metabolization of patulin to other unknown compounds has
been reported previously. Several research groups also observed
that strains of Saccharomyces cerevisiae were able to cause the
disappearance of patulin from fermented apple juice over
time.22,23 In recent years, the degradation products have been
identified in the case of the biodegradation carried out by the
fermentative yeast Saccharomyces cerevisiae in anaerobic
conditions.12 The final products are two different isomers of
ascladiol, E-ascladiol and Z-ascladiol. E-Ascladiol is the predo-
minant degradation compound. Not much data exist on the
toxicity of ascladiol. It is allegedly less toxic than patulin but still
retains a quarter of the toxicity of patulin, and can be considered a
mycotoxin in itself.24 The results of TLC analyses suggest that
the major product of aerobic patulin degradation by the biocon-
trol yeast R. kratochvilovae LS11 has an Rf value of 0.46, which is
distinct from that of ascladiol.21,25 Therefore, a novel pathway
could be responsible for the metabolization of patulin by R.
kratochvilovae LS11.
’ MATERIALS AND METHODS
Materials. A commercial standard of patulin was purchased from
Sigma-Aldrich (Milan, Italy). Immediately prior to use, weighed aliquots
of the mycotoxin were dissolved in water that had been acidified with
acetic acid (pH 4). All solvents employed were double-distilled or HPLC
grade. All solvents and reagents were obtained from SigmaꢀAldrich
(Milan, Italy). Sodium [1-13C] acetate (isotopic purity 99 atom % 13C)
was obtained from the Euriso-Top Co. (Saint Aubin, France).
All microbial stains used in this study were obtained from the culture
collection of the Dipartimento di S.A.V.A., Universitꢁa del Molise. Cells
of the yeast R. kratochvilovae LS11, originally isolated from an olive tree,
were maintained viable in 20% glycerol at ꢀ80 ꢀC. Cultures of P.
expansum FS-7 were preserved on potato dextrose agar (PDA, 200 g of
potatoes, 20 g of D-glucose, and 20 g of agar per liter of distilled water) on
slant tubes at 4 ꢀC.
Growth of R. kratochvilovae LS11 in the Presence of
Unlabeled Patulin. For the analysis of patulin metabolization by R.
kratochvilovae LS11, cells of the yeast were scraped from a Petri dish,
added to 50 mL of Lilly-Barnett medium (LiBa, 10.0 g of D-glucose, 2.0 g
of L-asparagine, 1.0 g of KH2PO4, 0.5 g of MgSO4 7H2O, 0.01 mg of
3
FeSO4 7H2O, 8.7 mg of ZnSO4 7H2O, 3.0 mg of MnSO4 H2O, 0.1 mg
3
3
3
of biotin, and 0.1 mg of thiamine per liter of medium),26 and incubated at
23 ꢀC on a rotary shaker at 150 rpm for 24ꢀ36 h. The culture was
centrifuged for 20 min at 6000 rpm, the cell concentrations were
adjusted to 1.0 ꢁ 105 CFU/mL, corresponding to values of 0.01 optical
density (OD) at 595 nm, and the cells were transferred to three flasks,
each containing 50 mL of LiBa supplemented with 150 μg/mL patulin.
Three flasks each containing 50 mL of LiBa with 150 μg/mL patulin, and
not inoculated with the yeast, were used as the control. All of the flasks
were incubated on a rotary shaker at 150 rpm and at 23 ꢀC for 72 h. At 0,
24, 48, and 72 h, samples of 200 μL were withdrawn from each flask for
subsequent HPLC analyses of crude samples. The same experiments
were also carried out in macerated browning apple tissue, supplemented
with patulin, and inoculated with R. kratochvilovae LS11 as previously
described.21 The only difference from the previously reported biode-
gradation procedure21 was the volume of liquid used and the application
of gentle shaking in the present experiments.
For NMR analysis of the major biodegradation product of patulin by
R. kratochvilovae LS11, yeast cells were grown as described above, but
after centrifugation they were transferred to a single flask containing
50 mL of LiBa with 250 μg/mL patulin. During the course of the
incubation, the decrease in intensity of the spot corresponding to patulin
and the increased intensity of two spots with Rf 0.46 and Rf 0.38, which
appeared during incubation, were monitored by TLC. Forty microliter
aliquots of culture filtrate, which had previously been extracted with
ethyl acetate and concentrated,21,25 were applied on 0.25 mm thick
plates (Merck Kiesegel 60 F254, Darmstadt, Germany), and chromatog-
raphy was performed by using tolueneꢀethyl acetateꢀformic acid 5:4:1
(v/v/v) as the solvent system. TLC results were first observed under UV
light and then visualized by spraying with a 0.5% solution of 3-methyl-2-
benzothiazolinone hydrazone (Sigma-Aldrich, Milan, Italy) (w/v) and
heating at 120 ꢀC for 15 min. The biodegradation reaction was stopped
after 96 h, at which time the patulin spot and the one having Rf 0.38 could
no longer be detected. The culture was centrifuged, cells were discarded,
and the supernatant was lyophilized and then dissolved in acidified water
(pH 4) prior to purification of the major biodegradation product.
HPLC Analyses of Patulin Metabolization by R. kratochvi-
lovae LS11. HPLC analyses were performed as previously described27
with slight modifications. The HPLC apparatus was a Dionex
(Sunnyvale, CA) analytical system consisting of a P680 solvent delivery
system and a 20 μL injector loop (Rheodyne, Cotati). The UVD170
detector (Dionex, Sunnyvale) set at 276 nm was connected to a data
integration system (Dionex Chromeleon Version 6.6). The column used
In this work, we analyzed by HPLC the metabolization of
patulin operated by R. kratochvilovae LS11, characterized by
NMR the major metabolite produced, and analyzed by HREIMS
isotope incorporation into this compound following incubation
of strain LS11 with 13C-labeled patulin, to provide unequivocal
evidence that the produced metabolite derives from the biode-
gradation of patulin by the yeast. Furthermore, we confirmed that
a detoxification had occurred by comparing the toxicity of the
new compound to that of patulin in a bioassay based on cultured
human lymphocytes.
11572
dx.doi.org/10.1021/jf203098v |J. Agric. Food Chem. 2011, 59, 11571–11578

Journal of Agricultural and Food Chemistry
ARTICLE
was a 250 mm ꢁ 4.6 mm i.d., 5 μm, Agilent Zorbax C18 (Agilent
technologies Italia s.p.a, Milan, Italy). The mobile phase was acidified
water (with 1% acetic acid v/v) and methanol 95:5 (v/v) with a flow rate
of 1 mL/min and a total run of 18 min. Standard serial solutions of
patulin in acidified water were injected, and peak areas were determined
to generate a standard curve for quantitative analyses. Quantitation of
the major metabolite of patulin metabolization was also carried out as
described for patulin, following purification and characterization as
desoxypatulinic acid. The quantity of desoxypatulinic acid was deter-
mined by using a calibration curve with injected amounts, ranging from
0.02 to 0.4 μg. The linear fit was: y = 63.145x ꢀ 0.0521 [Pearson’s
coefficient (R2) = 1]. The limit of quantitation (LOQ) was 20 ng (1 μg/
mL), and the limit of detection (LOD) was 6.7 ng (0.333 μg/mL), with
signal/noise (S/N) ratios of 9 and 3, respectively. The experiments were
performed three times, and each experiment consisted of three repli-
cates. Data from the experiments were pooled, because they were similar
in the three repetitions, and expressed as μg/mL of patulin or des-
oxypatulinic acid ( standard deviation.
was evaporated under reduced pressure. The residue was purified by
column chromatography on silica gel (Merck, Darmstadt, Germany), by
using toluene/ethyl acetate/formic acid 5:4:1 (v/v/v) as the solvent
system, to afford 55 mg of labeled patulin.
A suspension of fresh cells of R. kratochvilovae LS11 (1.0 ꢁ 105 CFU/
mL) was added to an Erlenmeyer flask (500 mL) containing 200 mL of
Lilly-Barnett medium and 150 μg/mL of labeled patulin. The yeast was
incubated for 4 days at 23 ꢀC, at 150 rpm. At this time point, the culture
medium and the yeast cells were separated by centrifugation for 20 min
at 6000 rpm. The medium was acidified to pH 2 with an aqueous
solution of HCl 2 M, saturated with NaCl, and extracted three times with
ethyl acetate. The ethyl acetate extract was washed three times with
H2O, dried over anhydrous Na2SO4, and the solvent was evaporated
under reduced pressure. Purification of labeled desoxypatulinic acid was
carried out as described for labeled patulin, to afford 26 mg of this pure
compound. The isotopic composition of patulin and desoxypatulinic
acid was calculated by GC-HREIMS on VG-Autospec spectrometer fol-
lowing correction for natural abundance levels, which were determined
experimentally using authentic and unlabeled standard compounds.
Effect of Patulin and Desoxypatulinic Acid on Cultured
Human Lymphocytes. Peripheral lymphocytes were withdrawn from
two healthy nonsmoking males belonging to AVIS (Italian Association
of Voluntary Blood donors) who were less than 40 years old. Immedi-
ately after withdrawal, lymphocytes were separated from whole blood
using a density gradient (Histopaque 1077 Sigma-Aldrich), and cultured at
a concentration of 2 ꢁ 106 cells/mL in 5 mL (i.e., 4 ꢁ 105 cells/mL) of
RPMI 1640 medium (Sigma-Aldrich) supplemented with 15% fetal calf
serum, 1% phytohemagglutinin, 1% penicillinꢀstreptomycin solution
(v/v), and 1 mM L-glutamine (Sigma-Aldrich). The cultures were
incubated at 37 ꢀC in a humid atmosphere in the presence of 5% CO2
(v/v). After 48 h, lymphocyte cultures were treated with patulin or with
desoxypatulinic acid (the purified major product of patulin metaboliza-
tion by the yeast, which had been identified by NMR analyses). Both
desoxypatulinic acid and patulin were individually dissolved in sterile
double-distilled water and added to lymphocyte cultures to obtain final
concentrations of 0.1, 0.5, 1, 5, 10, 50, and 100 μM. Cell concentrations
were measured as a function of time by trypan blue-exclusion test. Cell
aliquots withdrawn from each culture at 3 and 24 h from the beginning of
the experiment were mixed with an equal volume of trypan blue solution
(diluted at 0.8 mM in PBS) for 2 min at room temperature, and the cells
that stained positive were counted under a light microscope. Living cells
with membrane integrity do not take up trypan blue, while dead cells
with damaged membranes stain blue due to the uptake of trypan blue.
The percentage of live lymphocytes was determined as the ratio of the
number of nontrypan blue (negative) cells (i.e., living cells) to the total
number of cells, that is, the sum of trypan blue (positive) cells (i.e., dead
cells) and living cells.29,30 Results were expressed as the percentage of
viable cells in treated cultures, which was compared to that of untreated
control cultures. Each treatment was performed in duplicate with
separate cultures of cells from two donors (i.e., four cultures were setup
for each treatment). The experiment was performed twice. Only the
results from one experiment are reported because the outcome was
similar in both cases.
Characterization of the Major Biodegradation Product of
Patulin. Purification of the patulin degradation product was carried out
using an HPLC apparatus series 200 Perkin-Elmer, equipped with a
binary pump and a UV/vis 785 A detector set at 276 nm. The column
used was a 250 ꢁ 4.6 mm i.d., 5 μ, Phenomenex Luna C18 100 A
(Torrance, CA). The mobile phase and flow were the same as for HPLC
analyses. Twenty aliquots of 100 μL were injected with an autosampler
(Perkin-Elmer, USA), and fractions of 500 μL each from the peak with
retention time of 15.2 min were collected in the dark. Twenty fractions
were pooled, and the solvent was evaporated under a N2 stream in a
preweighed glass vial. After being dried, the weight of the purified sample
was determined to 2.20 mg, and it was stored at ꢀ20 ꢀC.
Prior to NMR analysis, both this sample and a sample of patulin were
dissolved in D2O with 0.17 M deuterated acetic acid with pH adjusted to
4.0 by the addition of potassium carbonate. NMR spectra were
performed at 300 K on an AVANCE AQS600 spectrometer (Bruker
Biospin GmbH Rheinstetten, Karlsruhe, Germany) operating at the
proton frequency of 600.13 MHz. The 1H spectra were referred to the
1
residual H signal of CHD2COOD set at 2.08 ppm. 13C spectra were
referred to the 13C methyl carbon of residual CHD2COOD set at
21.4 ppm.
1
13
2D NMR experiments, that is, 1Hꢀ H COSY, 1Hꢀ C HSQC, and
1Hꢀ C HMBC, were performed using the same experimental condi-
13
tions as previously reported by Mannina et al.;28 the delay for the
evolution of long-range couplings in 1Hꢀ C HMBC experiments
13
was 80 ms.
Mass spectrum was recorded by GCꢀMS with a VG-Autospec
instrument. Calculated mass for C7H8O4 [M]+ was 156.0423, while
the one we found experimentally was 156.0415.
Isotope Incorporation Studies. Labeled patulin was produced
as follows: P. expansum strain FS-7 was grown at 25 ꢀC, at 150 rpm, in
four Erlenmeyer flasks (500 mL) each containing 300 mL of potato
dextrose broth (PDB) medium (200 g of potatoes and 20 g of D-glucose
per liter of distilled water), diluted with H2O to a final ratio of 1:10. Each
flask was inoculated with fresh conidia to a final concentration of 4 ꢁ 106
conidia/mL. After 4 days of incubation (the time interval that was
identified as the optimum one, following a time-course study on the in
vitro formation of patulin), the mycelia from the four flasks were
transferred into the same number of new Erlenmeyer flasks (500 mL),
each containing 300 mL of PDB medium, diluted as above. A filter-
sterilized (by using 0.22 μm filters) aqueous solution of sodium [1-13C]
acetate was added at a final concentration of 300 μg/mL. Three days
after addition of the labeled precursor, the culture medium and mycelia
were separated by filtration. The broth was saturated with NaCl and
extracted three times with ethyl acetate. The organic extract was washed
three times with H2O, dried over anhydrous Na2SO4, and the solvent
Fate of Patulin and Desoxypatulinic Acid in the Presence
of Glutathione in Vitro. These experiments were performed follow-
ing the method described by Pfeiffer et al.31 with slight modifications.
Patulin or desoxypatulinic acid (10 mM) was incubated with a 5-fold
molar excess of GSH (50 mM) in 67 mM potassium phosphate buffer,
pH 7, at 37 ꢀC for 24 h. Controls were patulin and desoxypatulinic acid
incubated in buffer under the same conditions as above in the absence of
GSH. Aliquots of 100 μL were taken from the incubation mixtures at 0,
1/12 (i.e., 5 min), 1, 6, and 24 h, acidified with 10 μL of formic acid, and
analyzed for the presence of patulin and desoxypatulinic acid by HPLC.
Analyses were performed using a Shimadzu LC10 HPLC instrument
11573
dx.doi.org/10.1021/jf203098v |J. Agric. Food Chem. 2011, 59, 11571–11578

Journal of Agricultural and Food Chemistry
ARTICLE
(Shimadzu, Kyoto, Japan) equipped with a photodiode array detector.
Separation was carried out using the same column and the same initial
mobile phase [acidified water (with 1% acetic acid v/v) and methanol
95:5 (v/v) with a flow rate of 1 mL/min] as those used for patulin and
desoxypatulinic acid analysis in the biodegradation experiments, with the
following linear gradient: 5% methanol for 20 min, 5ꢀ100% methanol in
40 min.
Statistical Analysis. Data from all of the experiments reported in
this study were analyzed with the SPSS program (SPSS Inc., Chicago, IL,
release 15 for Windows). Data obtained from the three experiments of
patulin metabolization by R. kratochvilovae LS11 were similar, so they
were pooled prior to statistical analysis. Likewise, the results from the
three experiments of the in vitro binding of patulin and desoxypatulinic
acid to glutathione were pooled because the data were similar. Results
from the experiments of patulin metabolization were expressed as μg/
mL of patulin or desoxypatulinic acid ( standard deviation (n = 9).
Those assessing the fate of patulin and desoxypatulinic acid in the
presence of glutathione were expressed as millimolar concentrations of
the two compounds ( standard deviation (n = 9). The results were
analyzed by Student’s t test (P < 0.001). Results of the effect of patulin
and desoxypatulinic acid on cultured human lymphocytes were ex-
pressed as percentage of viable cells ( standard deviation in treated
cultures (n = 8). Percentages were compared to those of the untreated
control by Student’s t test (at P < 0.05).
’ RESULTS AND DISCUSSION
Analytical HPLC chromatograms demonstrating the typical
fate of patulin after 0, 24, 48, and 72 h of in vitro incubation with
the yeast R. kratochvilovae LS11 are presented in Figure 1AꢀD.
In the presence of the yeast, the concentration of patulin
progressively decreased, and only 3.7 ( 1.2 μg/mL of the
mycotoxin could be detected 72 h after the beginning of the
experiment. Conversely, approximately the same concentration
of patulin added to the noninoculated growth medium (control)
at the beginning of the experiment (150 μg/mL) was recorded at
the same time point. Interestingly, in the presence of the yeast, a
peak with Rt 14.70 min appeared over the first time interval
(0ꢀ24 h) (Figure 1B) and progressively increased with time
paralleling the decrease of patulin (Figure 1C,D). This new
peak corresponds to desoxypatulinic acid, as shown by NMR
(Figure 2A), and to the spot in thin layer chromatography ana-
lyses with the Rf 0.46 (data not shown).21 The concentration
of desoxypatulinic acid progressively increased and reached the
value of 87.0 ( 11.7 μg/mL at 72 h. Similar results were obtained
when patulin was incubated with strain LS11 in browning and
macerated apple tissue (data not shown).21 The quantitative
analyses of patulin and desoxypatulinic acid show that the yeast
metabolizes patulin almost completely within 72 h and confirm
its ability to resist high patulin concentrations.21 Because patulin
is a broad-spectrum toxin and antifungal agent, it is potentially
lethal also to R. kratochvilovae LS11, which may convert it to
desoxypatulinic acid to protect itself against the toxin. Scott et al.
reported that 46 μg/mL of this compound was not toxic to a
number of bacteria and yeasts, whose growth was completely
inhibited by patulin under the same conditions.32 In fruit wounds,
the basidiomycetous yeast R. kratochvilovae LS11 can utilize its
capacity to convert patulin to desoxypatulinic acid to protect itself
and the fruit from one of the weapons of the green mold fungus.
Desoxypatulinic acid was identified as the major compound
deriving from patulin biodegradation in vitro by LS11 following
purification and subsequent NMR analysis. The chromato-
graphic conditions for HPLC quantitative analyses of patulin
Figure 1. Time-course HPLC chromatograms of in vitro patulin
degradation and production of desoxypatulinic acid (DPA) by the
biocontrol yeast Rhodosporidium kratochvilovae strain LS11 following
incubation for 0 (A), 24 (B), 48 (C), and 72 h (D).
were similar to those used for the purification of desoxypatulinic
acid and subsequent NMR analysis. A yield of 2.20 mg of
desoxypatulinic acid was obtained and used for NMR analysis
and structural characterization.
1
The assignment of H and 13C NMR spectra of patulin in
D2O/acetic acid is reported in Table 1. The high-resolution mass
spectrum of the major biodegradation product indicated a molec-
ular formula C7H8O4. To determine its molecular structure, a
1
complete NMR study, including 1D and 2D experiments, 1Hꢀ H
13
13
COSY, 1Hꢀ C HSQC, and 1Hꢀ C HMBC (Figure 2C), was
performed. The 1H spectrum of this stable metabolite shows the
presence of three CH2 groups labeled as 2, 3, and 7 and one CH
group labeled as 6. The CH2-2 at 4.61 ppm and the CH2-3 at 2.72
ppm correlate in COSY experiment and therefore belong to a
CH2ꢀCH2 fragment, while CH2-7 is a doublet with a very small
coupling constant, indicating the absence of the vicinal CHx groups.
The 1H/13C chemical shifts of CH2-2 group in this metabolite
are similar to chemical shifts of CH2-6 of patulin (Figure 2A,B),
suggesting a structural similarity (OꢀCH2), but the multiplicity
11574
dx.doi.org/10.1021/jf203098v |J. Agric. Food Chem. 2011, 59, 11571–11578

Journal of Agricultural and Food Chemistry
ARTICLE
1
13
Figure 2. Structures of (A) desoxypatulinic acid (arrows indicate key long-range correlations in HMBC spectrum) and (B) patulin. (C) Hꢀ C
HMBC spectrum of desoxypatulinic acid in D2O/acetic acid at 27 ꢀC.
1
Table 1. H and 13C Assignments of Patulin and Desoxypatulinic Acid in D2O/Acetic Acid
patulin
multiplicity: J, (Hz)
desoxypatulinic acid
multiplicity: J, (Hz)
group
1H (ppm)
13C (ppm)
group
1H (ppm)
13C (ppm)
CH (3,7)
CH (4)
CH2 (6, 60)
6.20ꢀ6.22
6.14
m
110.2
88.3
59.5
CH (6)
CH2 (2)
CH2 (3)
7.62
4.61
2.72
t: <0.7
t: 7.1
t: 7.1
164.7
67.9
34.6
s
4.70
dd: 17.5, 3.0
dd: 17.5, 3.8
4.53
CdO (2)
C (7a)
172.2
145.9
151.3
CH2 (7)
3.18
d: <0.7
30.5
195.6
111.8
176.5
CdO (4)
CdC (5)
COOH (8)
C (3a)
of 1H signals is different. Geminal protons of the patulin CH2-6
group are not chemically equivalent (Table 1), being close to the
asymmetric C-4 carbon atom, whereas the protons of the CH2-2
group of the biodegradation product of patulin are equivalent,
indicating the transformation of chiral CH(OH)-4 group into a
nonchiral one.
of desoxypatulinic acid by a microorganism other than Penicillium
sp., as a biodegradation product of patulin.
Isotope incorporation experiments show that biodegradation
of 13C-labeled patulin by R. kratochvilovae LS11 led to the
emergence of 13C-labeled desoxypatulinic acid, the major biode-
gradation product, with a similar distribution of label as in the
original 13C-labeled patulin. The specific 13C incorporation into
patulin and desoxypatulinic acid was analyzed by HREIMS. This
analytical method involves obtaining the spectra of the labeled
compound and the unlabeled analogue under conditions as
identical as possible. The isotopic composition of the labeled
compound is determined by subtracting the relative abundance
ratios of the labeled and unlabeled species in the ratio found for
the unlabeled compound.
The carbon skeleton of the patulin biodegradation product
1
13
was unequivocally assigned using long-range Hꢀ C correla-
tions (Figure 2C). Long-range contacts between protons of
CH2-2 and carbon C-6 and between proton CH-6 and carbon
C-2 in the HMBC spectrum, together with diagnostic chemical
shifts, suggested that the CH(OH)-4 group of patulin was
transformed into a CHd (6) group in this metabolite. The
signals of the COOH (8) and CdO (4) groups in the 13C NMR
spectrum indicate the opening of the lactone ring. These results
GCꢀMS analysis of patulin revealed that up to three 13C
labels were incorporated in 0.3% of the molecules, following
administration of sodium [1-13C] acetate to P. expansum FS-7,
whereas 14.7% and 1.5% of the molecules carried one and two
13C atom(s), respectively. Isotope incorporation was also de-
tected in desoxypatulinic acid, following incubation of R. kra-
tochvilovae LS11 with 13C-labeled patulin. Most importantly, a
similar percentage of the molecules of desoxypatulinic acid
(14.4%) carried one 13C label. However, no incorporation of
more than one 13C atom was detected in desoxypatulinic acid
1
13
and the Hꢀ C long-range correlations of protons with three
quaternary carbons in the HMBC experiment (Figure 2A)
enabled us to identify the metabolite as desoxypatulinic acid.
The corresponding NMR assignments for desoxypatulinic acid are
presented in Table 1. This compound had previously been isolated
by Scott et al. from Penicillium patulum.32 The spectroscopic data
of the metabolite resulting from patulin biodegradation agreed
with those published in the literature for desoxypatulinic
acid.32 The present study is the first to report the production
11575
dx.doi.org/10.1021/jf203098v |J. Agric. Food Chem. 2011, 59, 11571–11578

Journal of Agricultural and Food Chemistry
ARTICLE
Table 2. Effect of Different Concentrations of Patulin (PAT)
and Desoxypatulinic Acid (DPA) on the Viability of Cultured
Human Lymphocytes
duration of treatment
treatment
viable cells (%), 3 h
viable cells (%), 24 h
PAT (μM)
94.00 ( 1.41
0.1
0.5
1
92.50 ( 2.12
89.50 ( 3.54
68.00 ( 7.07a
46.00 ( 9.90a
38.00 ( 14.14a
30.00 ( 11.10a
25.00 ( 5.66a
92.00 ( 4.24
89.00 ( 14.14
78.50 ( 12.02
78.00 ( 1.41a
77.00 ( 4.24a
66.50 ( 9.21a
5
10
50
100
DPA (μM)
95.50 ( 0.71
94.50 ( 0.71
93.00 ( 1.41
91.00 ( 7.07
90.00 ( 5.56
89.00 ( 8.49
87.50 ( 7.78
95.50 ( 2.12
Figure 3. Time-course of the persistence of 10 mM patulin (PAT) and
10 mM desoxypatulinic acid (DPA), following incubation for 24 h at
37 ꢀC and pH 7 in the presence of 50 mM of the thiol-bearing tripeptide
glutathione (GSH). Values (mM) are the means ( standard deviation
(n = 9, using three measurements in three experiments). Symbols (**)
indicate highly significant differences (P < 0.001) between the resulting
PAT and DPA concentrations in the presence of GSH at the same time
points, when analyzed by Student’s t test.
0.1
93.50 ( 2.12
92.00 ( 1.41
90.00 ( 1.41
88.00 ( 2.83
87.50 ( 4.95
74.50 ( 4.95a
45.00 ( 4.24a
96.00 ( 1.41
0.5
1
5
10
50
100
7 has been demonstrated to bind covalently to thiol groups.6,7
The biotransformation of patulin into desoxypatulinic acid by R.
kratochvilovae LS11 leads to the loss of the hydroxyl group at C-4
of patulin, thus hindering the nucleophilic substitution at the
corresponding carbon in the molecule of desoxypatulinic acid
(C6). Desoxypatulinic acid could possibly undergo a Michael-
like addition of thiol groups at C-6 because of the presence of an
α,β-unsaturated carbonyl system. However, reports show that
similar α,β-unsaturated ketones do not add thiol nucleophiles to
this position, possibly because of deactivation by the ring oxygen.34
The proposed inability of desoxypatulinic acid to react with
thiol groups is supported by the results reported in Figure 3,
showing the fate of patulin and desoxypatulinic acid in the
presence of glutathione at 37 ꢀC and pH 7. In these experiments,
the mycotoxin reacted so rapidly with glutathione that even at
time 0 the concentration of patulin in the presence of glutathione
was lower than in the respective control (i.e., patulin in the
absence of glutathione). After 5 min and until the end of the
experiment, very low concentrations of patulin were present in
the reaction buffer, whereas in the absence of glutathione the
concentration of the mycotoxin remained unchanged at all time
points. The concentration of desoxypatulinic acid, in contrast to
that of patulin, remained almost unaffected over time in the
presence of glutathione. A slight decrease of desoxypatulinic acid
concentrations was only detected at 24 h, both in the presence
and in the absence of glutathione.
The absence of thiol-binding activity of desoxypatulinic acid
could provide an explanation for the low toxicity of this com-
pound. The low toxicity of desoxypatulinic acid could be due to
the loss of the hemiacetal group35 and the hydrolysis of the
lactone ring. The well-known instability of patulin in alkaline
conditions, which leads to loss of biological activity,36 is a
consequence of the opening of the lactone ring, suggesting that
the lactone component of patulin is essential to its toxicity.
Moreover, desoxypatulinic acid is more hydrophilic than patulin,
and the increased polarity would facilitate its excretion from the
body after ingestion.
control
a Significantly different from control according to Student’s test
(P < 0.05).
because of the low relative intensities of the isotopic peaks in the
HREIMS spectrum for the labeled and unlabeled compound.
These results prove that desoxypatulinic acid derives from
metabolization of patulin by the yeast.
Table 2 shows the percentage of viable human lymphocytes
following incubation with 0.1ꢀ100 μM of patulin or desoxypa-
tulinic acid for 3 and 24 h. The harvested cells were counted. The
percentage of viable cells in control cultures was similar at both
time points. Treatment with patulin caused a significant decrease
in the number of viable cells in a time- and dose-dependent
manner. The most rapid patulin-induced effect on lymphocyte
viability was observed after 3 h of incubation with patulin at 10
μM, when a decrease of viable cells slightly above 20% was
recorded. The lowest patulin concentration that had a significant
effect on the viability of lymphocytes was 1 μM, which caused a
decrease of approximately 30% of viable cells after 24 h. The most
dramatic effect was recorded after 24 h of incubation and with the
highest concentration of patulin, that is, 100 μM, which caused a
decrease of 70% in lymphocyte viability. The observed cytotoxi-
city of patulin in the present study agrees with that observed by
other authors on other cell lines.6,33 Conversely, desoxypatulinic
acid, on the other hand, was not cytotoxic to lymphocytes
following 3 h of incubation, for the whole range of concentrations
used. Desoxypatulinic acid was slightly toxic only at the highest
concentrations tested (50 and 100 μM) and over the longest
period of incubation (24 h).
The mechanism of patulin toxicity is usually attributed to its
reactivity to thiol groups, which are present in cysteine, glu-
tathione, and in cellular enzymes that contain thiol groups in
their active sites. Patulin thus destroys and inactivates these
compounds and blocks the active sites of enzymes containing
sulfhydryl groups.7,34 Within the patulin molecule, carbon 3, 4, or
11576
dx.doi.org/10.1021/jf203098v |J. Agric. Food Chem. 2011, 59, 11571–11578

Journal of Agricultural and Food Chemistry
ARTICLE
The ascomycetous yeast Saccharomyces cerevisiae is known to
metabolize patulin to E-ascladiol under anaerobic conditions.
This explains why patulin contamination in fermented drinks
such as cider is usually lower than in juices and other nonfer-
mented pome fruit-based products.12,22,23 E-Ascladiol has no
hemiacetal group (which would probably account for its slightly
lower toxicity as compared to patulin), but retains the integrity of
the lactone ring in its molecular structure. It is in itself considered
to be a mycotoxin, although with less acute toxicity than
patulin.24 The biodegradation of patulin by R. kratochvilovae
LS11 to desoxypatulinic acid has the additional advantage that it
is an aerobic process. From a practical perspective, an aerobic
degradation pathway is advantageous for the development of a
large-scale apple juice detoxification process, because costly
removal of oxygen becomes unnecessary.
The present study shows that a biocontrol yeast, R. kratochvi-
lovae LS11, forms a novel degradation product, desoxypatulinic
acid, from the mycotoxin patulin. Desoxypatulinic acid is much
less toxic than patulin. This conversion by strain LS11 can
therefore be considered to be a detoxification process. Future
studies will include toxicity and genotoxicity tests of desoxypa-
tulinic acid to cultured human cells, and the elucidation of the
degradation pathway. The future identification of the enzymes
involved in the degradation process will enable the development
of tools for a more effective prevention and diagnosis of patulin
contamination in pome fruit-derived products.
Dr. Josꢀe Paulo Sampaio (UNL, Portugal) for taxonomic reclassi-
fication of strain LS11 as Rhodosporidium kratochvilovae, and to
Dr. Alison Hill (University of Exeter, UK) and Dr. Rebecca Goss
(UEA, UK) for their helpful suggestions.
’ REFERENCES
(1) Castoria, R.; Logrieco, A. Mycotoxins in fruits and major fruit-
derived products ꢀ an overview. In Microbial Biotechnology in Horticul-
ture; Ward, O. P., Ray, R. C., Eds.; Science Publishers: New Hampshire,
2007; Vol. II, pp 305ꢀ344.
(2) Moake, M. M.; Padilla-Zakour, O. I.; Worobo, R. W. Compre-
hensive review of patulin control methods in foods. Compr. Rev. Food Sci.
Food Saf. 2005, 1, 8–21.
(3) Bourdiol, D.; Escoula, L. Effect of patulin on microbicidal activity
of mouse peritoneal macrophages. Food Chem. Toxicol. 1990, 28, 29–33.
(4) IARC. Patulin. Monograph No. 40 on the Evaluation of Carcino-
genic Risk of Chemicals to Man; International Agency for Research on
Cancer (IARC): Lyon, France, 1986; pp 83ꢀ98.
(5) Si-min, Z.; Li-ping, J.; Cheng-yan, G.; Jun, C.; Lai-fu, Z. Patulin-
induced genotoxicity and modulation of glutathione in HepG2 cells.
Toxicon 2009, 53, 584–586.
(6) Mahfoud, R.; Maresca, M.; Garmy, N.; Fantini, J. The mycotoxin
patulin alters the barrier function of the intestinal epithelium: Mechan-
ism of action of the toxin and protective effects of glutathione. Toxicol.
Appl. Pharmacol. 2002, 181, 209–218.
(7) Fliege, R.; Metzler, M. Electrophilic properties of patulin.
N-Acetylcysteine and glutathione adducts. Chem. Res. Toxicol. 2000,
13, 373–381.
(8) FAO. Worldwide Regulations for Mycotoxins. A compendium. Food
and Nutrition Paper No. 64, Food and Nutrition Division; FAO: Rome,
Italy, 1997.
(9) European Commission (EC). Regulation (EC) No. 455/2004
amending Regulation (EC) No. 466/2001 as regards patulin. Off. J. Eur.
Union L74, 11.
’ ASSOCIATED CONTENT
S
Supporting Information. Figure 1: COSY of desoxypa-
b
1
13
tulinic acid. Figure 2: Hꢀ C HSQC of desoxypatulinic acid.
This material is available free of charge via the Internet at http://
pubs.acs.org.
(10) FAO/WHO. Proposed draft code of practice for the prevention
of patulin contamination in apple juice and apple juice ingredients in
other beverages. Joint FAO/WHO Food Standards Programme reports,
Codex Committee on Food Additives and Contaminants, 34th session;
Rotterdam, The Netherlands, 2002; pp 1ꢀ8.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: +39(0)8744004698. Fax: +39(0)8744004652. E-mail:
castoria@unimol.it.
(11) Karlovsky, P. Biological detoxification of fungal toxins and its use
in plant breeding, feed and food production. Nat. Toxins 1999, 7, 1–23.
(12) Moss, M. O.; Long, M. T. Fate of patulin in the presence of
yeast Saccharomyces cerevisiae. Food Addit. Contam. 2002, 19, 387–399.
(13) Janisiewicz, W. J.; Korsten, L. Biological control of postharvest
diseases of fruits. Annu. Rev. Phytopathol. 2002, 40, 411–441.
(14) Castoria, R.; Wright, S. A. I.; Droby, S. Biological control of
mycotoxigenic fungi in fruits. In Mycotoxins in Fruits and Vegetables;
Barkai-Golan, R., Paster, N., Eds.; Elsevier: San Diego, CA, 2008; pp
311ꢀ333.
Present Addresses
rDipartimento di Scienze e Tecnologie Agro-Alimentari,
Ambientali e Microbiologiche, Universitꢁa degli Studi del Molise,
Via F. De Sanctis snc, 86100 Campobasso, Italy.
ODipartimento di Chimica e Tecnologie del Farmaco, Sapienza
Universitꢁa di Roma, 00185 Rome, Italy.
(15) Castoria, R.; De Curtis, F.; Lima, G.; De Cicco, V. β-1,3-
glucanase activity of two saprophytic yeasts and possible mode of action
involved as biocontrol agents against postharvest diseases. Postharvest
Biol. Technol. 1997, 12, 293–300.
Funding Sources
This research was funded by the Italian Ministry of University
and Scientific Research (MIUR): Project PRIN no. 2006072204
“Pilot study on innovative systems for the reduction of patulin
contamination in pome fruits”, and bilateral cooperation be-
tween Italy and Spain “Studio del pathway di biodegradazione
della micotossina patulina operata da un lievito basidiomicete”
no. IT088MB951. S.A.I.W. participated in this work thanks to the
grant obtained by MIUR “Incentivazione alla mobilitꢁa di studiosi
stranieri e italiani residenti all’estero” (DM 1.2.2005, no. 18).
(16) Castoria, R.; De Curtis, F.; Lima, G.; Caputo, L.; Pacifico, S.; De
Cicco, V. Aureobasidium pullulans (LS-30) an antagonist of postharvest
pathogens of fruits: study on its modes of action. Postharvest Biol.
Technol. 2001, 22, 7–17.
(17) Castoria, R.; Caputo, L.; De Curtis, F.; Lima, G.; De Cicco, V.
Resistance to oxidative stress of postharvest biocontrol yeast: a possible
new mechanism of action. Phytopathology 2003, 93, 564–572.
(18) Ippolito, A.; Nigro, F. Impact of preharvest application of
biological control agents on postharvest diseases of fresh fruits and
vegetables. Crop Prot. 2000, 19, 715–723.
’ ACKNOWLEDGMENT
(19) de Felice, D. V.; Solfrizzo, M.; De Curtis, F.; Lima, G.;
Visconti, A.; Castoria, R. Strains of Aureobasidium pullulans can lower
ochratoxin A contamination in wine grapes. Phytopathology 2008,
98, 1261–1270.
We wish to thank A. Spina and V. Morena for their collabora-
tion in carrying out the preliminary experiments of this work. We
are also indebted to Dr. Alexander Idnurm (UMKC, MI) and
11577
dx.doi.org/10.1021/jf203098v |J. Agric. Food Chem. 2011, 59, 11571–11578

Journal of Agricultural and Food Chemistry
ARTICLE
(20) Lima, G.; De Curtis, F.; Castoria, R.; De Cicco, V. Integrated
control of apple postharvest pathogens and survival of biocontrol yeasts
in semi-commercial conditions. Eur. J. Plant Pathol. 2003, 109, 341–349.
(21) Castoria, R.; Morena, V.; Caputo, L.; Panfili, G.; De Curtis, F.;
De Cicco, V. Effect of the biocontrol yeast Rhodotorula glutinis strain
LS11 on patulin accumulation in stored apples. Phytopathology 2005,
95, 1271–1278.
(22) Harwig, J.; Scott, P. M.; Kennedy, B. P. C.; Chen, Y. K.
Disappearance of patulin from apple juice fermented by Saccharomyces
spp. Can. Inst. Food Sci. Technol. J. 1973, 6, 45–46.
(23) Stinson, E. E.; Osman, S. F.; Huhtanen, C. N.; Bills, D. D.
Disappearance of patulin during alcoholic fermentation of apple juice.
Appl. Environ. Microbiol. 1978, 36, 620–622.
(24) Suzuki, T.; Takeda, M.; Tanabe, H. A new mycotoxin produced
by Aspergillus clavatus. Chem. Pharm. Bull. 1971, 19, 1786–1788.
(25) Cole, R. J., Cox, R. H., Eds. Toxic lactones. Handbook of Toxic
Fungal Metabolites; Academic Press Inc.: New York, 1981; pp 510ꢀ526.
(26) Lilly, V. G.; Barnett, H. L. Physiology of the Fungi; McGraw-Hill:
New York, 1951.
(27) MacDonald, S.; Long, M.; Gilbert, J. Liquid Chromatographic
method for determination of patulin in clear and cloudy apple juices and
apple puree: collaborative study. J. AOAC Int. 2000, 83, 1387–1394.
(28) Mannina, L.; Viel, S.; Duprꢁe, S.; Pecci, L.; Fontana, M.; Pinnen,
F.; Antonucci, A.; Segre, A. L. Structural elucidation of the oxidation
product of aminoethylcysteine ketimine decarboxylated dimer by per-
oxynitrite. Tetrahedron 2004, 60, 4151–4157.
(29) Patterson, M. K., Jr. Measurement of growth and viability of
cells in culture. Methods Enzymol. 1979, 58, 141–152.
(30) Uliasz, F.; Hewett, S. J. A microtiter trypan blue absorbance
assay for the quantitative determination of excitotoxic neuronal injury in
cell culture. J. Neurosci. Methods 2000, 100, 157–163.
(31) Pfeiffer, E.; Diwald, T. T.; Metzler, M. Patulin reduces glu-
tathione level and enzyme activities in rat liver slices. Mol. Nutr. Food Res.
2005, 49, 329–336.
(32) Scott, P. M.; Kennedy, B.; Van Walbeek, W. Desoxypatulinic
acid from a patulin-producing strain of Penicillium patulum. Experientia
1972, 28, 1252.
(33) Ferrer, E.; Juan-García, A.; Font, G.; Ruiz, M. J. Reactive oxygen
species induced by beauvericin, patulin and zearalenone in CHO-K1
cells. Toxicol. in Vitro 2009, 23, 1504–1509.
(34) Fliege, R.; Metzler, M. Electrophilic properties of patulin.
Adducts structures and reaction pathways with 4-bromothiophenol
and other model nucleophiles. Chem. Res. Toxicol. 2000, 13, 363–372.
(35) Wallen, L. L.; Lyons, A. J.; Pridham, T. G. Antimicrobial activity
of patulin derivatives: a preliminary report. J. Antibiot. 1980,
33, 767–769.
(36) Singh, J. P. In Antibiotics; Gottleib, D., Shaw, P. D., Eds.;
Springer-Verlag: New York, 1967; Vol. I, pp 621ꢀ630.
11578
dx.doi.org/10.1021/jf203098v |J. Agric. Food Chem. 2011, 59, 11571–11578