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Journal ArticleDOI

Mechanisms underlying the effect of commercial starter cultures and a native yeast on ochratoxin A production in meat products

01 Jan 2020-Lwt - Food Science and Technology (Academic Press)-Vol. 117, pp 108611

TL;DR: In conclusion, ochratoxigenic fungi do not all respond to antagonistic microorganisms in the same way, and this study sheds some light on the mechanisms behind the different effects of microorganisms.
Abstract: Processed meat products are of worldwide importance, but they are highly prone to fungal and ochratoxin A (OTA) contamination. In previous studies, several Lactic Acid Bacteria (LAB) and yeasts have been tested as biocontrol agents against P. nordicum growth and OTA production in meat products, with promising results. However, A. westerdijkiae has been poorly studied for this matrix. The aim of this work was to evaluate in vitro the mechanisms underlying the effects of a commercial starter culture and of a meat-native Candida zeylanoides strain on the growth and OTA production of P. nordicum and A. westerdijkiae, by co-culture in ham and sausage-based media under different conditions. In ham medium, C. zeylanoides live cells, cell broth and diffused compounds significantly inhibited OTA production by P. nordicum, but live cells of the starter culture significantly increased it. For A. westerdijkiae strong and significant stimulation was observed by direct contact in both media. In conclusion, ochratoxigenic fungi do not all respond to antagonistic microorganisms in the same way. This study sheds some light on the mechanisms behind the different effects of microorganisms.
Topics: Candida zeylanoides (53%)

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LWT - Food Science and Technology
journal homepage: www.elsevier.com/locate/lwt
Mechanisms underlying the eect of commercial starter cultures and a
native yeast on ochratoxin A production in meat products
Sana Meftah
a
, Salwa Abid
b
, Teresa Dias
a
, Paula Rodrigues
a,
a
Centro de Investigação de Montanha, Instituto Politécnico de Bragança, Campus de Santa Apolónia, 5300-253 Bragança, Portugal
b
Laboratory for Research on Biologically Compatible Compounds, Faculty of Dentistry, Rue Avicenne, 5019, Monastir, Tunisia
ARTICLE INFO
Keywords:
Mycotoxins
Biocontrol
Food safety
Pork meat products
ABSTRACT
Processed meat products are of worldwide importance, but they are highly prone to fungal and ochratoxin A
(OTA) contamination. In previous studies, several Lactic Acid Bacteria (LAB) and yeasts have been tested as
biocontrol agents against P. nordicum growth and OTA production in meat products, with promising results.
However, A. westerdijkiae has been poorly studied for this matrix.
The aim of this work was to evaluate in vitro the mechanisms underlying the eects of a commercial starter
culture and of a meat-native Candida zeylanoides strain on the growth and OTA production of P. nordicum and A.
westerdijkiae, by co-culture in ham and sausage-based media under dierent conditions.
In ham medium, C. zeylanoides live cells, cell broth and diused compounds signicantly inhibited OTA
production by P. nordicum, but live cells of the starter culture signicantly increased it. For A. westerdijkiae strong
and signicant stimulation was observed by direct contact in both media.
In conclusion, ochratoxigenic fungi do not all respond to antagonistic microorganisms in the same way. This
study sheds some light on the mechanisms behind the dierent eects of microorganisms.
1. Introduction
Mycotoxins are natural substances produced as secondary metabo-
lites of several lamentous fungi. They have worldwide distribution and
aect a signicant part of food and feed products. Mycotoxins pose a
health risk to humans and animals due to their harmful biological
properties. They also have a very wide economic impact resulting from
health and veterinary care costs, reduction in livestock production,
investment in research to reduce risks of mycotoxin problems (Zain,
2011).
Processed meat products such as dry-cured ham, fermented sausage
and others are foods of major importance in several European countries,
both nutritionally and economically. However, due to their character-
istics, they are highly exposed to mycotoxin producing fungi.
Ochratoxin A (OTA) is the most signicant mycotoxin found in
processed meat products. Because it is strongly adapted to salt and
protein-rich matrices and is moderately psychrotrophic, Penicillium
nordicum has been associated with OTA contamination of these pro-
ducts, but Aspergillus westerdijkiae has been recently associated with
high levels of OTA in meat products (Meftah, Abid, Dias, & Rodrigues,
2018; Merla et al., 2018; Vipotnik, Rodríguez, & Rodrigues, 2017).
The development of ecient strategies to avoid it from entering the
food chain is on the top of research. One of the most promising stra-
tegies under study is to prevent its accumulation by creating the best
conditions to inhibit OTA production. Several studies have reported
Lactic Acid Bacteria (LAB) and yeasts as promising biocontrol agents
(BCAs) against P. nordicum growth and OTA production in meat pro-
ducts (Andrade, Thorsen, Rodríguez, Córdoba, & Jespersen, 2014;
Iacumin, Manzano, Andyanto, & Comi, 2017; Rodriguez et al., 2015;
Simoncini, Virgili, Spadola, & Battilani, 2014; Virgili et al., 2012), but
only one of these studies has considered the eect of these micro-
organisms on A. westerdijkiae (Meftah et al., 2018).
The mechanisms by which BCAs aect fungal growth can be due to
competition for nutrients and space (Hernández-Montiel, Ochoa, Troyo-
Diéguez, & Larralde-Corona, 2010), production of hydrolytic enzymes
or killer toxins (Masih & Paul, 2002) and secretion of volatile com-
pounds (Masoud, Poll, & Jakobsen, 2005; Taczman-Brückner Mohácsi-
Farkas, Balla & Kiskó, 2005; Fialho, Toano, Pedroso, Augusto, &
Pascholati, 2009; Fiori et al., 2014; Farbo et al., 2016). Several yeasts
such as Debaryomyces hansenii, Hyphopichia burtonii and Candida zeyla-
noides
were reported as strong inhibitors of P.
nordicum growth in dry-
cured ham and dry fermented sausage (Andrade et al., 2014; Nuñez
et al., 2015; Virgili et al., 2012). Bacteria of the Pediococcus and Lac-
tobacillus genera have also been studied as potential BCAs against fungi
https://doi.org/10.1016/j.lwt.2019.108611
Received 30 May 2019; Received in revised form 5 August 2019; Accepted 9 September 2019
Corresponding author.
E-mail address: prodrigues@ipb.pt (P. Rodrigues).
LWT - Food Science and Technology 117 (2020) 108611
Available online 10 September 2019
0023-6438/ © 2019 Elsevier Ltd. All rights reserved.
T

and mycotoxins in several food matrices, with inhibiting eects being
observed (Abrunhosa et al., 2014; Ngang et al., 2015; Pereira et al.,
2016).
Our group has previously studied the eect of a commercial starter
culture composed by LAB, CNC and D. hansenii and of several
Portuguese dry-sausage endogenous yeasts on P. nordicum and A. wes-
terdijkiae growth and OTA production ability in a Portuguese-style dry-
fermented sausage-based medium (Meftah et al., 2018). That study
concluded that A. westerdijkiae was signicantly stimulated to produce
OTA in meat products by the native yeast C. zeylanoides and by the
commercial starter culture. The same starter culture also stimulated
OTA production by P. nordicum. The present work aimed to evaluate in
vitro the mechanisms underlying the observed eects of the yeast and
the starter culture on these ochratoxigenic fungi.
2. Materials and methods
2.1. Microorganisms and inocula preparation
2.1.1. Yeasts and starter culture
A strain of C. zeylanoides (Cz) previously isolated from Portuguese
traditional dry-fermented sausage and identied molecularly (Meftah
et al., 2018) was sub-cultured from stock vials onto Potato Dextrose
Agar (PDA, Liolchem-ITALY) and incubated at 28 °C. The preparation
of the pre-inoculum followed the procedure described by Meftah et al.
(2018). Briey, one colony from 3 day old cultures was suspended in
Potato Dextrose Broth (PDB, Liolchem-ITALY) and incubated at 28 °C
for 24 h in a rotary shaker (120 rpm). Optical density of the suspension
was determined by spectrophotometry at 600 nm wave length and an
inoculum with approximately 10
5
cells/mL was prepared for all assays.
Cell concentration was estimated by interpolation of absorbance values
measured at 600 nm using the relationship OD600 = 1.0 corresponding
to 3 × 10
7
cells/mL, as described by Day, Schneider, and Schneider
(2004).
The commercial freeze-dried starter culture (Texel®ELCE Br,
Danisco) composed of Pediococcus pentosaceus, Lactobacillus sakei,
Staphylococcus carnosus, Staphylococcus xylosus and Debaryomyces han-
senii was also used. The freeze-dried starter culture (0.01% w/v) was
inoculated in MRS (de Man, Rogosa, Sharpe) broth and incubated at
37 °C for 24 h. After that, 300 μL were used to inoculate 150 mL of each
meat extract media.
2.1.2. Ochratoxigenic fungi
Two species of OTA-producing fungi previously isolated from cured
pork meat were used: A. westerdijkiae MUM16.142 (previously identi-
ed as strain 6B/131) and P. nordicum strain PN 44 (provided by Dr.
Alicia Rodríguez, University of Extremadura, Spain). Fungi were in-
oculated in Malt Extract Agar (MEA, Liolchem-ITALY) and incubated
for 10 days at 25 °C in the dark. After incubation, 2 mL of 0.05% Tween
80 solution were added to the culture and spores were rubbed to obtain
a suspension, adjusted to 10
7
spores/mL with the aid of a Neubauer
counting chamber.
2.2. Meat extract media preparation
Media based on traditional sausage (TSM) and on ham (HM) were
prepared as previously described in Meftah et al. (2018). Thirty grams
of minced and lyophilized meat product were boiled in 1000 mL of
distilled water (3% meat) during 30 min and ltered through a cheese
cloth. Meat extract was supplemented with 3% NaCl, and the volume
was made up to 1000 mL. Media were solidied with 2% of bacter-
iological agar (HiMedia), and were autoclaved at 121 °C for 15 min.
pH was measured in triplicate with a pH-meter (METTLER
TOLEDO) and corrected to 5.5 whenever necessary. Water activity was
measured in triplicate using a water activity meter (Aqualab 4 TE).
2.3. Mechanisms of action of biocontrol agents on fungal growth and OTA
production
To understand the mechanisms of action of the yeast C. zeylenoides
and of the starter culture, fungi were co-inoculated with each test or-
ganism in dierent ways: 1) Eect of live cells was tested by in-
corporation of living cells in the medium, as positive control (the same
condition as previously tested); 2) Eect of dead cells, was tested by
incorporation of dead cells in the medium to determine if the eect on
fungi was caused by a structural compound of the cell; 3) Eect of cell-
free culture ltrate, by using the ltered broth where yeast/starter were
previously grown as culture medium, to determine if the eect was
caused by a compound previously produced by the yeast/starter and
diused to the medium; 4) Eect of diusible compounds, fungi and
yeast/starter where co-inoculated at a given distance, to study if the
eect was caused by yeast/starter compounds being produced si-
multaneously with the fungus and being diused towards the fungus;
and 5) Eect of volatile compounds, fungi and yeast/starter were co-
inoculated without direct contact (cut culture medium), to verify if the
microorganisms produced a volatile compound which inuenced fungal
growth and OTA production.
Two culture media TSM and HM supplemented with 3% NaCl
and one temperature 20 °C were used with C. zeylanoides and starter
culture. For all assays, each fungus was inoculated in 9 cm Petri dishes
by two-point inoculation with 2 μL spore suspension, in triplicate. After
co-inoculation, all petri dishes were incubated at 20 °C for 14 days.
2.3.1. Eect of live cells, dead cells and cell-free broth
C. zeylanoides and starter culture cell suspensions were prepared as
previously described. The suspensions were inoculated in 300 mL of
TSM and HM broths, and incubated in a rotary shaker (120 rpm) at
28 °C and at 37 °C, respectively. After 24 h of incubation, the broth was
homogenized and divided into 2 portions. One portion was directly
used for the live cells test. From the other portion, cells were cen-
trifuged at 5000 rpm for 10 min. The resulting pellet was resuspended
in 10 mL of sterile water and autoclaved at 120 °C for 15 min to kill the
cells. Dead cells were incorporated in new a portion of each media. The
supernatant was further
ltered with 0.22 μm lters and used for the
cell-free broth test. For each media portion, 2% of agar were aseptically
added and the media with the live cells, dead cells and cell-free were
distributed in 9 cm Petri dishes. Fungi were then inoculated as de-
scribed.
2.3.2. Eect of diusible compounds
To verify the inuence of the yeast and the starter on fungal growth
and OTA production by producing diusible compounds, 2 μL of spore
suspension and 2 μL of starter or C. zeylanoides were inoculated in op-
posite quadrants of the Petri dish.
2.3.3. Eect of volatile compounds
Twenty mL of each meat-based medium were plated in 9 cm petri
dishes and then divided into 4 quadrants by cutting 3 mm lanes of agar
perpendicularly, to avoid the contact of diusible compounds poten-
tially produced by test microorganisms with the fungus. Then, 2 μ Lof
spore suspension were added in two opposite quarters and 2 μL of yeast
or starter suspension were added to the other 2 opposite parts.
2.4. Fungal growth assessment
Fungal growth was determined throughout the incubation period
every two days until colony coalescence, by orthogonal measurements
of fungal colonies diameter. At the end of incubation period, all Petri
dishes were submitted to OTA analysis as described below. Petri dishes
without test microorganisms (fungi only) were used as control. All tests
were run in triplicate.
S. Meftah, et al.
LWT - Food Science and Technology 117 (2020) 108611
2

2.5. OTA analysis
2.5.1. OTA extraction
OTA was extracted from plates after 14 days of incubation. Three
agar plugs were removed from the inner, middle and outer areas of the
colony, weighted and extracted with 1.5 mL of methanol as described
by Bragulat et al. (2001). The agar plugs were maintained in methanol
for 1 h and vortexed every 15 min. The extracts were ltered by PTFE
0.2 μm syringe lters and stored at 4 °C until further analysis.
2.5.2. OTA detection and quantication by High Performance Liquid
Chromatography (HPLC)
OTA was analysed as described by Meftah et al. (2018), with a High
Performance Liquid Chromatography (HPLC) system equiped with:
Smartline pump (1000, Knauer, Berlin, Germany), degasser system
(Smartline manager 5000), auto-sampler (AS-2057, Jasco, Easton, MD,
USA), and a uorescence detector (FP-2020, Jasco) set to λex 330 nm
and λem 463 nm. Data were analysed using Clarity 2.4 Software (Da-
taApex, Prague, Czech Republic). The chromatographic separation was
achieved using an isocratic elution with a C18 reverse-phase column
(100 × 4.6 mm, Merck Chromolith Performance, Darmstadt, Germany)
operating at 35 °C (7971 R Grace oven). The mobile phase consisted of
an isocratic programme of water: acetonitrile: acetic acid (29.5:70:0.5,
v/v/v) and was pumped at 0.8 mL/min for a total run time of 4 min.
The injection volume was 10 μL. Under these conditions, retention time
for OTA was 2.2 min.
2.6. Statistical analysis
Statistical analysis was performed using IBM® SPSS® Statistics
v.22.0 software (Armonk, NY: IBM Corp.). For the comparison of
means, samples were rst tested for normal distribution by Shapiro-
Wilk test and for homogeneity of variances by Levene's test. Since
samples generally followed these criteria, t-student test and One-way
ANOVA were used for comparison of means for 2-level variables and for
more than 2-level variables, respectively. Post-hoc analyses were per-
formed with Dunett test to create condence intervals for dierences
between the mean of each factor level and the mean of a control group.
In all cases, statistical signicance was established at p < 0.05.
3. Results
3.1. Candida zeylanoides
The results of fungal colony diameter of P. nordicum and A. wes-
terdijkiae growth co-cultured with C. zeylanoides under dierent
methods in TSM and HM with 3% NaCl incubated at 20 °C for 14 days
are represented in Figs. 1 and 2. The results of OTA production are
represented in Fig. 3. C. zeylanoides had a similar eect on growth of
both P. nordicum and A. westerdijkiae. On the contrary, growth was
generally increased with the incorporation of dead cells and of cell
broth.
3.2. Starter culture
The results of P. nordicum and A. westerdijkiae, as well as OTA
production relative to dierent
methods of co-culture with the com-
mercial starter culture, in TSM and HM with 3% NaCl incubated at
20 °C for 14 days are represented respectively in Figs. 4 and 5. Results
of P. nordicum growth showed that the incorporated live cells reduced
fungal growth signicantly only in HM (p < 0.05), whereas cell broth
and dead cells signicantly stimulated fungal growth in both media. In
the case of A. westerdijkiae growth, no signicant eect was detected
between control and test conditions and also between treatments. On
the other hand, a signicant stimulation of OTA production in both
media occurred with the incorporation of live cells and in the volatiles
test.
4. Discussion and conclusion
The mechanisms by which BCAs aect fungal growth and myco-
toxin production are not always well understood, and this can be a
limitation to their industrial use. BCAs have been reported to act by
competition for nutrients and space (Hernández-Montiel et al., 2010;
Zhao, Tu, Shao, Jing, & Su, 2008), production of hydrolytic enzymes or
killer toxins (Marquina, Barroso, Santos, & Peinado, 2001; Masih &
Paul, 2002; Hernández et al., 2010) and, also frequently reported, se-
cretion of volatile compounds (Masoud et al., 2005; Taczman-Brückner,
Mohácsi-Farkas, Balla, & Kiskó, 2005; Fialho et al., 2009; Fiori et al.,
2014; Farbo et al., 2016). OTA adsorption to the yeast cell wall
(Bejaoui, Mathiue, Taillandier, & Lebrihi, 2004; Shetty, Hald, &
Jespersen, 2007) and the inuence on regulation of the mycotoxin
biosynthesis at a transcriptional level have been described (Gil-Serna,
Patiño, Cortés, González-Jaén, & Vazquez, 2011; Peromingo, Núñez,
Rodríguez, Alía, & Andrade, 2018) have also been previously described.
Several yeasts such as D. hansenii, H. burtonii and C. zeylanoides were
reported as strong inhibitors of P. nordicum growth in dry-cured ham
and dry fermented sausage (Andrade et al., 2014; Nuñez et al., 2015;
Virgili et al., 2012), but their eect on A. westerdijkiae remains without
information. Bacteria of the Pediococcus and Lactobacillus
genera have
also
been studied as potential BCAs against fungi and mycotoxins in
several food matrices, with inhibiting eects being observed
(Abrunhosa et al., 2014; Ngang et al., 2015; Pereira et al., 2016).
In the present study, the mechanisms of action of the yeast C. zey-
lanoides and of a commercial starter culture used for sausage fermen-
tation on growth and OTA production by P. nordicum and A. wester-
dijkiae were tested. The results obtained for P. nordicum growth showed
that the incorporated live cells reduced fungal growth in HM, whereas
cell broth and dead cells signicantly stimulated fungal growth in both
media. For P. nordicum, growth patterns are similar for both media
suggesting that cell broth or a cell component can be exerting the sti-
mulation eect. In respect to OTA production, P. nordicum produced
detectable levels only in HM. While the incorporation of live cells sig-
nicantly stimulated OTA production, a signicant decrease was ob-
served for both cell broth and incorporated dead cells. No signicant
eect was observed on OTA production in diusible and volatile tests.
Direct contact between the starter culture and the fungus seems to exert
a signicant eect on the fungus, maybe as a result of competitive
exclusion, based on competition for nutrient and space (Andrade et al.,
2014; Hernández-Montiel et al., 2010; Zhao et al., 2008). This stress,
while reducing fungal growth, seems to induce secondary metabolism
with consequent mycotoxin production, as reported frequently
(Vipotnik et al., 2017). In fact, fungal secondary metabolism is strongly
stimulated by sub-optimal conditions like nutrient depletion or meta-
bolites produced by other organisms. Since OTA is inhibited by cell
broth and by dead cells we can assume that the starter metabolites
potentially responsible for OTA induction are only produced as re-
sponse to the presence of the fungus in co-culture with live starter cells.
In the cell broth and in contact with dead cells, not only starter meta-
bolites are not produced, but also extra nutrients are being oered to
the fungus, thus reducing fungal stress with consequent increased
growth and decreased OTA production. In a biocontrol test with D.
hansenii against P. nordicum, Andrade et al. (2014) compared the eect
of cell-free supernatants with those from live cells and concluded that
the main mode of the antagonistic activity was based on competition for
nutrients and space and, to a minor extent, the production of extra-
cellular compounds such as volatile compounds or killer proteins.
In the case of A. westerdijkiae, there was no signicant eect of the
starter culture on fungal growth between control and test conditions
and also between treatments. On the other hand, a signicant
stimu-
lation of OTA production in both TSM and HM occurred with the in-
corporation of live cells and in the volatiles test. The direct contact of
S. Meftah, et al.
LWT - Food Science and Technology 117 (2020) 108611
3

live starter cells seems to have a signicant impact on A. westerdijkiae,
which is stronger in TSM. This could be explained by the fact that the
starter culture is more adapted to this matrix assuming higher or faster
growth, and consequently stronger eect in this medium. Cell super-
natant and dead cells do not seem to exert any type of eect on A.
westerdijkiae. On the contrary, volatile compounds being produced by
the starter had a signicant negative impact by increasing OTA pro-
duction by this fungus.
Growth data for the incorporation test for both fungi are in
agreement with the results previously obtained (Meftah et al., 2018),
where the incorporation of live cells signicantly inhibited P. nordicum
and A. westerdijkiae growth in both media. These eects on growth are
similar to those registered and discussed for the starter culture.
As previously demonstrated, P. nordicum does not produce detect-
able amounts of OTA in TSM (Meftah et al., 2018). However, in HM it
produced 123 ng/g. C. zeylanoides inhibited signicantly OTA produc-
tion by P. nordicum under three test conditions: incorporation of live
cells, cell broth and diusion. Since no OTA was detected in the control
Fig. 1. Growth of P. nordicum and A. westerdijkiae in co-culture with C. zeylanoides, cultured in ham and traditional sausage media at 20 °C.
Fig. 2. Growth of P. nordicum and A. westerdijkiae in co-culture with C. zeylanoides, in ham medium (top; black bars) and traditional sausage medium (bottom; light
bars) at 20 °C. In all cases, results are the average of six replicates; standard deviation is indicated as vertical thin lines. * Signicantly dierent from control of each
situation (without C. zeylanoides), p < 0.05.
S. Meftah, et al.
LWT - Food Science and Technology 117 (2020) 108611
4

Fig. 3. OTA production by P. nordicum and A. westerdijkiae in co-culture with C. zeylanoides, in ham medium (top; black bars) and traditional sausage medium
(bottom; light bars) at 20 °C. In all cases, results are the average of six replicates; standard deviation is indicated as vertical thin lines. * Signicantly dierent from
control of each situation (without C. zeylanoides), p < 0.05.
Fig. 4. Growth of P. nordicum and A. westerdijkiae in co-culture with starter culture, in ham medium (top; black bars) and traditional sausage medium (bottom; light
bars) at 20 °C. In all cases, results are the average of six replicates; standard deviation is indicated as vertical thin lines. * Signicantly dierent from control of each
situation (without starter culture), p < 0.05.
S. Meftah, et al.
LWT - Food Science and Technology 117 (2020) 108611
5

Figures (5)
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Abstract: Recently, specific dry-cured hams have started to be produced in San Daniele and Parma areas. The ingredients are similar to protected denomination of origin (PDO) produced in San Daniele or Parma areas, and include pork leg, coming from pigs bred in the Italian peninsula, salt and spices. However, these specific new products cannot be marked as a PDO, either San Daniele or Parma dry cured ham, because they are seasoned for 6 months, and the mark PDO is given only to products seasoned over 13 months. Consequently, these products are called short-seasoned dry-cured ham (SSDCH) and are not branded PDO. During their seasoning period, particularly from the first drying until the end of the seasoning period, many molds, including Eurotium spp. and Penicillium spp., can grow on the surface and work together with other molds and tissue enzymes to produce a unique aroma. Both of these strains typically predominate over other molds. However, molds producing ochratoxins, such as Aspergillus ochraceus and Penicillium nordicum, can simultaneously grow and produce ochratoxin A (OTA). Consequently, these dry-cured hams may represent a potential health risk for consumers. Recently, Aspergillus westerdijkiae has been isolated from SSDCHs, which could represent a potential problem for consumers. Therefore, the aim of this study was to inhibit A. westerdijkiae using Debaryomyces hansenii or Lactobacillus buchneri or a mix of both microorganisms. Six D. hansenii and six L. buchneri strains were tested in vitro for their ability to inhibit A. westerdijkiae. The strains D. hansenii (DIAL)1 and L. buchneri (Lb)4 demonstrated the highest inhibitory activity and were selected for in situ tests. The strains were inoculated or co-inoculated on fresh pork legs for SSDCH production with OTA-producing A. westerdijkiae prior to the first drying and seasoning. At the end of seasoning (six months), OTA was not detected in the SSDCH treated with both microorganisms and their combination. Because both strains did not adversely affect the SSDCH odor or flavor, the combination of these strains are proposed for use as starters to inhibit OTA-producing A. westerdijkiae.

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26 May 2020
TL;DR: The aim of this work was to select GCC+ isolates with antifungal activity to study its effectiveness in a dry-cured ham model system at the environmental conditions reached during the ripening, and showed that the inoculation of S. xylosus completely inhibited the growth of most fungi.
Abstract: Toxigenic moulds can develop on the surface of dry-cured meat products during ripening due to their ecological conditions, which constitutes a risk for consumers. A promising strategy to control this hazard is the use of antifungal microorganisms usually found in these foods. However, to date, the effectiveness of gram-positive catalase-positive cocci (GCC+) has not been explored. The aim of this work was to select GCC+ isolates with antifungal activity to study its effectiveness in a dry-cured ham model system at the environmental conditions reached during the ripening. Forty-five strains of GCC+ were evaluated and the isolate Staphylococcus xylosus Sx8 was selected to assess its efficacy at two different concentrations (106 and 104 cfu/mL) against Penicillium nordicum, Aspergillus flavus, Aspergillus parasiticus, and Penicillium griseofulvum at 15, 20, and 25 °C. The results showed that the inoculation of 106 cfu/mL of S. xylosus completely inhibited the growth of most fungi. In addition, in the presence of this strain at 104 cfu/mL, a significant reduction in fungal growth and mycotoxins production was observed at the three temperatures studied. In conclusion, S. xylosus Sx8 possesses great potential as a biological agent to control toxigenic moulds in dry-cured meat products.

1 citations


Cites background from "Mechanisms underlying the effect of..."

  • ...In fact, fungal secondary metabolism is strongly stimulated by sub-optimal conditions like nutrient depletion or metabolites produced by other organisms [56]....

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Journal ArticleDOI
26 Oct 2020
TL;DR: Number of studies finding report indicate that Ochratoxin A was existed in several processed and unprocessed food stuffs, species and different alcoholic beverage.
Abstract: Ochratoxin A (OTA) is a mycotoxin produced by several fungal species including Aspergillus ochraceus, A. carbonarius, A. niger and Penicillium verrucosum. Various studies report shown that Ochratoxin A can be leads several health problems for both animal and human health through the consumption of Ochratoxin A contaminated plant and animal origin foods. For instance, OTA has been shown to be nephrotoxic, teratogenic, immunotoxic, and carcinogenic in human health. Therefore, the main aim of this review focused on the occurrence, analytical methods and the condition for the formation of Ochratoxin A. Number of studies finding report indicate that Ochratoxin A was existed in several processed and unprocessed food stuffs, species and different alcoholic beverage. Primarily, cereals and cereals contained food products have highly vulnerable for Ochratoxin A due the presence of high moisture contents. On the other hand, several environmental conditions are playing an important for the formation of Ochratoxin A in different food stuffs. For example, most important abiotic factors which influence the growth and OTA production by such Spoilage fungi include water availability, temperature and gas composition. Finally, several analytical methods are used for detection of Ochratoxin A from different plant and animal origin foods such as Thin layer chromatography, Enzyme linked immunosorbent assay and High performance liquid chromatography. However, based on the sensitivity, resolution and efficiency currently high performance liquid chromatography techniques are more popular and advanced techniques for Ochratoxin A detection.

1 citations


Cites background from "Mechanisms underlying the effect of..."

  • ...For instance, in cereal, milk, meat and species [12-15] in alcoholic beverage such as beer and wine [16, 17]....

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Journal ArticleDOI
Miao Zhang1, Haijun Qiao1, Weibing Zhang1, Zhongming Zhang1  +2 moreInstitutions (1)
Abstract: This study aimed to the variations of fungal diversity and community structure in different parts of traditional homemade Sichuan pork bacon. A total of seven phyla and 91 fungal genera were identified. Among them, Ascomycota and Basidiomycota were the first and second most abundant phyla in the bacon samples. In addition, five dominant genera (Debaryomyces, Aspergillus, Candida, Malassezia, and Penicillium) were shared by all bacon samples. The numbers of OTUs unique to individual groups were 14, 67, and 65 for the muscle tissue, the adipose tissue, and pork skin, respectively. Linear discriminant analysis showed that a total of 31 taxa significantly differed among the groups. Results of Nonmetric Multidimensional Scaling and unweighted pair-group analysis indicated that physicochemical characteristics of bacon tissue were a major factor in shaping the bacon microbial communities. Results of network analysis also indicated that tissue type was a crucial factor influencing the fungal interactions in different samples. This study can lay a foundation for further isolation and identification of fungi in the product and provides a basis for further research of food ecology in homemade traditional pork bacon.

References
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Journal ArticleDOI
Abstract: Mycotoxins are secondary metabolites of molds that have adverse effects on humans, animals, and crops that result in illnesses and economic losses. The worldwide contamination of foods and feeds with mycotoxins is a significant problem. Aflatoxins, ochratoxins, trichothecenes, zearalenone, fumonisins, tremorgenic toxins, and ergot alkaloids are the mycotoxins of greatest agro-economic importance. Some molds are capable of producing more than one mycotoxin and some mycotoxins are produced by more than one fungal species. Often more than one mycotoxin is found on a contaminated substrate. Mycotoxins occur more frequently in areas with a hot and humid climate, favourable for the growth of molds, they can also be found in temperate zones. Exposure to mycotoxins is mostly by ingestion, but also occurs by the dermal and inhalation routes. The diseases caused by exposure to mycotoxins are known as mycotoxicoses. However, mycotoxicoses often remain unrecognized by medical professionals, except when large numbers of people are involved. Factors influencing the presence of mycotoxins in foods or feeds include environmental conditions related to storage that can be controlled. Other extrinsic factors such as climate or intrinsic factors such as fungal strain specificity, strain variation, and instability of toxigenic properties are more difficult to control. Mycotoxins have various acute and chronic effects on humans and animals (especially monogastrics) depending on species and susceptibility of an animal within a species. Ruminants have, however, generally been more resistant to the adverse effects of mycotoxins. This is because the rumen microbiota is capable of degrading mycotoxins. The economic impact of mycotoxins include loss of human and animal life, increased health care and veterinary care costs, reduced livestock production, disposal of contaminated foods and feeds, and investment in research and applications to reduce severity of the mycotoxin problem. Although efforts have continued internationally to set guidelines to control mycotoxins, practical measures have not been adequately implemented.

830 citations


Journal ArticleDOI
TL;DR: To assess, for the first time, the efficiency in removing ochratoxin A from laboratory medium, synthetic grape juice medium, and natural grape juice by viable and dead oenological Saccharomyces strains compared with a commercial yeast walls additive.
Abstract: Aims: To assess, for the first time the efficiency in removing ochratoxin A (OTA) from laboratory medium [yeast peptone glucose (YPG)], synthetic grape juice medium (SGM) and natural grape juice by viable and dead (heat and acid-treated) oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) compared with a commercial yeast walls additive. Methods and Results: Levels of OTA during its interaction with six oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) or with a commercial yeast walls additive in YPG medium, in SGM or in natural grape juices was assessed by HPLC after appropriate extraction methods. A significant decrease of OTA levels in YPG medium and SGM was observed for many of the growing strains reaching a maximum of 45%, but no degradation products were detected. With both heat and acid pretreated yeasts, OTA removal was enhanced, indicating that adsorption, not catabolism, is the mechanism to reduce OTA concentrations. Adsorption was also improved when the yeast concentration was increased and when the pH of the medium was lower. Approximately 90% of OTA was bound rapidly within 5 min and up to 72 h of incubation with heat-treated cells of either S. cerevisiae or S. bayanus. A comparative study between heat-treated cells (HC) and commercial yeast walls (YW) (used as oenological additive), introduced at two different concentrations (0·2 and 6·7 g l−1) in an OTA-contaminated grape juice, showed the highest efficiency by HC to adsorb rapidly within 5 min the total amount of the mycotoxin. Conclusions: Oenological S. cerevisiae and S. bayanus were able to remove ochatoxin A from synthetic and natural grape juices. This removal was rapid and improved by dead yeasts having more efficiency than commercial yeast walls. Significance and Impact of the Study: The efficiency of heat-treated yeasts to remove OTA gives a new hope for grape juice and must decontamination avoiding negative impacts on human health.

196 citations


Journal ArticleDOI
TL;DR: The results obtained show that some strains of S. cerevisiae, viable or non-viable, are effective aflatoxin binders and these properties should be considered in the selection of starter cultures for relevant indigenous fermented foods where high a Flatoxin level is a potential health risk.
Abstract: Saccharomyces cerevisiae constitutes one of the most important microorganisms involved in food fermentations throughout the world. Aflatoxin B(1) binding abilities of S. cerevisiae strains isolated from indigenous fermented foods from Ghana, West Africa were tested in vitro. Results show that aflatoxin binding was strain specific with 7 strains binding 10-20%, 8 strains binding 20-40% and 3 strains binding more than 40% of the added aflatoxin B(1) when grown and incubated under standard conditions. Binding by two of the strains was further characterized. Highest binding capacity was seen with cells collected at the exponential growth phase with the strains A18 and 26.1.11 binding 53.0 and 48.8% of the total toxin respectively and the binding reduced towards the stationary phase. Aflatoxin B(1) binding increased steadily when the cells were incubated with 1 to 20 microg/ml of aflatoxin B(1). Binding was not affected by the cells grown at temperatures ranging from 20 to 37 degrees C, but was significantly reduced at 15 degrees C. Binding seems to be a physical phenomenon with cells treated at 52, 55 and 60 degrees C for 5 and 10 min or 120 degrees C for 20 min binding significantly higher quantities (more than 2-fold in 120 degrees C treated cells) of aflatoxin B(1) than their viable counterpart. Similarly, when the cells were treated with 2 M HCl for 1 h, up to 2-fold increase in binding was observed. The results obtained show that some strains of S. cerevisiae, viable or non-viable, are effective aflatoxin binders and these properties should be considered in the selection of starter cultures for relevant indigenous fermented foods where high aflatoxin level is a potential health risk.

164 citations


Journal ArticleDOI
Yan Zhao1, Kang Tu1, Xingfeng Shao1, Wei Jing1  +1 moreInstitutions (1)
TL;DR: Tomato fruit is capable of responding to the yeast P. guilliermondii, which could activate defensive enzymes and thereby induce host disease resistance and could be one of the mechanisms for biocontrol.
Abstract: The yeast Pichia guilliermondii was examined for its ability to control Rhizopus nigricans on tomato fruit during storage, and in order to highlight the reason for biocontrol, a possible mode of action is discussed. Results showed that autoclaved yeast culture and culture filtrate had no effect on controlling the postharvest disease caused by R. nigricans, although inoculation of P. guilliermondii prior to R. nigricans resulted in enhanced biocontrol efficacy. Moreover, rapid colonization of the yeast on wound sites was observed during the initial 3 days at 20 °C, and then the population stabilized for the remaining 4 days. This phenomenon indicated that at room temperature, P. guilliermondii could acclimatize itself to the environment of tomato fruit wounds and occupy the living space quickly. The results indicate that P. guilliermondii did not produce an antifungal substance, however, competition for nutrients and space on wounds appeared to play a role in the activity of the biocontrol and could be one of the mechanisms. In addition, the fruit inoculated with P. guilliermondii demonstrated changes in peroxidase (POD), polyphenoloxidase (PPO), superoxide dismutase (SOD), catalase (CAT), phenylalanine ammonia-lyase (PAL), chitinase (CHI) and β-1,3-glucanase activities, all of which were correlated with the onset of induced resistance. This result suggests that tomato fruit is capable of responding to the yeast P. guilliermondii, which could activate defensive enzymes and thereby induce host disease resistance.

109 citations


Journal ArticleDOI
Emmanuel Isaac Masih1, Bernard Paul1Institutions (1)
TL;DR: In vitro experiments confirm that this yeast can be used as a biological control organism against B. cinerea and an account of this antagonism and the production of β-1,3-glucanases by P. membranifaciens is given here.
Abstract: Pichia membranifaciens strain FY-101, isolated from grape skin, was found to be antagonistic to Botrytis cinerea, the causal organism of the grey mold disease of the grapevine. When grown together on solid as well as in liquid media, the yeast brings about the inhibition of Botrytis cinerea, which in turn loses its ability to produce the grey mold symptoms on the grapevine plantlets. The secretion of β-1,3-glucanases by P. membranifaciens is one of the possible mechanisms related to this antagonism. In vitro experiments confirm that this yeast can be used as a biological control organism against B. cinerea. An account of this antagonism and the production of β-1,3-glucanases by P. membranifaciens is given here.

108 citations


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20205