39th OIV Congress, Brazil 2016
Contribution for food safety in wine: the application of oenological
fining agents to remove ochratoxin A from contaminated white and
red wines
Fernanda Cosme1*, Filipa Carvalho2, Davide Silva2, Luís Filipe-Ribeiro2, Fernando M. Nunes3, Luís
Abrunhosa4, António Inês1
1
Chemical Research Centre - Vila Real (CQ-VR), University of Trás-os-Montes and Alto Douro, School of Life
Sciences and Environment, Department of Biology and Environment, Edificio de Enologia, 5000-801, Vila Real,
Portugal
2
Chemical Research Centre - Vila Real (CQ-VR), University of Trás-os-Montes and Alto Douro, School of Life
Sciences and Environment, 5000-801, Vila Real, Portugal
3
Chemical Research Centre - Vila Real (CQ-VR), University of Trás-os-Montes and Alto Douro, School of Life
Sciences and Environment, Chemistry Department, 5000-801, Vila Real, Portugal
4
CEB-Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal
*Email:[email protected]
Abstract. The occurrence of mycotoxins in wine is of concern for food safety. Mycotoxins are toxic
secondary metabolites produced by certain species of moulds, being ochratoxin A (OTA) one of the
most important. Thus, this work aimed to know the effect of different commercial fining agents on
OTA removal, as well as, their impact on white and red wine physicochemical characteristics. To
assess their effectiveness, 11 commercial oenological products were tested in white and red wines
artificially supplemented with OTA. In white wine, a commercial formulation (MIX) that contains
gelatine, bentonite and activated carbon was the most effective in removing OTA (80%). Eliminations
between 10-30% were obtained with potassium caseinate, mannoprotein and pea protein. With
bentonites, carboxymethylcellulose, chitosan and polyvinylpolypyrrolidone (PVPP) no considerable
OTA was removed. Concerning to red wine, the most effective fining agents were an activated carbon
(66%) and the MIX formulation (55%). Less efficient fining agents were egg albumin, mannoprotein,
pea protein, isinglass, gelatine, PVPP and chitosan, as they removed <19% of OTA present in red
wine. The results obtained provide useful information for winemakers, namely for the selection of the
most appropriate oenological product for the removal of OTA.
Keywords: Red wine, white wine; ochratoxin A, fining, wine physicochemical characteristics, wine
quality
1 Introduction
The occurrence of mycotoxins in foodstuffs including
wine is of concern for food safety. Mycotoxins are toxic
secondary metabolites produced by certain species of
moulds, being ochratoxin A (OTA) one of the most
important [1]. In Europe, wine is estimated to be the
second source of human OTA intake, after cereals. This
mycotoxin is a dihydro-isocoumarin linked at the 7carboxy group to a molecule of L-β-phenylalanine via an
amide bond. Contamination of wines with OTA may
constitute a hazard to consumer health, thus treatments are
necessary to achieve safe levels of OTA for human
consumption [2]. According to the Regulation No.
1881/2006 of the European Commission, the maximum
limit for OTA in wine is 2 µg/kg [3]. Therefore, it is
important to prevent and control their occurrence in wines.
With the purpose to eliminate this toxin, several chemical,
microbiological and physical methods are described in the
literature [1, 4-6]. In the particular case of the wine,
effective technologies that can be applied are more
limited. The use of adsorbents at present is the technology
more used. Thus, the aim of this work is to know the effect
of different commercial fining agents in the removal of
OTA in wine, as well as, their impact on white and red
wine physicochemical characteristics. These results can
provide useful information for winemakers, namely for the
selection of the most appropriate oenological product for
39th OIV Congress, Brazil 2016
OTA removal, reducing wine toxicity and simultaneously
enhancing wine food safety and quality.
comparison of peak areas with a calibration curve made
with an OTA standard (Sigma-Aldrich).
2 Material and methods
2.4 Quantification of non-flavonoids, flavonoids
and total phenols
2.1 Wine characteristics
The white wine used in this work was from the Douro
Valley (vintage 2014) and presented the following
characteristics: alcohol content (10.0%, v/v), specific
gravity at 20 ºC (0.9915 g/mL), titratable acidity (6.7 g/L
tartaric acid), pH 3.00, volatile acidity (0.14 g/L acetic
acid). The red wine used was also from the Douro Valley
(vintage 2014) and their characteristics were as follow:
alcohol content (13.4%, v/v), specific gravity at 20 ºC
(0.9914 g/mL), titratable acidity (5.0 g/L tartaric acid), pH
3.49, volatile acidity (0.35 g/L acetic acid). Wines were
supplemented with OTA at a final concentration of 10
g/L before the fining experiments.
2.2 Experimental design
The commercial oenological products with different
characteristics used in white wine were sodium bentonite B1, calcium bentonite - B2; casein - C;
carboxymethylcellulose - CMC1 and CMC2; chitosan - Q;
polyvinylpolypyrrolidone - PVPP; pea protein - PE;
mannoproteins - MP1 and MP2; and commercial
formulation composed by gelatine, bentonite and activated
carbon – MIX. In red wine, the commercial oenological
products with different characteristics used were chitosan
– Q1 and Q2; PVPP; PE; MP1 and MP2; MIX; egg
albumin – A; isinglass – I; gelatine – G; and activates
carbon - CA. The oenological products were all applied to
the wine at the average dose recommended by the
manufacturer. Each oenological product was added to 250
mL of wine in a graduated cylinder, mixed and allowed to
remain in contact with the wines for 7 days at 20 ºC and
then the supernatant was collected and processed as
described below. All the experiments and analyses were
performed in duplicate.
The phenolic content of the white and red wine was
quantified using the absorbance at 280 nm before and after
precipitation of the flavonoid phenols, through reaction
with formaldehyde, according to Kramling and Singleton
[7]. The results were expressed as gallic acid equivalents
by means of calibration curves with standard gallic acid.
The total phenolic content was also determined by a
spectrophotometric
method,
using
a
UV-Vis
spectrophotometer according to Ribéreau-Gayon et al. [8].
All analyses were performed in duplicate.
2.5 Colour intensity
White wine colour was quantified by measuring
absorbance at 420 nm using a 1 cm path length cell, and
the red wine colour intensity was quantified by measuring
absorbance at 420 nm, 520 nm and 620 nm using a 1 mm
path length cell as described in the O.I.V. methods [9]. All
analyses were performed in duplicate.
2.6 Total anthocyanins
Total anthocyanins were determined using the sulphur
dioxide bleaching method described by Ribéreau-Gayon
and Stonestreet [10]. All analyses were performed in
duplicate.
2.7 Statistical treatment
The data are presented as means ± standard deviation.
Physicochemical data were statistically tested by analysis
of variance (ANOVA) and a post-hoc Tuckey test, using
the Statistica 10 software (Statsoft, OK, USA).
3 Results and discussion
2.3 OTA analysis in white and red wine
3.1 OTA removal from white and red wine
After white and red wine treatment with the different
oenological products, the supernatants were centrifuged at
4000 rpm for 15 min. Then, 2 mL of the supernatant was
collected and added to an equal volume of
acetonitrile/methanol/acetic acid (78:20:2 v/v/v). After 12
hours, the extracts were filtered through a syringe filter
with 0.45 m of porosity and stored at 4 °C until analysed
by
HPLC
with
fluorescence
detection.
The
chromatographic separation was performed on a C18
reversed-phase YMC-Pack ODS-AQ analytical column
(250 x 4.6 mm I.D., 5 mm), fitted with a pre-column with
the same stationary phase. The samples were eluted at a
flow rate of 1 mL/min during 20 min with a mobile phase
consisting of water/acetonitrile/acetic acid (99:99:2 v/v/v).
The injection volume was 50 L and parameters for
detection: λexc = 333 nm, λem = 460 nm and gain = 1000.
The OTA retention time was approximately 16 min. The
OTA concentration in the samples was determined by
The most effective oenological product in removing
OTA from white wine (approx. 80%) was the commercial
formulation Mix. The effect is due to the presence of
activated carbon in the product, a well-known mycotoxin
adsorbent. Reductions of mycotoxin from 10 to 30% were
also obtained in the samples treated with casein, pea
protein
and
mannoproteins.
Bentonites,
carboxymethylcellulose, polyvinylpolypyrrolidone and
chitosan did not produce a considerable reduction of OTA
concentration in white wine (Figure 1). The chromatogram
obtained from the analysis of OTA in the white wine
without treatment is shown is Figure 2A and from the
white wine treated with the commercial formulation MIX
is shown in Figure 2B. There, it can be observed the
reduction in the OTA peak area after the wine treatment.
39th OIV Congress, Brazil 2016
treated with activated carbon is shown in Figure 4B.
There, it can be observed the reduction in the OTA peak
area after the wine treatment.
B
A
OTA
OTA
Figure 1. OTA removal efficiency (%) obtained in white wine
for the evaluated oenological products.
Unfined wine (T), Bentonite (B1 and B2), Casein (C),
Carboxymethylcellulose (CMC1 and CMC2), Commercial
formulation composed by gelatine, bentonite and activated
carbon (MIX), Mannoprotein (MP1 and MP2), Pea protein (PE),
Polyvinylpolypyrrolidone (PVPP) and Chitosan (Q).
Figure 4. (A) Chromatogram from the red wine without
treatment and (B) red wine treated with activated carbon.
3.2 Effect of oenological products on wine total
phenolic compounds, flavonoid and nonflavonoid compounds
OTA
OTA
The impact of oenological fining agents on the total
phenolic compounds, flavonoids and non-flavonoids in
white wine is shown in Figure 5. After white wine fining,
total phenols, non-flavonoid and flavonoid phenols did not
decreased significantly.
Figure 2. (A) Chromatogram from the white wine without
treatment and (B) white wine treated with MIX
In red wine, OTA removals between 6-19% were
obtained with egg albumin, mannoprotein, pea protein,
isinglass, gelatine, polyvinylpolypyrrolidone and chitosan.
The most effective fining agents in removing OTA from
red wine were an activated carbon (66%) followed again
by the commercial formulation composed by gelatine,
bentonite and activated carbon (55%) (Figure 3).
Figure 5. Total phenols, non-flavonoid and flavonoid phenols of
white wine after treatment with different oenological products.
Unfined wine (T), Carboxymethylcellulose (CMC1 and CMC2),
Commercial formulation composed by gelatine, bentonite and
activated carbon (MIX), Mannoprotein (MP1 and MP2),
Chitosan (Q), Pea protein (PE), Bentonite (B1 and B2),
Polyvinylpolypyrrolidone (PVPP) and Casein (C). Means within
a column for each wine followed by the same letter are not
significantly different (Tukey, p<0.05).
Figure 3. OTA removal efficiency (%) obtained in red wine for
the evaluated oenological products.
Unfined wine (T), Egg albumin (A), Mannoprotein (MP1 and
MP2), Isinglass (I), Polyvinylpolypyrrolidone (PVPP), Gelatine
(G), Chitosan (Q1 and Q2), Pea protein (PE), Activated carbon
(CA) and commercial formulation composed by gelatine,
bentonite and activated carbon (MIX).
The chromatogram from the OTA analysis of the red wine
without treatment is shown in Figure 4A and the
chromatogram of the OTA analysis of the same red wine
The impact of oenological fining agents on the total
phenolic compounds, flavonoids and non-flavonoids in red
wine is shown in Figure 6. In red wine, after fining total
phenols and flavonoid compounds decreased significantly.
The non-flavonoid compounds did not decrease
significantly after fining with exception of the wine fined
with activated carbon.
39th OIV Congress, Brazil 2016
reduced colour intensity of red wine in 9.9 % and 4.1 %,
respectively.
Figure 6. Total phenols, non-flavonoid and flavonoid
phenols of red wine after treatment with different
oenological products.
Unfined wine (T), Mannoprotein (MP1 and MP2), Commercial
formulation composed by gelatine, bentonite and activated
carbon (MIX), Polyvinylpolypyrrolidone (PVPP), Pea protein
(PE), Gelatine (G), Isinglass (I), Egg albumin (A), Chitosan (Q1
and Q2) and Activated carbon (CA). Means within a column for
each wine followed by the same letter are not significantly
different (Tukey, p<0.05).
3.3 Effect of oenological fining agents on wine
colour
As it can be observed in Figure 7, that the colour of
white wine after fining with the different oenological
products, decreased significantly only after the addition of
PVPP. In Figure 7, it can also be observed that the
oenological products with capacity to remove OTA from
white wine did not change the colour of the white wine
after their application.
Figure 8. Colour intensity of red wine after treatment with
different oenological products.
Unfined wine (T), Mannoprotein (MP1 and MP2), Commercial
formulation composed by gelatine, bentonite and activated
carbon (MIX), Polyvinylpolypyrrolidone (PVPP), Pea protein
(PE), Gelatine (G), Isinglass (I), Egg albumin (A), Chitosan (Q1
and Q2) and Activated carbon (CA). Means within a column for
each wine followed by the same letter are not significantly
different (Tukey, p<0.05).
3.4 Effect of oenological products on total
anthocyanins of red wine
The influence of oenological products on total
anthocyanins of red wine is shown in Figure 9. The total
anthocyanins of red wine decreased significantly after the
addition of all oenological products. In Figure 7, it also
can be observed that the oenological product with capacity
to remove OTA from red wine (Figure 3), such as MIX
and activates carbon, reduced the total anthocyanins
content in 35.8 %, and 9.7 %, respectively. These results
are in line with the decrease observed for these
oenological products in red wine colour intensity (Figure
8).
Figure 7. Colour of white wine after treatment with
different oenological products.
Unfined wine (T), Carboxymethylcellulose (CMC1 and CMC2),
Commercial formulation composed by gelatine, bentonite and
activated carbon (MIX), Mannoprotein (MP1 and MP2),
Chitosan (Q), Pea protein (PE), Bentonite (B1 and B2),
Polyvinylpolypyrrolidone (PVPP) and Casein (C). Means within
a column for each wine followed by the same letter are not
significantly different (Tukey, p<0.05).
As can be observed in Figure 8, the colour intensity of red
wine after fining with the different oenological products,
decreased significantly after the addition of MIX, PVPP,
pea protein, mannoprotein 1 and activated carbon. In
Figure 8, it can also be observed that the oenological
products with higher capacity to remove OTA from red
wine (Figure 3), such as the MIX and activates carbon
Figure 9. Total anthocyanins of red wine after treatment
with different oenological products.
Unfined wine (T), Mannoprotein (MP1 and MP2), Commercial
formulation composed by gelatine, bentonite and activated
carbon (MIX), Polyvinylpolypyrrolidone (PVPP), Pea protein
(PE), Gelatine (G), Isinglass (I), Egg albumin (A), Chitosan (Q1
and Q2) and Activated carbon (CA). Means within a column for
each wine followed by the same letter are not significantly
different (Tukey, p<0.05).
39th OIV Congress, Brazil 2016
4 Conclusions
The results obtained indicate that some oenological
products could decrease considerably the level of OTA
from white and red wine. The commercial formulation
MIX, composed by gelatine, bentonite and activated
carbon showed the best performances in removing OTA
from wine. In white wine, it removed 80% of the OTA, but
its performance was compromised in red wine as only
55% of the mycotoxin was removed. The most effective
product in red wine was the activated carbon, as it
removed 66% of the OTA. It was also observed that fining
agents without activated carbon in their composition have
little effect in removing OTA from wine. However, in red
wine the oenological products containing activated carbon
in their composition reduced total anthocyanins (CA- 9.7%
and Mix-35.8%), and consequently the colour intensity
(CA- 4.1% and Mix-9.9%). In white wine, total phenols,
non-flavonoid and flavonoid phenols did not decreased
significantly after application of all oenological products.
Those results provide useful information for winemakers,
namely for the selection of the most appropriate
oenological product for the removal of OTA from wines,
enhancing the food safety and wine quality.
Acknowledgements
This work was funded by FEDER funds through the
COMPETE and by national funds through FCT, Ref.
FCOMP-01-0124-FEDER-028029
and
PTDC/AGRTEC/3900/2012, respectively. Luís Abrunhosa received
support through grant nº UMINHO/BPD/51/2015 from
project UID/BIO/04469/2013 financed by FCT/MEC
(OE). This study was also supported by the FCT under the
scope of the strategic funding of UID/BIO/04469/2013
unit and COMPETE 2020 (POCI-01-0145-FEDER006684), by the Project RECI/BBB-EBI/0179/2012
(FCOMP-01-0124-FEDER-027462), and by BioTecNorte
operation (NORTE-01-0145-FEDER-000004) funded by
the European Regional Development Fund under the scope
of Norte2020 - Programa Operacional Regional do Norte.
This work was also partially funded by Chemical Research
Centre of Vila Real (CQ-VR), University of Trás-osMontes and Alto Douro, School of Life Sciences and
Environment.
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