Vitality and detoxification ability of yeasts

in naturally As-rich musts

Daniela Bertoldi, Toms Romn,

Raffaele Guzzon, Alessandro Santato,
Mario Malacarne, Giorgio Nicolini &
Roberto Larcher
European Food Research and
Technology
Zeitschrift fr LebensmittelUntersuchung und -Forschung A
ISSN 1438-2377
Eur Food Res Technol
DOI 10.1007/s00217-016-2664-6

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Eur Food Res Technol
DOI 10.1007/s00217-016-2664-6

ORIGINAL PAPER

Vitality anddetoxification ability ofyeasts innaturally Asrich

musts
DanielaBertoldi1 TomsRomn1 RaffaeleGuzzon1 AlessandroSantato1
MarioMalacarne1 GiorgioNicolini1 RobertoLarcher1

Received: 12 November 2015 / Revised: 2 February 2016 / Accepted: 13 February 2016

Springer-Verlag Berlin Heidelberg 2016

Abstract Considering the carcinogenic risk to human

health, it is necessary to carry out research into arsenic (As)
content in agro-food products and the impact of food processing on the final content. Yeast fermentation may represent a strategy for detoxifying some widespread beverages
such as wine, beer and rice wine. A preliminary study of
some commercial yeast species showed different viability
responses to the presence of As. Yeasts had a noteworthy
detoxification capability during fermentation, reducing
the initial As content by about 75% on average (minimummaximum: 4592%), making it possible to produce
wines with a considerably reduced content as compared
to the corresponding grape juices from naturally As-rich
soils. Nevertheless, significant differences between strains
were observed in relation to resistance to arsenic toxicity
and As removal capability. The choice of yeast strain can
determine a difference of 40% on the As content remaining in the wine after fermentation. Arsenic content of up
to 1000g/L did not significantly worsen the fermentation
of some wine yeasts, suggesting that the use of specific
yeasts may represent an effective tool for reducing As in
fermented beverages.
Keywords Arsenic Yeast strain Fermented beverages
Wine As-detoxification

* Daniela Bertoldi
daniela.bertoldi@fmach.it
1

Centro Trasferimento Tecnologico, IASMA Fondazione

Edmund Mach, via E. Mach, 1, 38010San Michele
allAdige, Italy

Introduction
The International Agency for Research on Cancer classifies arsenic (As) as a substance carcinogenic to humans
belonging to group 1 [1]. The intake and the exposure
period determine the toxicity of this element for people and
the linked diseases: fever, diarrhoea, vomiting/nausea for
acute or semi-acute poisoning, dermatological and haematological disorders, hepatic inflammation or cardiovascular problems, besides the genesis of cancer due to chronic
poisoning [2, 3]. For this reason, in 1988 the World Health
Organization (WHO) established a provisional tolerable
weekly intake (PTWI) of 15g/kg of body weight. Following other studies, this PTWI was withdrawn because it
was in the region of the lower limit of the benchmark dose
for a 0.5% increased incidence of lung cancer [4]. A provisional guideline value for arsenic in drinking water was
set at 10g/L [5], a level also confirmed by the United
State Environmental Protection Agency and established by
Council Directive 1998/83/EC and Commission Directive
2003/40/EC in water intended for human consumption and
mineral water, while the International Organisation of Vine
and Wine (OIV) has set the maximum allowable content
in wines at 200g/L [6]. Recently, the FDA confirmed its
intention to consider 10g/kg of inorganic As as an action
level for apple juices [7].
Arsenic is an ubiquitous element, present in water and
foodstuffs of vegetal and animal origin, so its intake by
humans is unavoidable, but strategies for the reduction of
As levels in food and consequently dietary exposure to
inorganic As are recommended, as also stated by the European Food Safety Authority [8]. Both natural and anthropogenic factors have an impact on As content in water,
food and fermented beverages. In particular, in fermented

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beverages As content depends mainly on As in raw materials such as water for beer, and grapes or rice for wine and
rice wine.
As regards the natural input of As, the geological origin, composition and physico-chemical properties of soil
largely determine the content in vines and grapes.
In relation to anthropogenic pollution, the use of fossil fuels is significant, in particular coal, as its combustion
leads to powdery As compounds that subsequently precipitate [9]. Chromated copper arsenate treatments are used
for wood preservation, and treated poles can be found in
vineyards. Arsenic leaching from wood to soil has been
reported [10].
During the second half of the twentieth century, treatments with arsenic pesticides and insecticides in vineyards, and the use of copper vessels and tools in wineries,
were the main cause of the high levels found in wines [11,
12], when As concentration could exceed 500g/L [13,
14]. As regards the modern winemaking practices, wine
protein stabilisation with bentonite can affect the As content, contributing a few g/L to the final concentration
[15]. Standard treatments with fossil shell flours are also
reported to raise the final As concentration by 2.6g/L on
average [16], while the contribution of gum arabic is marginal [17].
Today, As levels in grapes range between less than 0.1
and 70g/kg [10, 1820]. The total content is affected by
exogenous As, located in the outer part of the skin, which
even without arsenic spray treatments can amount to up to
37% of the total [21], probably as a consequence of dust
deposition, especially in areas naturally rich in As [20].
However, endogenous levelsmainly located in the pulp
and skin [19]are predominant and seem to be related to
the content in growing soils. Indeed, grapes grown in plots
close to an old mining area showed As levels up to 6 times
higher than those from outside the mine area [20].
Despite its common presence in grapes, the As content
in wine is usually low and rarely exceeds 30g/L, in contrast to the past [2226].
Arsenic can be present as different species: inorganic
forms [i.e. As(III), the most toxic species, and As(V),
slightly less toxic species], which are reported to be the
main As species present in wines [2732], and organic
forms (mainly monomethylarsonic acid and dimethylarsinic acid), less toxic than inorganic forms and generally not
detectable or present only in trace amounts. Only HercePagliai etal. [23] found a considerable quantity of organic
species in Spanish table wines and sherry.
Of the large numbers of foods contaminated with this
element, wine is an interesting case study. Despite possible significant input of As during vine cultivation and winemaking, its final content in wine is usually low. A thorough
study of practices allowing a reduction in As during the

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production of the wine can therefore offer general guidance

for the detoxification of many fermented beverages.
In winemaking, the relationship between the presence
of As in juice and the yeast used to ferment can be studied from two different points of view: the effect of As content on yeast vitality and fermentation performances, and
the As-detoxification ability of yeast in wine. As regards
the former, very few data are reported in the literature [33]
even if Saccharomyces cerevisiae cells proved to be able to
bioremediate contaminated water removing As(III) [34].
As regards the latter, to the best of our knowledge, a comparison between yeast strain performances in terms of As
reduction during alcoholic fermentation of grape juice in
winery conditions has never previously been carried out.
This work investigates both the possible impact of As
concentration on the vitality and performance of fermenting yeasts, and the detoxification ability of different yeast
strains in terms of reducing As in wines produced from naturally polluted grapes.

Materials andmethods
Sensitivity ofoenological yeasts toAs
Nine species of yeast usually found in the oenological
environment were assayed to verify their sensitivity with
increasing As concentrations in the growth medium.
The yeast strains considered were: S. cerevisiae ATCC
9763, Saccharomyces bayanus ATCC 48556, Saccharomycodes ludwigii ATCC 11313, Schizosaccharomyces pombe
CBS 356, Brettanomyces bruxellensis ATCC 36234, Candida stellata ATCC 10673, Pichia anomala ATCC 8168,
Torulaspora delbrueckii ATCC 10662 and Kloeckera apiculata ATCC 10634. All the strains were cultured in 10mL
of Yeast Peptone Dextrose (YPD) Broth (Sigma-Aldrich,
ST Louis, MO, USA) until a cell concentration of about 108
colony-forming unit (CFU)/mL was obtained. Then 0.2mL
of cell suspension was utilised to inoculate the tests for As
tolerance. Tests were performed on 200mL of YPD broth,
supplemented with 20, 200 and 1000g/L of As using a
certified standard As solution (CertPUR, Merck, Darmstadt, Germany) and incubated at 20C for 5days with
continuous agitation. YPD medium without As addition
was used as a reference. Cell density was assayed using
flow cytometry measurement, as previously described by
Guzzon and Larcher [35]. Flow cytometry analysis of samples after 5days of yeast growth provided quantification
of both live and dead cells, basing the identification of the
two sub-populations on cell staining with two dyes [36].
Fluorescein diacetate, which only shows florescence if processed by metabolically active cells, was employed for live
cells. In contrast, dead cells were essayed using propidium

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iodide, which penetrates inside yeast with a damaged membrane and therefore compromised activity.
Impact ofAs concentration ingrape juice
oncommercial yeast strains
The yeast strains S. cerevisiae Laffort Zymaflore VL1
(code: VL1), La Claire EM2 (EM2) and SP665 (SP665)
and S. cerevisiae var. bayanus Anchor Yeast Vin13 (VIN13)
were used, after rehydration with distilled water for 30min
at 37C, at a dose of 200mg/L, to ferment a previously
frozen, clear and sulphited (50mg/L sulphur dioxide)
Chardonnay juice (natural As content 20.1g/L), spiked
with 0, 5, 10, 500 and 1000g/L of As using a certified
standard As solution (CertiPUR, Merck, Darmstadt, Germany). Alcoholic fermentation took place at 25C. The
fermentation time course was monitored by measuring the
weight decrease twice a day. After fermentation, the wine
was cold settled (4C7days) and analysed after centrifugation (centrifuge model 4226, ALC International,
Cologno Monzese, Italy; 5500rpm5min).
Comparison ofyeast strain performance onAs removal
fromwine
Seven samples of Chardonnay grapes (Vitis vinifera L.),
each made up of 50kg, were harvested at ripening conditions suitable for fizzy or sparkling wines in different
plots in Valsugana (Trentino, North-East Italy). Plots were
located in areas naturally rich in As, being close to ancient
pyrite mines.
The grapes were pressed (90 L Hydropress, Speidel
Tank- und Behlterbau GmbH, Ofterdingen, Germany),
and the juice was sulphited (50mg/L sulphur dioxide),
treated with pectic enzyme (2mL/L) and accurately settled (24h12C; nefelometric turbidity units <50). After
devatting, the clear juice was divided into 10 homogeneous
fractions, each fermented with different commercial yeast
strains (200mg/L) available on the Italian market, previously rehydrated in distilled water (37C 30min). The
yeast strains were: EM2, VL1, Blastosel FR95 (FR95),
AEB Fermol Arom plus (FAP), Ferrari WP (WP) and SN6
(SN6), Oliver Ogar VP5 (VP5) and Springer Oenologie CK
S102 (CK102), all Saccharomyces cerevisiae. Moreover,
a S. cerevisiae var. bayanus Lallemand Vitilevure DV10
(DV10) and a hybrid S. cerevisiae S. kudriavzevii AWRI
1503 (AW1503) were used. Fermentation took place at
1819C. Chemical analysis of wine was carried out by
sampling at the end of fermentation, before any addition of
sulphur dioxide and clarifying agent.
Winemaking processes were performed, protecting juices
and wines from any oenological cross-contamination related
to the use of settling, filtration or fermentation adjuvants.

ICPMS analysis
All wine samples were filtered, acidified (1% v/v HNO3)
and diluted three times for As analysis, performed with
inductively coupled plasma mass spectrometry (ICP-MS,
Agilent 7500ce, Agilent Technologies, Tokyo, Japan),
equipped with a collision cell (He mode) to remove polyatomic interference on mass 75. The instrument was tuned
daily with a solution containing Li Y, Ce and Tl (1g/L;
Agilent Technologies, Tokyo, Japan) following the manufacturers specifications and calibrated against external
standard solutions prepared by diluting certified As standard material (CertiPUR, Merck, Darmstadt, Germany)
with ultrapure ethanol (5% v/v, Carlo Erba, Milan, Italy).
All the data were automatically corrected using a Sc solution (3mg/L, Aristar, BDH, Poole, England) as on-line
internal standard.
The accuracy of the method was checked using 12 wine
samples spiked with a known amount of As standard solution. The average recovery was 106%. The detection limit,
calculated as three times the standard deviation of 10 blank
samples in a sequence, was 0.01g/L.
Statistical analysis
Statistical analysis was performed using the software package STATISTICA 8.0 (Statsoft Inc., Tulsa, OK, USA).
Normal distribution and homogeneity of variance were
checked with the KolmogorovSmirnov and Levene tests
(p<0.05), respectively. The t test (p<0.05) was used to
verify differences in cell concentration in the reference and
in the test spiked with As. Post hoc Fishers least significant
difference test (LSD test, p<0.05) was used to verify differences between groups.

Results anddiscussion
Sensitivity ofoenological yeasts toAs
Considering the lack of extensive literature about the effect
of As on oenological yeast, a survey was performed, taking into account nine species of yeast, from those most frequently isolated in the oenological environment [37]. We
tested the ability of yeasts to grow in a synthetic medium
containing an increasing concentration of As: 20g/L, a
value commonly found in wine; 200g/L, the limit fixed
by OIV for wines and 1000g/L, a very high concentration
that according to our preliminary hypotheses could interfere with yeast growth. The results for live cell concentrations presented in Table1 suggest the possible interference
of As with the growth of most yeast species investigated
in this work. At 20 and 200g/L of As, the differences

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Table1Cell concentration reached after 5-day incubation with different yeast species in media containing an increasing concentration
of As

impact of As on fermentative activity must be assayed. This

is one of the objectives of the subsequent chapters of this
work.

As (g/L)
0

20

200

1000

Pa

Brettanomyces
bruxellensis

8.4104 6.7104 9.3104 5.9104 *

Saccharomyces
cerevisiae

7.7106 6.1106 6.7106 2.6106 ***

Saccharomyces
bayanus

5.0106 5.7106 4.1106 3.7105 ***

Saccharomycodes 1.3105 1.1105 9.3104 5.7104 ***

ludwigii
Schizosaccharomyces pombe

3.0104 2.7104 3.5104 3.7104 ns

Candida stellata

1.0106 1.0106 9.7105 7.8105 *

Pichia anomala

2.8104 3.5104 2.8104 3.3104 ns

Torulaspora
delbrueckii

1.0106 1.3106 8.8105 1.0106 ns

Kloeckera
apiculata

7.8104 6.3104 5.1104 2.6104 ***

Data refer to live cells measured using flow cytometry with a standard
deviation of 10%
a

Significant differences (t test) in cell concentration in the reference (As 0g/L) and As 1000g/L (*=p<0.05; ***=p<0.001;
ns=not significant)

observed in microbial growth (number of live cells) were

not significant and probably due to the variability of microbiological analysis. In contrast, with an As concentration
of 1000g/L a general decrease in the yeast population
was observed, with a statistically significant variation as
compared to the reference (As 0g/L, Table1). With the
exception of three species (S. pombe, P. anomala and T.
delbruekii), where even 1000g/L of As did not affect cell
growth, the decrease in the cell population ranged between
90% for S. bayanus and 30% for B. bruxellensis and C.
stellata. The population of dead cells did not show any
significant variation and remained below 10% of the total
yeast population in all tests on average. Some hypotheses
could be proposed to explain the behaviour of yeast in relation to the increasing presence of As in the growth medium.
With up to 200g/L of As, the known detoxification ability of yeast [13, 38, 39] prevails, minimising the adverse
effects of As on the cellular metabolism; at 1000g/L,
the presence of As exceeds the resistance capacity of the
cells, slowing down their multiplication due probably to
strong oxidative stress and the negative impact on protein
synthesis and DNA replication [39]. In conclusion, this preliminary evidence suggests the possible interference of As
concentration on yeast growth. However, considering the
ability of yeast to detoxify the environment and exponential cellular growth, typical of microorganisms, the actual

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Impact ofAs concentration ingrape juice

oncommercial yeast strains
The grape juice considered for this experiment had a relatively high natural As content (20.1g/L). Wines produced
without pre-fermentative addition of As had 5075%
lower As content as compared to juice, ranging from 4.95
to 10.25g/L. The fermentation time courses expressed
as the % of developed fermentation over time observed in
natural juices and juices with the addition of 5, 10, 500 and
1000g/L of As are displayed in Fig.1. All yeast strains
properly completed fermentation, in spite of the high As
content added. No notable differences in the shape of the
fermentation curves due to the As level were observed. This
is also confirmed by a statistical approach (Fishers least
significant difference test, p<0.05) in which no significant
differences were found at the initial (1924h), half-way
(4248h) and final (240332h; 9899% of fermentation)
phase of fermentation in the five levels of As.
Our results confirm the assumptions made in the previous paragraph, namely that the rapid cell replication typical of many yeasts reduces the technological effects of the
damage induced by high concentrations of As. In contrast,
the previous observations of Amin etal. [33] as regards an
increased lag time in the presence of As, observed in batch
or immobilised cell systems, were not confirmed and this
could be explained by the substantially different experimental plan and fermentative conditions. Our experimental conditionsi.e. yeast dose, fermentation temperature,
presence of available nitrogen (215mg/L) and the relative
possibility of producing high levels of glutathione able to
link and detoxify As in the cell vacuolewere probably
not sufficiently drastic to highlight the As-related toxicity
mechanisms reviewed by Wysocki and Tams [39] on S.
cerevisiae cells. Nevertheless, given the As concentration
found in natural grape juices, we decided it was not justified to test yeast behaviour with content exceeding 1mg/L.
Comparison ofyeast strain performance interms
ofremoving As fromwine
Arsenic content varied from 0.09 to 10.3g/L in the wines
considered in this experiment, starting from natural juices
and without artificial As addition. These levels confirmed
the wide range reported in recent literature, shown to be
<0.5g/L in Cretan wines [12], 2.114.6g/L in Spanish
wines [23], 1.6615.2g/L in Czech wines [25] and 1.77
9.66g/L in Croatian wines [26]. Similar As levels were
also determined in other fermented beverages such as beer

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Fig.1Fermentation time
courses for grape juice (TQ) and
juice spiked with 5, 10, 500 and
1000g/L of arsenic

(1.523.8g/L) [4042] and apple cider (5.4115.27g/L)

[43], while fruit juices and above all grape juice had higher
values (6.9647.59g/L) [43]. Nevertheless, in contrast to
wine, the As content in these beverages is strongly influenced by the As content in the water used in the production
process.
Our results confirmed that the final As content in wine
was low, the highest level being roughly equal to 5% of the
limit established by OIV, even when starting from grapes
grown on soils naturally rich in As. Although this level did
not seem to be a matter of concern, the WHO and EFSA
requirement to reduce dietary exposure to As encourages
more in-depth knowledge of the role of the fermentation
process in the final concentration of As in wine.
The As content in juices was calculated from the As content previously determined in the seven grapes [20], assuming a transformation yield from grape to juice of 70% [44].
The As content in juices and in the corresponding wines
after alcoholic fermentation is shown in Fig.2.
A positive correlation between the As content in juice
and wine (average content in 10 winemaking processes)
was observed. Only 1 out of 7 juicewine couples (number 4 in Fig.2) did not follow the general trend. If these
data are removed from the processing, a highly significant
correlation can be highlighted (Pearsons test, p<0.05).
A particular profile of As species in this sample could
lead to different yeast detoxification efficacy. Indeed, this
juicefor which a low reduction in As concentration following fermentation was found (see below)came from
grapes grown on calcareous soil poor in AlFeMn oxides,
where a large fraction of available As out of the total was
observed [20].

Fig.2As content (meanstandard deviation) in grape juice and

corresponding wines

On average, the As content decreased by about 75%

from the juice to the wine (minmax 4592%), this being
consistent with our previous results on the As tolerance of
yeasts and the little data available in the literature [38].
Figure3 shows the % As reduction achieved in the final
wines with the 10 different yeast strains. Strains CK102,
FR 95 and VP5, with an average reduction of over 80%,
provided the best depletion performance statistically (LSD
test, p<0.05). They were followed by the yeast strains
VL1, FAP, SN6, DV10, AW1503, WP and finally EM2
(average reduction 68%), albeit not statistically different
from WP, AW1503, DV10 and SN6.
Considering the performance obtained with each starting
grape must, an average reduction of >70% was observed in
6 out of 7 cases, while in only one case (starting from grape
no 4 in Fig.2) an average reduction of <60% was observed.

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Conclusions

Fig.3Percentage reduction in As content from grape juice to wine

by yeast strains. Boxes represent 25% and 75 with median point.
Whiskers represent not-outlier interval; circles and asterisks are outliers and extremes, respectively

A reduction of less than 60% was also shown starting from

grape no. 7 using strains EM2 and WP. Considering the As
content remaining in the wine after fermentation, in percentage terms, the range between strains was considerable,
the lowest meanbelonging to the strain CK102being
39% below that of the EM2 strain. The percentage differences between strains observed here agree with those in a
preliminary experiment (roughly 40%), in which partially
different strains were used, reported by Romn [45]. In this
case too, the FR95 strain showed an As reduction capability higher than FAP. Better performance by strain VL1 as
compared to EM2 was also observed in the section above,
with a slightly higher percentage difference between strains
(52%). The differences observed between strains could be
due to their different metabolisms also in relation to the different characteristics of juices. According to the literature,
electrostatic interaction or absorption of As with the yeast
cell wall, the protein sulphide groups and the wine colloids
could occur [13, 38]. Moreover, vacuolar sequestration of
As by the yeast cell as a detoxification mechanism (bioaccumulation) has been reported [39].
As regards the wines obtained after the addition of As
to the juice, yeast performance was very different depending on the dose. With final content of up to 30g/L in the
juice, a reduction of 5070% from the juice to the wine
was shown, whereas with a final content of about 500
1000 g/L in the juice a reduction of only 120% was
achieved. For these wines, a level higher than the OIV limit
was observed, ranging between 421 and 912g/L. However, such a high content in grape juice is very improbable, the maximum value reported in the literature being
47.59g/L [43].

13

This work proved that, despite the unfavourably high

As content in grapes, the bio detoxification activity of
yeasts effectively reduced the final level in wines, this
being decidedly lower than the limit suggested by OIV
(200g/L). Considering the repeated recommendations of
WHO, FDA and EFSA on the health risks of dietary exposure to As, this limit could be considered too high, given
that current good manufacturing practices make it possible to obtain wine with a decidedly lower level, even when
starting from grapes naturally rich in As.
In our tests, S. cerevisiae, a widespread microorganism
involved in the production of the majority of fermented
food, showed good resistance to As. Elemental content
of up to 1mg/L did not cause stuck fermentation or the
lengthening of fermentation. The evidence presented in
this paper showed a significant reduction in the initial content of As mediated by yeasts involved in fermentation.
From grapes juice to wine, on average yeast detoxification reduced As content by about 75%, but the use of a
specific yeast strain in winemaking significantly affected
the amount of residual arsenic in wines. Considerable differences were noted in terms of the resistance to this contaminant and the ability to remove it from the fermentation
medium, with differences of around 40% between strains.
Yeasts are therefore an effective tool for reducing As in
food chains, and careful choice of the strain involved in
the fermentation process is advisable, in order to improve
the healthiness of fermented beverages. Resistance and
detoxification ability should then be carefully considered
during the process to select new yeast strains for bioremediation purposes.
Acknowledgments We thank the technical staff of the Consulting
and Services Centre (Viticulture Area) of the Fondazione E. Mach for
support during sampling and A. Versari (University of Bologna) for
critical review.
Compliance with ethics standards
Conflict of interest The authors declare that they have no conflict
of interest.
Compliance with ethics requirements This paper does not contain
any studies with human or animal subject.

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