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Effects of Non- Saccharomyces Yeasts on Color, Anthocyanin and Anthocyanin-

Derived Pigments of Tannat Grapes during Fermentation

Article  in  American Journal of Enology and Viticulture · December 2017

DOI: 10.5344/ajev.2017.17055

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 AJEV American
Journal of

69/2 Enology and

Viticulture

PUBLISHED BY THE AMERICAN

SOCIETY FOR ENOLOGY AND
VITICULTURE SINCE 1950 ajevonline.org
 Effects of Non-Saccharomyces Yeasts on Color,
Anthocyanin, and Anthocyanin-Derived Pigments
of Tannat Grapes during Fermentation
Karina Medina,1 Eduardo Boido,1 Eduardo Dellacassa,2 and Francisco Carrau1*

Abstract: Phenolic compounds can affect the sensory characteristics of quality wines, yet the chemical modifica-
tions induced by yeasts on phenolic compounds have been studied only to a limited extent. Forty-nine non-Saccharo-
myces yeast strains belonging to different genera were screened for their effects on color and fermentation capability
in a synthetic must supplemented with anthocyanins from Tannat grapes. Total anthocyanins, color intensity, hue, and
total polyphenol index were significantly affected (p < 0.001) by the different yeasts studied, and the effects on these
parameters were highly diverse in the yeasts’ capacity for improving color indexes of the red grape model wines. Six
strains belonging to the species Metschnikowia pulcherrima, Hanseniaspora guilliermondii, H. opuntiae, H. vineae,
and H. clermontiae were selected to evaluate the production of anthocyanin-derived pigments; significant variations
in the production of four compounds were found between them and compared with Saccharomyces cerevisiae. The
formation of vitisin A, vitisin B, malvidin-3-glucoside-4-vinylphenol, and malvidin-3-glucoside-4-vinylguaiacol
are reported for the first time for species of genera Hanseniaspora and Metschnikowia.
Key words: anthocyanin, anthocyanin-derived pigments, non-Saccharomyces yeasts, Tannat, wine color

Wine color depends on grape pigments and how those
pigments are modified during vinification, storage, and ag-
ing (Boulton 2001, Vivar-Quintana et al. 2002, Schwarz et al.
2003). Yeasts have been demonstrated to interact with antho-
cyanins in different ways during fermentation; for example,
the interaction of yeast cell-wall material producing antho-
cyanin adsorption is a well-known phenomenon (Vasserot et
al. 1997, Morata et al. 2003b, Medina et al. 2005). However,
in the last decade, yeast secondary metabolic products re-
leased into the medium, such as pyruvic acid and acetalde-
hyde, were identified as potential precursors in the formation
of new pigments. These compounds were demonstrated for
Saccharomyces strains to react with anthocyanins, producing
derivatives such as vitisin A, vitisin B, and ethyl-linked an-
thocyanin-flavanol pigments (Asenstorfer et al. 2003, Eglinton
et al. 2004, Lee et al. 2004, Medina et al. 2005, Morata et al.
2003a, 2007a, 2007b, Benito et al. 2009). These findings sug-
gest that yeast strain selection strongly affects color intensity
(CI) and the final concentration of anthocyanins (Morata et
al. 2006, Monagas et al. 2007) and other phenolics (Monagas
et al. 2007).
1
Universidad de la República, Sección Enología, Food Science and Technology
Department, Facultad de Química, 11800 Montevideo, Uruguay; and 2Univer-
sidad de la República, Aroma Biotechnology Laboratory, Organic Chemistry
Department, Facultad de Química, 11800 Montevideo, Uruguay.
*Corresponding author (fcarrau@fq.edu.uy; tel: [598 2] 9248194)
Acknowledgments: The authors thank the following agencies for financial
support: Grants ANII Postgraduate BE_POS_2010_1_2525, CSIC-CAP 2014,
and PEDECIBA Program (Biology and Chemistry Areas).
Manuscript submitted June 2017, revised Sept 2017, accepted Dec 2017
Copyright © 2018 by the American Society for Enology and Viticulture. All
rights reserved.
doi: 10.5344/ajev.2017.17055
148
Am. J. Enol. Vitic. 69:2 (2018)
Furthermore, non-Saccharomyces yeasts have attracted
much attention from wine producers because their metabolic
properties may contribute in different ways than conventional
Saccharomyces cerevisiae (Romano et al. 1997, Fleet 2008).
Several studies have evaluated the involvement of non-Sac-
charomyces yeasts during alcoholic fermentation and their
role in vinification by increasing aroma complexity of the
final product (Carrau et al. 2016); however, changes in the
chemical composition of phenolic compounds have been stud-
ied only to a limited extent. Recently, studies have proven
that some species may be involved in wine-color stabilization
(Morata et al. 2012, Loira et al. 2015). Some of these reactions
could be attributed to the variable levels of acetaldehyde syn-
thesis by different yeast species. For example, Pichia species
produced significantly higher levels of acetaldehyde compared
with Saccharomyces (Clemente-Jimenez et al. 2005), and ac-
etaldehyde has been shown to increase linearly with increas-
ing cell biomass concentration (Duff and Murray 1988).
Although non-Saccharomyces yeast strains account for
>99% of the grape native flora, limited information has been
presented about the effect of non-Saccharomyces strains on
wine pigment composition, except for a few reports on Pichia
and Schizosaccharomyces species (Benito et al. 2011, 2014,
Morata et al. 2012).
The color of wines obtained from Vitis vinifera L. cv Tan-
nat, a widely grown cultivar in Uruguay and one of the rich-
est varieties in polyphenolic compounds (Carrau et al. 2011,
Da Silva et al. 2013), is important; therefore, we developed
a program to select native yeasts to increase yeast diversity
for fermentation without affecting the wine color. The Tan-
nat grape juice model medium utilized here allowed us to
screen the strains’ capacity to synthetize anthocyanin-derived
compounds while avoiding the interference of grape solids,
such as the skin and seeds, as was demonstrated previously

for S. cerevisiae strains (Medina et al. 2005). Consequently,
we present the results obtained from studying the effects of
fermentation with different non-Saccharomyces yeast strains
on color and the anthocyanin profile of Tannat in model wine.
Materials and Methods
Yeast strains. Forty-nine non-Saccharomyces yeast
strains, isolated from different varieties of red grapes
from different Uruguayan regions, were evaluated accord-
ing to their effect on wine color. The yeasts were identi-
fied as Metschnikowia pulcherrima (M00/09G, M00/19G,
T00/23G, SB00/03G, M00/08G, T00/21G, T00/25G, T03/10G,
CH03/29G, T03/28G, A03/19G, M03/02G, CH03/08G,
M03/26G, PV03/16G, PV/18G, M03/25G), Hanseniaspora
vineae (T02/05F, T02/19F, T02/25F), H. uvarum (T06/05G,
C06/30G, T06/13G, T06/04G, C02/27G, C06/35G, CF10/33G,
PV10/67F, C10/47F, C10/49F, PV10/71F, C10/57F), H. opun-
tiae (T06/19G, T06/01G, T06/12G, CF10/38F), H. clermontiae
(C10/54F, A10/82F), H. guilliermondii (T06/09G), Torulas-
pora delbrueckii (Td 1550), Starmerella bacillaris (T06/11G,
T06/20G, T06/14G, T06/08G, C10/46F), Issatchenkia terri-
cola (C10/58F, 06/21G), Candida railenensis (T00/22G), and
C. shehatae (C 12878). All the isolated yeasts used in this
work were genetically identified by sequencing the variable
D1/D2 region of the 26S rDNA gene. An S. cerevisiae na-
tive strain (Sc 882), which is characterized by its increased
production of anthocyanin-derived compounds and its ability
to improve color in red wine using the same red grape juice
medium (RGJM), was used as a control (Medina et al. 2005).
Yeast were maintained on yeast extract peptone dextrose
medium slants (1% yeast extract, 2% peptone, 2% glucose,
2% agar, pH 4.5 using 0.1 M citrate-phosphate buffer) at 5°C
and incubated for 12 hr at 20°C on fresh medium before use
in fermentation experiments.
RGJM. RGJM was prepared using a previously defined
fermentation medium based on the nutrient composition of
grape juice (Carrau et al. 2005), with a yeast assimilable ni-
trogen content of 150 mg N/L, 3.25 g/L H 2SO4 total acidity,
and 20.6 Brix. The resulting synthetic must was then sup-
plemented with an anthocyanin extract obtained from grape
skins of V. vinifera cv. Tannat and prepared according to Me-
dina et al. (2005), resulting in an RGJM with a total anthocy-
anin concentration of 800 mg/L. The final pH of the medium
was adjusted to 3.5 with HCl, and then filtered through a 0.22
µm pore-size membrane (Merck Millipore Corp).
Fermentation conditions. Inoculums were prepared in
RGJM by incubation for 12 hr on a rotary shaker at 150 rpm
and 20°C. Fermentations were conducted in sterilized 125
mL Erlenmeyer flasks filled to 50 mL with RGJM and stop-
pered with Muller valves containing sulfuric acid. Triplicate
flasks were inoculated with 5 × 105 cfu/mL at 20°C to simu-
late winemaking conditions.
Fermentation activity was measured by CO2 weight loss,
expressed in grams per 100 mL (Carrau et al. 2008). Samples
were taken once a day to measure cell growth in an improved
Neubauer chamber, and the number of dead cells was deter-
mined by methylene blue staining technique. Purity control of
Am. J. Enol. Vitic. 69:2 (2018)
Effects of Non-Saccharomyces Yeasts on Color – 149
the different assays was determined at the end of the fermen-
tations using Wallerstein Laboratory nutrient medium to test
for growth in a yeast differential culture medium (Pallmann
et al. 2001).
Color analysis. All chemical determinations were per-
formed immediately after a constant vessel weight was ob-
tained. Absorbance at 420, 520, and 620 nm was determined
using a Spectronic Genesys 2 spectrophotometer (Spectronic
Instruments) equipped with a 1 cm path length quartz cell. To
avoid modifications from pH differences and copigmentation
(Boulton 2001), the wines were diluted 1:10 (v/v) in citrate
buffer, pH 3.2 (0.05 M Na 2HPO 4, 0.75 M citric acid). Hue
(H = A420/A520) and color intensity (CI = A420 + A520 + A620)
values were also calculated.
Determination of anthocyanin and total polyphenol con-
tent. Upon removal of yeast cells by centrifugation (10,000
RPM, 10 min), the total anthocyanin concentration in each
wine was spectrophotometrically measured at 540 nm after di-
lution in water-ethanol-hydrochloric acid (70:30:1) (Di Stefano
et al. 1989). Total polyphenol content was measured at 280
nm, after a 1:100 dilution in distilled water (Iland et al. 1993).
HPLC-DAD analysis of anthocyanins. Anthocyanins
were analyzed by high-performance liquid chromatography
(HPLC) using a Hewlett-Packard Series 1050 instrument
equipped with an HP Chem Station and a photodiode-array
detector (DAD). Gradients of solvent A (water-formic acid,
95:5, v/v) and solvent B (acetonitrile) were applied over a C18
reverse-phase 150 mm × 4.6 mm column (Phenomenex), as
follows: 10 to 30% B, linear (1.0 mL/min) from 0 to 40 min;
30 to 35% B (1.0 mL/min) from 40 to 48 min; 35 to 10% B (1.0
mL/min) from 48 to 49 min; then 10% from 49 to 55 min to
reequilibrate the column to initial conditions (Rivas-Gonzalo
et al. 1995).
Anthocyanins were detected by scanning from 280 to
600 nm. Quantification was performed against an external
standard at 530 nm and expressed as a function of malvidin-
3-glucoside concentration. Samples (20 μL) of previously
filtered wines (200 μL) were injected into the HPLC. Deter-
minations were made in duplicate. The following anthocya-
nins were identified in model wines: delphinidin, cyanidin,
petunidin, peonidin, and malvidin, as well as 3-glucosides,
6-acetylglucosides, 6-p-coumarylglucosides, and malvidin-
6-caffeoylglucosides.
Identification and quantification of anthocyanin-de-
rived pigments by HPLC-MS. HPLC-mass spectrometry
(HPLC-MS) analyses were performed using an HPLC-DAD
1200 (Agilent Technologies), equipped with a binary pump
and a triple Quad LC/MS 6410 LC detector, using electro-
spray ionization. Both the sheath gas and the auxiliary gas
were a mixture of nitrogen and helium. The experimental
conditions for the HPLC analyses were as indicated above,
and those for HPLC-MS as follows: sheath gas flow, 10 L/
min; capillary voltage, 4000 V; and capillary temperature,
300°C. Spectra were recorded in positive ion mode between
m/z 100 and 1600 atomic mass units. The anthocyanin-de-
rived pigments were identified by their M+ and fragmen-
tation patterns (Boido et al. 2006). The area of each M+
150 – Medina et al.

fragment was used for comparing the different strains’ pro-
duction between treatments.
Normalization of data measures. For fermentation trials
of each of the 49 strains, data were normalized by dividing
the measured variable value for each sample by the respec-
tive control treatment measurement (unfermented RGJM). The
four normalized parameters were total anthocyanin concentra-
tion (TA), CI, hue, and total polyphenol concentration (TPI).
Statistical analysis. Analysis of variance (ANOVA) was
performed to determine the significance of differences for in-
oculated strains in relation to TA, CI, hue, and TPI. Statistica
7.1 software was used for analysis.
Results
Yeast effect on TA, CI, hue, and TPI. For a better visu-
alization of the results, data were presented according to the
different yeast genera studied (Tables 1, 2, and 3).
Significant differences (p < 0.001) were found for the four
evaluated and previously normalized parameters in 17 M.
pulcherrima strains studied (Table 1). In particular, the dif-
ference between the strains presenting the highest and lowest
CI values was 54%. Similarly, the largest difference for TA
was 41%; for hue, 37%; and for TPI, 17%.
For the Hanseniaspora species, Table 2 shows that no
significant difference was observed for TPI values between
strains. As for M. pulcherrima, the parameter with the great-
est range was CI, followed by TA, and then hue. In Table 3,
significant differences for CI (p < 0.05) and TA (p < 0.001),
but not for hue and TPI, are presented for the species of Can-
dida, Torulaspora, and Issatchenkia.
As the above results show, CI and TA presented higher
variability across the 49 strains studied, suggesting they were
also the parameters most affected by yeast strain diversity,
whereas TPI was found to be the parameter least affected by
yeast strain. Considering that CI and TA are the main param-
eters to be considered for color behavior in wines, the follow-
ing yeast strains were selected to evaluate their effects on an-
thocyanin content and fermentation capacity in winemaking:
M. pulcherrima (M00/09G), H. guilliermondii (T06/09G), H.
opuntiae (T06/01G), H. vineae (T02/5F), and H. clermontiae
(A10/82F and C10/54F). These strains were compared with
the best wine-color improving S. cerevisiae strain from our
collection, Sc 882 (Medina et al. 2005).
Fermentation kinetics and cell growth. Figure 1A shows
the fermentation profile in RGJM for the six selected yeasts
compared with Saccharomyces strain Sc 882. As was expect-
ed, the best fermentation performance corresponded to strain
Sc 882, while H. vineae T02/05F (10.59 g CO2/100mL) and H.
clermontiae A10/82F (4.9 g CO2/100mL) showed the best non-
Saccharomyces performance. The other non-Saccharomyces
yeasts showed lower fermentation performance.
Significant differences were also found for the total cell
count at the end of fermentation among the seven yeast
strains studied. Saccharomyces Sc 882 showed the high-
est cell count (1.15 × 10 8 cell/mL ± 0.05), followed by Hv
T02/05F (8.75 × 107 cell/mL ± 0.25). A good correlation was
found between fermentation capacity and cell number (Fig-
ure 1B).
Effect of selected non-Saccharomyces strains on color
and phenolic parameters. When the effect on color was eval-
uated, the strains Ho T06/01G and Hc C10/54F showed the
highest CI values, comparable to Sc 882 (Table 4), but their
poor fermentation capacity made them ineligible for wine-
making (Figure 1A). Strains Hc A10/82F and Hv T02/05F

Table 1 Mean normalized value and standard deviation of color parameters for Metschnikowia species.
CI, color intensity; TA, total anthocyanins; TPI, total polyphenol index.
CI Hue TA TPI
Strain Code XXXa XXX XXX XXX
M. pulcherrima
M00/09G 1.05 (0.04) eb 1.00 (0.00) bc 1.04 (0.05) bc 0.98 (0.06) abc
M00/19G 1.07 (0.14) e 0.99 (0.02) abc 1.18 (0.0) c 0.99 (0.01) abcd
T00/23G 0.81 (0.0) bcd 1.06 (0.02) abc 1.01 (0.10) bc 0.93 (0.04) a
SB00/03G 0.85 (0.17) de 1.16 (0.17) abcde 0.95 (0.14) abc 0.98 (0.04) abc
M00/08G 0.66 (0.00) abc 1.24 (0.01) bcde 0.83 (0.01) abc 0.98 (0.02) abc
T00/21G 0.83 (0.01) bcd 1.15 (0.01) abdc 0.88 (0.01) abc 0.98 (0.01) abcd
T03/10G 0.49 (0.01) a 1.43 (0.02) e 0.69 (0.01) a 1.10 (0.01) bcd
CH03/29G 0.62 (0.03) abc 1.15 (0.01) abcd 0.78 (0.02) ab 1.09 (0.00) bcd
T03/28G 0.61 (0.01) abc 1.15 (0.02) abcd 0.78 (0.01) ab 1.07 (0.01) abcd
A03/19G 0.53 (0.02) a 1.35 (0.01) de 0.76 (0.01) ab 1.09 (0.01) bcd
M03/02G 0.59 (0.01) abc 1.2 (0.0) abcde 0.72 (0.03) a 1.04 (0.04) abcd
CH03/08G 0.55 (0.02) ab 1.28 (0.01) cde 0.72 (0.00) a 1.08 (0.01) abcd
M03/26G 0.63 (0.00) abc 1.08 (0.02) abcd 0.78 (0.01) ab 0.96 (0.03) ab
PV03/16G 0.59 (0.00) abc 1.29 (0.01) cde 0.79 (0.01) ab 1.13 (0.02) d
PV03/18G 1.00 (0.01) e 0.97 (0.00) a 1.08 (0.04) c 1.11 (0.02) bcd
M03/25G 0.7 (0.05) abc 1.17 (0.07) abcde 0.84 (0.07) abc 1.12 (0.03) cd
M. fructícola
T00/25G 0.84 (0.0) de 1.13 (0.04) abcd 0.93 (0.00) abc 1.09 (0.01) acd
a
Significance levels: xxx, 99.9%.
b
Letters indicate significant differences for each index between strain treatments by Tukey’s test (95%).

Am. J. Enol. Vitic. 69:2 (2018)

 Effects of Non-Saccharomyces Yeasts on Color – 151

were found to present good fermentation, resulting in good CI Progressive change in the wine spectrum is the most
and lower hue than the other non-Saccharomyces strains stud- obvious feature of aging reactions in red wines. Observa-
ied, which was attributable to lower absorbance at 420 nm. tions of wide variability in the extent of phenolic change in

Table 2 Mean normalized value and standard deviation of color parameters for Hanseniaspora species.
CI, color intensity; TA, total anthocyanins; TPI, total polyphenol index.
CI Hue TA TPI
Strain Code  XXXa XXX XXX NS
H. uvarum
T06/05G 0.70 (0.02) ab 1.12 (0.01) e 0.93 (0) def 0.99 (0.01)
C06/30G 0.81 (0.07) abc 1.09 (0.03) cde 0.90 (0.01) bcdef 0.99 (0.02)
T06/13G 0.90 (0.00) bcd 1.06 (0.01) bcde 0.92 (0.00) cdef 0.99 (0.01)
T06/04G 0.75 (0.03) abc 1.07 (0.02) bcde 0.93 (0) abcdef 0.99 (0.01)
C06/27G 0.73 (0.01) ab 1.10 (0.02) cde 0.89 (0.01) abcdef 0.95 (0.03)
C06/35G 0.79 (0.08) abc 1.09 (0.03) cde 0.90 (0.01) abcdef 0.99 (0.0)
CF10/33G 0.74 (0.02) ab 1.13 (0.02) e 0.72 (0.01) a 0.89 (0.05)
PV10/67F 0.86 (0.02) abcd 1.01 (0.01) bcde 0.88 (0.06) abcedef 0.83 (0.007)
C10/49F 0.87 (0.01) abcd 1.01 (0.02) bcde 0.85 (0.01) abcde 0.86 (0.00)
C10/47F 0.99 (0.02) d 1.01 (0.01) bcde 0.96 (0.01) def 1.0 (0.02)
PV10/71F 0.89 (0.00) bcd 1.01 (0.02) bcde 0.93 (0.02) def 0.87 (0.02)
C10/57F 0.98 (0.01) d 1.08 (0.02) bcde 0.90 (0.03) bcdef 1.01 (0.01)
H. vineae
T02/05F 0.87 (0.00) bcd 0.98 (0.00) abcd 0.88 (0.01) abcdef 0.94 (0.02)
T02/19F 0.92 (0.01) cd 1.02 (0.05) bcde 0.81 (0.02) abcd 0.93 (0.05)
T02/25F 0.74 (0.02) ab 1.11 (0.01) de 0.738 (0) ab 0.89 (0.01)
H. guilliermondii
T06/09G 0.96 (0.03) cd 0.95 (0.02) ab 0.92 (0.01) def 0.89 (0.03)
H. opuntiae
T06/19G 0.87 (0.04) abcd 0.85 (0.08) a 0.97 (0.00) def 0.96 (0.01)
T06/01G 0.92 (0.01) cd 0.97 (0.01) abc 0.93 (0.07) def 0.88 (0.04)
T06/12G 0.86 (0.03) abcd 1.1 (0.01) cde 0.75 (0.02) abc 0.87 (0.02)
CF10/38F 1.02 (0.02) d 1.02 (0.01) bcde 0.97 (0.05) ef 1.01 (0.01)
H. clermontiae
C10/54F 0.96 (0.03) cd 1.01 (0.02) bcde 0.99 (0.02) ef 0.95 (0.09)
A10/82F 0.93 (0.04) cd 1.05 (0.02) bcde 1.04 (0.07) f 0.99 (0.12)
a
Significance levels: xxx, 99.9%. NS indicates no significant difference.
b
Letters indicate significant differences for each index between strain treatments by Tukey’s test (95%).

Table 3 Mean normalized value and standard deviation of color parameters for genera Candida, Torulaspora, and Issatchenkia. CI, color
intensity; TA, total anthocyanins; TPI, total polyphenol index.
CI Hue TA TPI
Strain Code Xa NS XXX NS
C. zemplinina
T06/11G 0.72 (0.02) abb 1.09 (0.01) 0.90 (0.01) bc 0.99 (0.01)
T06/20G 0.74 (0.03) ab 1.09 (0.03) 0.93 (0.02) bc 1.02 (0.06)
T06/14G 0.88 (0.02) ab 1.05 (0.00) 0.93 (0.02) bc 1.01 (0.02)
T06/08G 0.81 (0.09) ab 1.09 (0.03) 0.92 (0.02) bc 1.03 (0.02)
C10/46F 0.90 (0.06) ab 1.11 (0.13) 0.95 (0.02) bc 0.98 (0.08)
C. railenensis
T00/22G 0.82 (0.08) ab 1.11 (0) 0.83 (0.03) b 0.93 (0.11)
C. shehatae
C12878 0.99 (0.01) b 1.01 (0.02) 0.99 (0.04) c 0.99 (0.06)
T. delbrueckii
TD1550 0.90 (0.04) ab 1.06 (0.01) 0.88 (0.05) bc 0.91 (0.06)
I. terricola
C10/58F 0.66 (0.06) a 1.20 (0.07) 0.65 (0.00) a 0.90 (0.10)
06/21G 0.77 (0.06) ab 1.06 (0.01) 0.86 (0.00) bc 0.91 (0.02)
a
Significance levels: x, 95%; xxx, 99.9%. NS indicates no significant difference.
b
Letters indicate significant differences for each index between strain treatments by Tukey’s test (95%).

Am. J. Enol. Vitic. 69:2 (2018)

152 – Medina et al.

commercial wines of equal age led to the concept of chemi-
cal age (CA) as a measure of wine maturation (Somers and
Evans 1977), where values for young red wines vary from
0.02 to 0.20.
In our study, the Hv T02/05F strain showed the highest
CA value (0.29), followed by Sc 882 (0.23) and Mp M00/09G
(0.22). Despite the fact that the CA measurements were per-
formed immediately after the alcoholic fermentation was
complete, it was possible to detect different behaviors of the
selected yeasts. Strain Sc 882 presented the highest values of
CI and TA. However, the non-Saccharomyces strains behave
differently—no correlation was found between CI and TA.
Strain effect on anthocyanin composition. ANOVA for
anthocyanin values obtained by HPLC-DAD-MS for the dif-
ferent yeast fermentations showed significant differences (p <
0.05) among treatments for all the glycosylated anthocyanins
found in wines. The highest variability between strains was
found for delphinidin-3-glucoside and peonidin-3-glucoside
(Figure 2). However, there were no significant differences for
delphinidin-3-coumaroylglucoside + malvidin-3-acetylglu-
coside, cyanidin-3-coumaroylglucoside, petunidin-3-couma-
roylglucoside, malvidin-3-coumaroylglucoside + peonidin-
3-coumaroylglucoside, or for malvidin-caffeoyl-3-glucoside.
Anthocyanin-derived compounds formed during yeast
fermentation. Anthocyanin derivatives were identified by
HPLC-DAD-MS analysis of the wine samples for all the yeast
strains studied (Figure 3). All strains showed formation of
vitisin B (Figure 3A), with Sc 882 being the leading producer
of precursors with approximately twice the highest value of
the best non-Saccharomyces strain (Mp M00/09G). No sig-
nificant differences were observed for this compound in the
other non-Saccharomyces yeasts studied. By contrast, higher
levels of vitisin A were found in fermentations for all the non-
Saccharomyces strains compared with Sc 882 (Figure 3B).
Regarding malvidin-3-O-glucoside-4-vinylguaiacol (Fig-
ure 3C), no significant differences were observed between
treatments according to Tukey’s test. All strains were found
to produce this compound; Mp M00/09G and the two H. cl-
ermontiae strains (Hc A10/82F and Hc C10/54F) showed the
highest concentrations.
Malvidin-3-O-glucoside-4-vinylphenol (Figure 3D) was
present in all yeast treatments except for strain Mp M00/09G.
Interestingly, although this strain does not produce this pig-
ment derived from vinylphenol, it produces one of the highest
levels of a pigment-derived compound from vinylguaiacol
(Figure 3C).
The last anthocyanin-derived compound identified in this
study was malvidin-3-O-glucoside-8-ethyl-catechin. This
compound only resulted from treatments with the Sc 882
strain.
Figure 4 shows the sum of all derived anthocyanin com-
pounds found, whereas only strain Ho T06/01G showed a sig-
nificantly lower value with respect to other yeast treatments.
A promising result for potential application of the other four
nonconventional strains is that no significant differences were
found compared with Sc 882, the best selected industrial S.
cerevisiae strain regarding wine color increases.

Discussion
Figure 1 (A) Fermentations of the selected yeast and Saccharomyces
cerevisiae 882 in red grape juice medium at 20°C. (B) Positive correlation TA, CI, hue, and TPI were significantly affected (p < 0.001)
between fermentation capacity and cell number at the end of fermentation. by the different yeast species studied. The fact that H. vineae

Table 4 Mean value and standard deviation of color spectrophotometer parameters for the selected yeast strains.
CI, color intensity; TA, total anthocyanins (mg/L); TPI, total polyphenol index; CA, chemical age index.
Hg T06/09G Ho T06/01G Mp M00/09G Hv T02/05F Hc C10/54F Hc A10/82F Sc 882 pa
CI 12 ± b
0.2 bc 12.5 ± 0.3 bc 10.3 ± 0.3 a 11.5 ± 0.1 b 12.4 ± 0.3 bc 11.9 ± 0.0 bc 12.7 ± 0.1 c xxx
Hue 0.64 ± 0.01 d 0.64 ± 0.01 d 0.63 ± 0.01 cd 0.60 ± 0.01 bc 0.64 ± 0.01 d 0.56 ± 0.01 a 0.58 ± 0.01 ab xxx
TA 530 ± 5a 537 ± 41 a 550 ± 34 ab 576 ± 8 ab 536 ± 9a 605 ± 13 b 609 ± 21 b xx
TPI 21 ± 0.7 ab 20.6 ± 0.1 a 21.3 ± 0.2 ab 22.5 ± 0.0 b 21 ± 0.3 ab 22 ± 0.3 ab 21 ± 0.2 ab x
CA 0.15 ± 0.00 ab 0.13 ± 0.00 a 0.22 ± 0.003 c 0.29 ± 0.00 d 0.15 ± 0.00 ab 0.19 ± 0.00 bc 0.23 ± 0.01 c xxx
a
Significance levels: x, 95%; xx, 99%; xxx, 99.9%.
b
Letters indicate significant differences for each index between strain treatments by Tukey’s test (95%).

Am. J. Enol. Vitic. 69:2 (2018)

 Effects of Non-Saccharomyces Yeasts on Color – 153

T02/05F showed the best fermentation performance within
the non-Saccharomyces strains studied, is in agreement with
previous work for H. vineae in white wine fermentations (Me-
dina et al. 2013).
The negative relationship between TA and CI, which some-
times appears in red wine, was previously reported by us for
Saccharomyces yeast fermentations (Medina et al. 2005). We
demonstrated that CI better represents the real wine color
value than does TA, as it includes anthocyanin-derived com-
pounds that contribute to the final color.
Thus far, vitisin B formation has been reported previously
only for Saccharomyces yeasts (Morata et al. 2003a, 2006,
2007a, Suarez-Lepe and Morata 2012). Results obtained for
anthocyanin-derived compound formation during fermenta-
tion in our model grape medium show that vitisin B can be
linked to the increased acetaldehyde levels produced by S.
cerevisiae when compared with non-Saccharomyces yeasts
(Medina et al. 2016). This behavior could be explained by
increased specialization of primary metabolism by S. cerevi-
siae yeasts that leads to increased production of acetaldehyde
and ethanol. According to our results, all non-Saccharomy-
ces strains studied showed vitisin B formation, even when
the concentrations obtained were relatively low in relation to
that formed by Saccharomyces yeast. In contrast, we show
a higher formation of vitisin A with non-Saccharomyces
strains, which is likely linked to the presence of pyruvic
acid in the medium (Morata et al. 2007a); some non-Sac-
charomyces strains produce higher levels of pyruvic acid,

Figure 2 Average and standard deviation for each of the anthocyanin compounds quantified in mg/L. Letters indicate significant differences (p < 0.05)
between strains, as determined by Tukey’s test (95%). NS indicates no significant difference.

Am. J. Enol. Vitic. 69:2 (2018)

154 – Medina et al.

Figure 3 Vinylphenolic pyranoanthocyanin pigments identified by HPLC-DAD-MS in wines fermented by select non-Saccharomyces yeast strains and
the Saccharomyces yeast control strain (Sc 882). (A) vitisin B; (B) vitisin A; (C) malvidin-3-O-glucoside-4-vinylguaiacol; (D) malvidin-3-O-glucoside-
4-vinylphenol. The area of each M+ fragment was used for the quantification. Letters indicate significant differences (p < 0.05) between strains, as
determined by Tukey’s test (95%).

Benito et al. 2009), and Pichia guilliermondii (Benito et al.

Figure 4 Total derived anthocyanin compounds produced by each of the
strains studied. Letters indicate significant differences (p < 0.05) between
strains, as determined by Tukey’s test (95%).
compared with S. cerevisiae (Morata et al. 2012). Moreover,
this behavior could be related to the so-called “crabtree ef-
fect” of different yeast species (González et al. 2013), which
defines a self-regulating system based on yeast respiration
repression and the occurrence of alcoholic fermentation. Sac-
charomyces strains show a positive crabtree effect and tend
to be more efficient in ethanol fermentation at high concen-
trations of sugars, compared with some non-Saccharomyces
strains (González et al. 2013). To our knowledge, the produc-
tion of vitisin A has been reported only for S. cerevisiae
(Morata et al. 2003a, 2003b, 2007a) and Schizosaccharomy-
ces pombe, for which a higher production of pyruvic acid was
noted, compared with Saccharomyces (Morata et al. 2012,
Benito et al. 2014).
Interestingly, the formation of another anthocyanin-de-
rived compound during alcoholic fermentation, malvidin-
3-O-glucoside-4-vinylguaiacol, was previously reported for
S. cerevisiae 882 (Medina et al. 2005), other S. cerevisiae
strains (Morata et al. 2006, 2007b, Vanbeneden et al. 2008,
2011). Therefore, ours is the first report on the formation of
this derived compound for the yeast genera Hanseniaspora
and Metschnikowia (Figure 3C). The moderate formation of
derived pigments from vinylphenol and higher formation
from vinylguaiacol by M. pulcherrima could be explained
if this species had a more specific hydroxycinnamate de-
carboxylase (HCDC) activity for ferulic acid than for cou-
maric acid. By contrast, an HCDC activity more specific
for coumaric acid has been reported for strains of genera
Pichia, Torulaspora, Zygosaccharomyces (Chatonnet et al.
1993a, 1993b, Dias et al. 2003, Suezawa and Suzuki 2007),
and more recently for P. guilliermondii (Benito et al. 2011).
However, the higher concentration of malvidin-3-glucoside-
4-vinylguaicol found for all yeast treatments might also be
a consequence of the different concentrations of each of the
respective hydroxycinnamic acids in the grapes used, which
constitutes a varietal difference (Boursiquot 1987), as was
also reported for Tannat grapes (Boido et al. 2011). The for-
mation of this compound during alcoholic fermentation is in
agreement with our previous studies with Sc 882 (Medina
et al. 2005). Malvidin-3-O-glucoside-8-ethyl-catechin was
detected only in Sc 882 treatments (data not shown). It is
an acetaldehyde-mediated condensation product, so this re-
sult correlates with that obtained for vitisin B (Figure 3A),
which was present in the highest concentration for the Sc
882 strain.
The promising results obtained for some of the non-Sac-
charomyces strains evaluated here are supported by the fact
that the Sc 882 strain, used as a control, arose from a previ-
ous selection made by comparison of commercial Saccha-
romyces strains (Montrachet 522 and Lalvin 254D) (Medina
et al. 2005).

Am. J. Enol. Vitic. 69:2 (2018)


Conclusion
In this study, we demonstrated a diversity of effects of non-
Saccharomyces yeast strains on polyphenols and, specifically,
on the final red color affecting wine quality. Higher variability
between the studied strains was found for color indexes, while
little difference was observed in total polyphenols and antho-
cyanins. Our results also report, for the first time, synthesis
of derived anthocyanin pigments by genera of Hanseniaspora
and Metschnikowia yeasts. Additionally, the RGJM reported
here, applied to studies of non-Saccharomyces strains, will
allow screening of other yeast strains’ effects on wine phe-
nolics and for different red grape varieties.
The reported results highlight the importance of yeast di-
versity studies and yeast selection as a tool to modulate red
wine composition and to contribute to improved wine qual-
ity. Further studies of non-Saccharomyces strains in mixed
cultures with S. cerevisiae will be the next step for wine
evaluation at an industrial level.
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