LWT - Food Science and Technology 123 (2020) 109072

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LWT - Food Science and Technology

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The impact of Saccharomyces and non-Saccharomyces yeasts on wine colour: T

A laboratory study of vinylphenolic pyranoanthocyanin formation and
anthocyanin cell wall adsorption
Jelena Topić Božiča, Lorena Butinarb, Alen Albrehtc, Irena Vovkc, Dorota Kortea,
Branka Mozetič Vodopivecb,∗
a
Laboratory for Environmental and Life Sciences, University of Nova Gorica, Vipavska 13, 5000, Nova Gorica, Slovenia
b
Wine Research Centre, University of Nova Gorica, Glavni Trg 8, 5271, Vipava, Slovenia
c
Department of Food Chemistry, National Institute of Chemistry, Hajdrihova 19, 1000, Ljubljana, Slovenia

A R T I C LE I N FO A B S T R A C T

Keywords: Wine fermentation is a complex process driven by yeasts, which influence the key properties of wine quality:
Non-Saccharomyces yeast aroma, flavour and colour. We investigated 95 different Saccharomyces and non-Saccharomyces strains for their
Hydroxycinnamate decarboxylase activity impact on wine colour through the synthesis of the stable pigments, pyranoanthocyanins. All strains were
Sequential fermentation screened for their hydroxycinnamate decarboxylase (HCDC) activity that varied from 0.0% to 91.1%. Eight
Vinylphenolic pyranoanthocyanins
strains that showed more than 40% HCDC activity were further studied for vinylphenolic pyranoanthocyanin
Anthocyanin adsorption
formation. Fermentations were carried out in deep well microtiter plates using synthetic grape must containing
Pinot Noir skin extract supplemented with p-coumaric acid. Two strains Pichia guilliermondii ZIM624 and
Wickerhamomyces anomalus S138 synthesized vinylphenolic pyranoanthocyanins in the highest concentration in
single culture fermentations, 40.2 and 38.5 mg L−1, respectively. The highest produced concentration of vi-
nylphenolic pyranoanthocyanins in co-inoculation experiments was 16.4 mg L−1 compared to 29.8 mg L−1 in
sequential fermentations. For the first time, S. paradoxus strains were assessed for pyranoanthocyanin formation.
Selected strains were also tested as mono-cultures for adsorption of anthocyanins on yeast cell walls and all
strains showed anthocyanin and pyranoanthocyanin adsorption to their cell wall with Metschnikowia reukafii
ZIM2019 showing the highest adsorbing capability (9.1%).

1. Introduction The reaction mechanism consists of decarboxylation of hydro-

Alcoholic fermentation in the production of wine is conventionally
performed by yeasts Saccharomyces cerevisiae. There are numerous
starters available, however because of the growing demand for wines
with specific characteristics, other Saccharomyces and non-
Saccharomyces species are being investigated (Masneuf-Pomarede, Bely,
Marullo, & Albertin, 2016). The colour of the wine can be affected by
metabolites produced by yeast during fermentation and may react with
grape anthocyanins to form highly stable pyranoanthocyanins such as
vitisins (Morata et al., 2016). Similar to vitisins, vinylphenolic pyr-
anoanthocyanin adducts also show great colour stability, although they
differ in absorption maximums (Benito, Morata, Palomero, González, &
Suárez-Lepe, 2011; Morata, González, & Suárez-Lepe, 2007). For ef-
fective formation of vinylphenolic pyranoanthocyanins yeast strains
with high hydroxycinnamate decarboxylase (HCDC) activity are used.
xycinnamic acids and formation of vinylphenols that afterwards con-
dense with grape anthocyanins and form stable vinyphenolic pyr-
anoanthocyanins (Escott, Morata, Loira, Tesfaye, & Suarez-Lepe, 2016).
It has already been demonstrated that some non-Saccharomyces strains
(Pichia guilliermondii, Schizosaccharomyces pombe) have positive HCDC
activity and can produce vinylphenolic pyranoanthocyanins in higher
concentrations when compared to S. cerevisiae (Benito et al., 2011;
Morata et al., 2016). The presence of HCDC activity is quite common in
Saccharomyces yeast and has been reported in non-Saccharomyces
strains as well, however it is strain-dependent and activity can vary
greatly between species (Benito, Palomero, Morata, Uthurry, & Suárez-
Lepe, 2009; Kosel, Čadež, & Raspor, 2014; Morata et al., 2013;
Shinohara, Kubodera, & Yanagida, 2000; Smit, Cordero Otero,
Lambrechts, Pretorius, & Van Rensburg, 2003). HCDC active strains
have also been successfully exploited for reducing volatile phenols

∗
Corresponding author.
E-mail address: branka.mozetic@ung.si (B.M. Vodopivec).

https://doi.org/10.1016/j.lwt.2020.109072
Received 14 October 2019; Received in revised form 10 January 2020; Accepted 20 January 2020
Available online 21 January 2020
0023-6438/ © 2020 Elsevier Ltd. All rights reserved.
J.T. Božič, et al. LWT - Food Science and Technology 123 (2020) 109072

precursors through formation of vinylphenolic pyranoanthocyanins Table 1

(Benito et al., 2009). HCDC activity of yeast strains studied.
Conversely, wine colour could also be affected by the adsorption of Genus Species N°strains HCDC activity [%]
anthocyanins on the yeast cell wall which is especially important in tested (average)a
grape varieties that have lower anthocyanin content in grapes, such as
Wickerhamomyces W. anomalus 1 91.1
Pinot Noir (Morata, Gómez-Cordovés, Colomo, & Suárez, 2005). There
Torulaspora T. delbrueckii 7 0.8–29.9 (12.1)
are two main approaches that have been taken to assess the ability of Starmerella S. bacillaris 3 7.2–16.0 (12.1)
yeast to adsorb anthocyanins on the cell wall: qualitative and quanti- Saccharomyces S. servazii 1 0.7
tative assessment (Morata et al., 2016). The qualitative technique is S. paradoxus 11 40.6–66.6 (49.8)
based on visual assessment of growing yeast colonies in a solid medium S. kudriavzevii 6 2.6–9.1 (6.8)
S. cerevisiae 18 5.1–66.0 (33.0)
enriched with grape anthocyanins such as yeast-peptone-dextrose
S. bayanus 2 27.0–30.9 (28.9)
(YEPD) medium (Caridi, 2013; Caridi, Sidari, Kraková, Kuchta, & Pichia Pichia sp. 1 8.4
Pangallo, 2015). The second approach is quantitative and gives in- P. membranifaciens 2 10.0–20.6 (15.3)
formation about the concentration of anthocyanins adsorbed on yeast P. manshurica 4 0–66.9 (26.0)
P. kudriavzevii 2 0–19.4 (9.7)
cell walls and their structures (Morata et al., 2016).
P. kluyveri 3 1.0–18.1 (10.3)
The aim of our work was to first screen yeast strains from Slovenian P. guilliermondii 2 88.5–88.7 (88.6)
yeast collections, comprising native isolates that mostly originated from P. anomala 1 9.9
the Vipava valley and Karst region, for their HCDC activity and then to Metschnikowia M. pulcherrima 5 12.9–23.9 (18.7)
select yeasts with high enzyme activity for further study following M. fructicola 1 1.7
M. reukaufii 1 86
different fermentation strategies under oenological conditions to assess
Lachancea L. thermotolerans 4 0–16.2 (9.8)
vinylphenolic pyranoanthocyanin formation and the capability of yeast Kregervanrija K. fluxuum 1 29.1
cell walls to adsorb anthocyanins. Kluyveromyces K. dobzhanskii 1 0
Kodamaea K. ohmeri 1 21.5
2. Materials and methods Issatchenkia I. terricola 1 11.8–22.6 (17.2)
Hanseniaspora H. uvarum 9 0–18.6 (10.8)
H. osmophila 1 3.1
2.1. Yeast strains and media Debaromyces D. hansenii 3 16.8–41.3 (27.3)
Candida C. sake 1 1.6
A total of 95 different yeast strains belonging to 29 species were C. rugosa 1 18.2
C. diversa 1 17.2
selected for the initial assessment of HCDC activity (Supp. Information
S1). Strains were selected from the Wine Research Centre (University of a
All experiments were performed in four replicates. HCDC activity was ex-
Nova Gorica, Vipava, Slovenia) yeast collection and the Collection of pressed as the ratio of p-coumaric acid concentration of the strain used at the
Industrial Microorganisms (ZIM; Biotechnical Faculty, University of end of experiment and p-coumaric acid concentration in the control sample.
Ljubljana, Slovenia). Two commercial S. cerevisiae strains were also
tested – Fermol Premier Cru (FPC) (AEB Group, Italy) and Lalvin dryness. The Pinot Noir skin extract was reconstituted in SGM in the
EC1118 (Lallemand, Canada). ratio 1:1 and supplemented with 100 mg L−1 of p-coumaric acid.

2.2. Screening for HCDC activity

2.4. Adsorption of anthocyanins and pyranoanthocyanins on yeast cell
walls
Yeast pre-cultures were prepared by inoculating single colonies into
3 mL of YPD medium in 15 mL sterile centrifuge tubes. Precultures were
Yeast's ability to adsorb colour compounds was assessed both qua-
incubated for 24 h at 25 °C with shaking (250 rpm). The optical density
litatively and quantitatively. For the qualitative assessment the visual
(OD600) of washed pre-cultures was checked and adjusted to
evaluation protocol by Morata et al. (2016) was used, adapted for the
OD600 = 1 with 0.85% sterile saline solution, if necessary. The sterile
present study as Pinot Noir skin extract was used as a source of an-
medium used in this study was composed of 0.67% (w/v) commercial
thocyanins (Morata et al., 2016). For quantitative assessment, 11 mL
yeast nitrogen base (YNB) media with (NH4)2SO4, 2% glucose (w/v) and
samples of SGM-PNe 15 mL sterile centrifuge tubes and inoculated with
100 mg L−1 of p-coumaric acid dissolved in double distilled water. This
1.2 mL of prewashed yeast preculture to achieve a final OD600 = 0.1.
medium (1.35 mL), distributed into deep well microtiter plates, was
The tubes were covered with a sterile cover to prevent evaporation and
inoculated with 150 μL of prewashed pre-culture (OD600 = 1), to
fermented for 10. Samples were prepared in triplicates. After 10 days,
achieve a final optical density equal to 0.1. Deep well plates were
the wines were centrifuged (10 min, 5000 rpm) and sediments with
covered with a sterile cover and incubated for 10 days in four re-
anthocyanins from the cell cultures extracted with 10% v/v formic acid
plicates.
in MeOH (Morata et al., 2016). Before HPLC analysis, the acid extract
was dried, reconstituted in mobile phase A and filtered through a
2.3. Preparation of synthetic grape must (SGM) supplemented with Pinot
0.45 μm PTFE filter.
Noir skin extract (SGM-PNe)

SGM medium was prepared according to the protocol by Henscke
and Jiranek (Henscke & Jiranek, 2002) with the pH of the media ad-
justed to 3.5 to mimic the pH of wine.
Pinot Noir grapes were collected from a wine estate in the Vipava
valley in Slovenia. Skins of Pinot Noir (PN) grapes were peeled from the
berries and freeze-dried (Kambič Lio5pb, Slovenia), followed by
grinding in liquid nitrogen and storage at −20 °C. MeOH (20 mL) was
added to 1 g of ground skin and the solution was ultrasonicated for
15 min. After ultrasonication, centrifugation (Eppendorf 5804,
Hamburg, Germany) followed (5 min, 6000 rpm), with procedure re-
peated four times. Supernatant was collected and evaporated to
2.5. Vinylphenolic pyranoanthocyanins formation
Selected yeasts with high HCDC activity were further assessed for
vinylphenolic pyranoanthocyanin formation using SGM-PNe media.
Yeasts were inoculated as mono- or mixed cultures, combining non-
Saccharomyces strains with Saccharomyces strains. Fermentations were
carried out in deep well microtiter plates. Mixed culture fermentations
were carried out as co-inoculation of non-Saccharomyces with
Saccharomyces strains or as sequential fermentation where
Saccharomyces strain was added on day three of fermentation. 1.35 mL
of SGM-PNe was distributed into deep well microtiter plates and

2
J.T. Božič, et al. LWT - Food Science and Technology 123 (2020) 109072

Fig. 1. The average concentration of p-coumaric acid (dark grey) and 4-vinylphenol (light grey) after 10-day incubation (mean ± SD; n = 4). Lower p-coumaric acid
concentration corresponds to higher HCDC activity. S. cerevisiae (FPC, ZIM2180, ZIM3253, Sm58RT, M106); S. paradoxus (F29, M101RT, Ma106, Gu95, Sut85, Sut86,
Sm10, Ma108, F67, F60, Sut99); P. manshurica (M49); M. reukafii (ZIM2019); P. guilliermondii (Ca81, ZIM624); W. anomalus (S138), D. hansenii (Sut116RT).

Table 2 each sample were prepared. Prior to HPLC analysis, samples were
4-ethylphenol production by yeast strains with HCDC activity higher than 40%. centrifuged (10 min, 5000 rpm). The production of pyranoanthocyanins
STRAIN SPECIES Concentration [mg L−1]
was monitored using HPLC-DAD and identity of the pyr-
anoanthocyanins was confirmed using HPLC-DAD-MS/MS.
FPC S. cerevisiae n.d
Ca81 P. guilliermondii 16.4 ± 1.3
F29 S. paradoxus n.d
F60 S. paradoxus n.d
2.6. HPLC analysis
F67 S. paradoxus n.d
Gu95 S. paradoxus n.d HCDC activity was determined by using Agilent Technologies 1100
M101RT S. paradoxus n.d (Palo Alto, California, USA) HPLC with diode array detector. Isocratic
M106A S. cerevisiae n.d
elution at flow rate 1 mL min−1 of mobile phase A (0.2% trifluoroacetic
M49 P. manshurica n.d
Ma106 S. paradoxus n.d acid in water) and mobile phase B (0.2% trifluoroacetic acid in me-
Ma108 S. paradoxus n.d thanol) in the ratio 80/20 A/B was used. The column used for separa-
S138 W. anomalus n.d tion was Phenomenex Luna C18 PFP (250 × 4.6 mm, 5 μm i.d.) with
Sm10 S. paradoxus n.d matching guard column (Phenomenex, Torrance, USA). The time of
Sm58RT S. cerevisiae n.d
Sut116RT D. hansenii n.d
analysis was 15 min. Quantification was performed by an external
Sut85 S. paradoxus n.d standard method at 320 nm (p-coumaric acid), 280 nm (4-ethylphenol)
Sut86 S. paradoxus n.d and 260 nm (4-vinylphenol). Calibration parameters are presented in
Sut99 S. paradoxus n.d Supplementary Table S2. Samples were centrifuged after the end of
ZIM2019 M. reukaufii n.d
incubation for 10 min at 5000 rpm. The supernatant was analysed by
ZIM2180 S. cerevisiae n.d
ZIM3253 S. cerevisiae n.d HPLC. The injection volume was 40 μL.
ZIM624 P. guilliermondii 15.0 ± 0.5 For the monitoring of pyranoanthocyanin formation, gradient elu-
tion of mobile phase A and mobile phase B was used as followed: 0 min
*All experiments were performed in four replicates and are reported as 20% B, 0–20 min 20–45% B, 20–30 min 45–55% B, 30–40 min 55–70%
means ± SD. n.d. is abbreviation for not detected. B, 40–50 min equilibration to initial conditions. Quantification was
performed by an external standard method with malvidin-3-O-gluco-
inoculated with 150 μL of prewashed preculture to achieve a final side (Mvd-3-glc) (Extrasynthese, France) at 520 nm and 320 nm for p-
OD600 = 0.1. The deep well plate was covered with sterile cover to coumaric acid (Sigma-Aldrich, USA). Pyranoanthocyanins and other
prevent evaporation during fermentation for 10 days. Four replicates of anthocyanins were quantified and expressed as malvidin-3-O-glucoside

3
J.T. Božič, et al.
Fig. 2. The average concentration of vinylphenolic pyranoanthocyanins after 10-day single culture fermentations (mean ± SD; n = 4). Values with the same letter
are not statistically different (p < 0.05). Black colour represents Saccharomyces yeasts and light grey non-Saccharomyces yeasts.
equivalents. Other chromatographic conditions were the same as for
determination of HCDC activity.
2.7. HPLC-DAD-MS/MS analysis
Anthocyanins and pyranoanthocyanins were identified with
UHPLC–DAD–MS system (Accela 1250 coupled to an LTQ Velos MS,
Thermo Fisher Scientific, Waltham, MA, USA) Gradient elution of mo-
bile phase A (2.2% formic acid in ddH2O) and mobile phase B (2.2%
formic acid, 85% acetonitrile and 12.8% ddH2O) was used. Gradient
elution at the rate 1 mL min−1 was as followed: 0 min 100% A, 0–5 min
4
LWT - Food Science and Technology 123 (2020) 109072
Fig. 3. Chromatograms of SGM-PNe (solid line) and
ZIM624 single strain fermentation (dashed line).
Numbers 1–5 present anthocyanins present in Pinot
Noir skin extract. Numbers 6–9 present vinylphe-
nolic pyranoanthocyanins formed during fermenta-
tion. Data about their names, retention times and
MS data are presented in Table 3.
10% B, 5–20 min 10–35% B, 20–22 min 35% B, 22–26 min 35–45% B,
26–27 min 45–60% B, 27–32 min equilibration to initial conditions.
The column used was a Kinetex EVO C18 (250 × 4.6 mm, 5 μm i.d.)
with matching guard column (Phenomenex, Torrance, USA). The
column temperature was set to 40 °C. 20 μL of sample was injected into
the HPLC. The ESI parameters were: heater temperature 400 °C, drying
gas (N2), sheath gas flow rate 60 a.u., auxiliary gas flow rate 10 a.u.,
sweep gas flor rate 2 a.u., spray voltage 3 kV, transfer capillary tem-
perature 350 °C. Mass spectrometry was performed in positive scan
mode (scan range m/z 250–1200). Fragmentation of precursor ions was
performed with a normalized collision energy of 35%.
J.T. Božič, et al.
Table 3
MS data for anthocyanins and pyranoanthocyanins detected.
Peak Compound Abbreviation
Anthocyanins
1 Delphinidin-3-O-glucoside Dp-3-glc
2 Cyanidin-3-O-glucoside Cy-3-glc
3 Petunidin-3-O-glucoside Pt-3-glc
4 Peonidin-3-O-glucoside Pn-3-glc
5 Malvidin-3-O-glucoside Mvd-3-glc
Vinylphenolic pyranoanthocyanins
6 Delphinidin-3-O-glucoside-4-vinylphenol Dp-3-glc-4-VP
7 Petunidin-3-O-glucoside-4-vinylphenol Pt-3-glc-4-VP
8 Peonidin-3-O-glucoside-4-vinylphenol Pn-3-glc-4-VP
9 Malvidin-3-O-glucoside-4-vinylphenol Mvd-3-glc-4-VP
2.8. Statistical analysis
Means and standard deviation were calculated. One-way ANOVA
and Tukey (HSD) tests were applied in order to determine the effects of
yeast on vinylphenolic pyranoanthocyanins formation. Calculations
were performed using XLSTAT (Addinsoft SARL, USA). Significance was
set at p < 0.05.
3. Results and discussion
3.1. Determination of HCDC activity
Out of the 95 strains tested, 73.7% were considered HCDC positive,
as they transformed more than 10% of p-coumaric acid. As already
reported, HCDC activity is strain-dependent (Benito et al., 2009; Morata
et al., 2013), and in some cases the difference in p-coumaric acid
transformation between two strains exceeded 70% (Table 1). HCDC
activity of the tested S. cerevisiae strains, species most commonly used
in the production of wine, varied between 5.1% and 66.1%. Among the
18 strains tested, seven showed HCDC activity higher than 40%. The
commercial strains FPC and EC1118 showed transformation rates of
43.9% and 21.5%, respectively. It was observed that some native strains
had higher HCDC activity than commercial ones, with strain ZIM2180
showing the highest HCDC activity among tested S. cerevisiae strains
(66.0%). The p-coumaric acid transformation rate and its variability
between strains is in accordance with published data (Benito et al.,
2011; Morata et al., 2013). M. reukafii strains showed high HCDC ac-
tivity, however other Metschnikowia species tested in our study were
found to have less than 24% transformation rates. M. pulcherrima strains
had on average a 19.2% conversion rate. Other authors have also re-
ported positive HCDC activity in M. pulcherrima strains, however no
data was provided about the transformation rates of p-coumaric acid as
the method described was qualitative (Escribano et al., 2017).
Of the 22 strains showing more than 40% HCDC activity, 11 of them
were S. paradoxus. Among them, Sut85 exhibited the highest HCDC
activity (66.6%). S. paradoxus strains are present in wine fermentations
in Slovenia and their use as starters should be investigated in more
depth (Dashko et al., 2016). Other species with more than 40% HCDC
activity include three strains of S. cerevisiae (ZIM2180, ZIM3253,
Sm58RT), D. hansenii Sut116RT, P. manshurica M49, M. reukafii
ZIM2019, W. anomalus S138 and two strains of P. guilliermondii (Ca81,
ZIM624).
3.2. Production of 4-vinylphenol and 4-ethylphenol
The highest concentration of 4-vinylphenol was produced by P.
guilliermondii strains, W. anomalus (S138) and S. paradoxus (Sut85)
producing more than 50 mg L−1 of 4-vinylphenol. These four strains
were also among the strains with the highest conversion rate of p-
coumaric acid (Fig. 1). The general trend was that species with high
conversion rate of p-coumaric acid produced 4-vinylphenol in larger
5
LWT - Food Science and Technology 123 (2020) 109072
Ret. time [min] MS [m/z] MS/MS MS/MS/MS
6.9 465 303 303
7.8 449 287 287
9.3 479 371 317,302
10.1 463 301 301,286
11.6 493 331 331,316,299
20.6 581 303,262,257
23.7 595 433 433,418
25.0 579 417 417,402,374
26.2 609 447 447,431,414
concentrations. Strains associated with less than 40% HCDC activity
rate produced less than 15 mg L−1 of 4-vinylphenol.
Out of the 95 yeast strains tested, only two of the strains were able
to synthesize 4-ethylphenol, showing that they have also vinylphenol
reductase enzyme activity. Both were P. guilliermondii strains (ZIM624,
Ca81), producing 15.0 mg L−1 (ZIM624) and 16.4 mg L−1 (Ca81) of 4-
ethylphenol, respectively (Table 2). These results are in accordance to
qualitative assessments of the media in which these two P. guilliermondii
strains were incubated as they possessed a distinctive horsey/barn
smell. The ability of P. guilliermondii to produce 4-ethylphenol was also
previously reported in grape juices (Barata, Nobre, Correia, Malfeito-
Ferreira, & Loureiro, 2006; Dias et al., 2003; Jensen, Umiker, Arneborg,
& Edwards, 2009). Although they have been able to produce 4-ethyl-
phenol in synthetic wines and liquid cultures, their oenological im-
portance in wines was not established (Benito et al., 2009).
Eight strains with high HCDC (S138, Sut116RT, ZIM624, M49,
ZIM2019, Sut85, ZIM2180 and commercial FPC) activity were selected
for further studies on their role in vinylphenolic pyranoanthocyanin
formation and anthocyanin cell wall adsorption under oenological
conditions.
3.3. Formation of vinylphenolic pyranoanthocyanins
Of the strains tested, all non-Saccharomyces strains except P. man-
shurica M49 synthesized higher concentrations of vinylphenolic pyr-
anoanthocyanins compared to Saccharomyces strains. P. guilliermondii
ZIM624 and W. anomalus S138 produced almost double the average
concentration synthesized by other strains tested, 40.2 mg L−1 and
38.5 mg L−1, respectively (Fig. 2). Results correlate with the two strains
having the highest HCDC activity out of the strains tested in this study.
Malvidin-3-O-glucoside-4-vinylphenol (Mvd-3-glc-4-VP) was synthe-
sized in the highest concentration by all strains (Fig. 3). Other pyr-
anoanthocyanins that were formed include delphinidin-3-O-glucoside-
4-vinylphenol (Dp-3-glc-4-VP), petunidin-3-O-glucoside-4-vinylphenol
(Pt-3-glc-4-VP) and peonidin-3-O-glucoside-4-vinylphenol (Pn-3-glc-4-
VP) (Table 3). P. guilliermondii species have shown increased production
of vinylphenolic pyranoanthocyanins in comparison to S. cerevisae
strains due to generally high HCDC activity (Benito et al., 2011). W.
anomalus has been exploited as a strain for improving the aromatic
profile of wines and as a strain for biocontrol against spoilage yeast
(Padilla, Gill and Manzanares 2016; 2018), however to the best of au-
thors’ knowledge, to date no data is available about the effect of W.
anomalus on the synthesis of vinylphenolic pyranoanthocyanins.
Though S. cerevisae strains produced slightly higher concentrations
of vinylphenolic pyranoanthocyanins in comparison to S. paradoxus,
there was no statistical difference among them. Nevertheless, S. para-
doxus yeasts have been found to possess oenological traits of interest,
making them interesting for wine production as they can contribute to
wine aroma, have good fermentation vigour, show low levels of volatile
acidity and a good tolerance to alcohol (Majdak, Herjavec, Orlic,
Redzepovic, & Mirosevic, 2002; Orlic, Redzepovic, Jeromel, Herjavec,
J.T. Božič, et al. LWT - Food Science and Technology 123 (2020) 109072

Fig. 4. The average concentration of vinylphenolic pyranoanthocyanins after 10-day co-inoculation fermentations (A) and sequential fermentations (B)
(mean ± SD; n = 4). Values with the same letter are not statistically different (p < 0.05). Sequential fermentations resulted in higher concentration of vinyl-
phenolic pyranoanthocyanins.

& Iacumin, 2007). Until now no data about their ability to produce
vinylphenolic pyranoanthocyanins has been reported.
Chen et al. tested five non-Saccharomcyes and S. cerevisae 7VA strain
in single fermentations with the addition of oenological tannins. The
results showed that prefermentative addition of oenological tannins
positively influenced the development of vinylphenolic pyr-
anoanthocyanins, promoting colour stability and protecting the wines
from oxidation (Chen et al., 2018). This approach, with the use of yeast
with high HCDC activity may even further promote vinylphenolic
pyranoanthocyanin formation. Although non-Saccharomyces strains
were able to produce higher concentrations of vinylphenolic pyr-
anoanthocyanins, they are generally incapable of completing fermen-
tation due to low fermentation power and low SO2 resistance. Non-
Saccharomyces yeasts are therefore used in mixed fermentations with S.
cerevisae strains (Comitini et al., 2011; Jolly, Augustyn, & Pretorius,
2017). Pure non-Saccharomyces culture fermentations may show

6
J.T. Božič, et al.
Fig. 5. Concentrations of adsorbed anthocyanins (dark grey) and vinylphenolic pyranoanthocyanins (light grey) on the yeast cell wall (mean ± SD; n = 4). Capital
letters present statistical difference between yeasts' ability to adsorb vinylphenolic pyranoanthocyanins. Bars with the same letter are not statistically different
(p < 0.05). Small case letters present statistical difference between yeasts' ability to adsorb anthocyanins. Bars with the same letter are not statistically different
(p < 0.05).
negative metabolite formation such as acetic acid, acetoin and ethyl
acetate. The use of mixed fermentation starters allows exploitation of
the desirable qualities of non-Saccharomyces yeasts (stable pigment
formation, influence on wine aroma), and when combined with S.
cerevisae it is possible to avoid stuck or sluggish fermentations (Padilla,
Gil, & Manzanares, 2016; Petitgonnet et al., 2019). Choosing mixed
fermentation can result in the absence of expression of negative oeno-
logical characteristics of non-Saccharomyces yeast or they can be
modified by S. cerevisae strains (Ciani, Comitini, Mannazzu, & Domizio,
2010).
Out of 15 co-inoculation fermentation trials, 46.7% produced vi-
nylphenolic pyranoanthocyanins at concentrations higher than
14 mg L−1 (Fig. 4). Co-inoculations of non-Saccharomyces strains with
S. paradoxus Sut85 resulted in the lowest concentrations of vinylphe-
nolic pyranoanthocyanins, which was not the case in sequential fer-
mentations. A possible explanation for this may be due to the fermen-
tation dynamics of yeasts and competition between non-Saccharomyces
and Saccharomyces yeasts for nutrients, the presence of yeast-yeast cell
contact and possible excretion of antimicrobial compounds (Bagheri,
Bauer, & Setati, 2017; Ciani & Comitini, 2015; Medina, Boido, Fariña,
Dellacassa, & Carrau, 2016). An important aspect of mixed fermenta-
tions is their complexity, and the interactions between yeast species are
still unclear, warranting further research into their dynamics (Bagheri
et al., 2017). Among 15 sequential fermentation trials, 87.7% synthe-
sized vinlphenolic pyranoanthocyanins in concentration above
20 mg L−1. P. guillermondii ZIM624 and W. anomalus S138 when used in
sequential fermentations, synthesized pyranoanthocyanins in the
highest concentrations. Results correlate with the same two strains
being able to produce the highest concentrations of vinylphenolic
pyranoanthocyanins in single culture fermentation trials. In sequential
fermentation, non-Saccharomyces yeast can be used as enhancers of the
formation of polymeric pigments precursor compounds from the yeast
metabolism origin (Escott et al., 2018). In single culture fermentations,
7
LWT - Food Science and Technology 123 (2020) 109072
highly HCDC active S138 and ZIM624 were able to grow in the media
without needing to compete for nutrients, which was also found to be
the case in sequential fermentations before the addition of Sacchar-
omyces yeast on day three. During co-inoculation, Saccharomyces most
likely dominated over non-Saccharomyces yeasts, resulting in lower
pyranoanthocyanin formation.
3.4. Adsorption of anthocyanins and pyranoanthocyanins on yeast cell
walls
By growing yeasts in YPD medium with added PNe it was possible to
observe by eye that M. reukafii ZIM2019 formed the darkest colonies in
comparison to other yeast strains. Furthermore, HPLC analysis showed
that ZIM2019 adsorbed 9.1% of colour compounds, more than two
times higher than other tested strains (Fig. 5), which showed less than
4% adsorption. Anthocyanins identified in the cell walls were Pt-3-glc,
Pn-3-glc and Mvd-3-glc, which corresponded to the Pinot Noir grape
skin major anthocyanin profile. Vinylphenolic pyranoanthocyanins
identified in yeast cell walls were Pt-3-glc-4-VP, Pn-3-glc-4-VP and
Mvd-3-glc-4-VP with the latter being present in the highest concentra-
tion. The strains which adsorbed higher concentrations of colour
compounds adsorbed both higher concentrations of anthocyanins and
pyranoanthocyanins, however the ratio among the type of adsorbed
colour compounds differed. The adsorption ratio was in favour of
pyranoanthocyanins by at least a factor of three. Sut85 and Sut116
strain were found to be an exception with a ratio of adsorbed antho-
cyanins to pyranoanthocyanins being 1.3 (41.7%: 58.3% and 44.1%:
55.9%, respectively). A possible explanation for this phenomena could
be yeast cell wall constitution, as its thickness and composition varies
among species and strains (Aguilar-Uscanga & François, 2003). Most of
the published data related to the adsorption capacity of yeasts is related
to S. cerevisae strains, which are the most commonly used in the pro-
duction of wine (Caridi, Sidari, Solieri, Cufari, & Giudici, 2007; Caridi
J.T. Božič, et al.
et al., 2015; Echeverrigaray, Menegotto, & Delamare, 2019; Escott
et al., 2016; Morata et al., 2003; 2005). Reported adsorption percen-
tages of anthocyanins and pyranoanthocyanins vitisins were below
12.0% (Morata et al., 2005, 2016). However, information about ad-
sorption capability of non-Saccharomyces yeast is of importance because
non-Saccharomyces strains are being investigated for their use as wine
starters (Fleet, 2008). The results showed that there is variation among
species in the adsorption potential of anthocyanins which should be
taken into account when selecting yeast starters.
4. Conclusions
In our study we showed that yeasts with higher HCDC activity
synthesized higher concentrations of vinylphenolic pyr-
anoanthocyanins. Our results report for the first time, the synthesis of
vinylphenolic pyranoanthocyanins in the species S. paradoxus, W.
anomalus and M. reukafii. Yeasts were also tested for cell wall adsorp-
tion of colour compounds. Our results indicate that yeasts can adsorb
not only anthocyanins, but also vinylphenolic anthocyanins.
Furthermore, co-inoculation and sequential fermentation experiments
showed that sequential fermentation yielded higher concentrations of
vinylphenolic pyranoanthocyanins. Further studies with selected yeast
in mixed fermentations with Pinot Noir grape pomace is be the next
step towards evaluating the effect that yeasts will have not only on
formation of vinylphenolic pyranoanthocyanins, but also on other
sensory characteristics of wines, in particular wine aroma and flavour.
Funding
This work was supported by Slovenian Research Agency, Slovenia
(ARRS) by grant for young researchers: ARRS-MR-LP-2017/393 and
research programme P1-0034; Analytics and Chemical Characterization
of Materials and Processes, (2009–2019).
CRediT authorship contribution statement
Jelena Topić Božič: Investigation, Formal analysis, Visualization,
Writing - original draft. Lorena Butinar: Conceptualization,
Methodology, Resources. Alen Albreht: Resources, Formal analysis,
Writing - review & editing. Irena Vovk: Resources, Writing - review &
editing. Dorota Korte: Writing - review & editing, Supervision. Branka
Mozetič Vodopivec: Conceptualization, Methodology, Writing - re-
view & editing, Supervision.
Acknowledgements
The authors acknowledge dr. Iain Robert White for help with proof
reading the article.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.lwt.2020.109072.
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