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NMR-based metabolic profiling of different yeast fermented apple juices
Journal Pre-proof NMR-based metabolic profiling of different yeast fermented apple juices Erica Cusano, Laura Ruth Cagliani, Roberto Consonni, Barbara Simonato, Giacomo Zapparoli PII:
S0023-6438(19)31113-2
DOI:
https://doi.org/10.1016/j.lwt.2019.108771
Reference:
YFSTL 108771
To appear in:
LWT - Food Science and Technology
Received Date: 19 June 2019 Revised Date:
11 October 2019
Accepted Date: 20 October 2019
Please cite this article as: Cusano, E., Cagliani, L.R., Consonni, R., Simonato, B., Zapparoli, G., NMRbased metabolic profiling of different yeast fermented apple juices, LWT - Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.108771. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
NMR-based metabolic profiling of different yeast fermented apple juices
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Erica Cusanoa, Laura Ruth Cagliania, Roberto Consonnia*, Barbara Simonatob, Giacomo Zapparolib
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a
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12, 20133 Milan, Italy
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b
Department of Biotechnology, Università degli Studi di Verona, 37134 Verona, Italy
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*
Corresponding author
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Email address: [email protected] (R. Consonni)
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Tel.: +39-02-23699578
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Fax: +39-02-23699620
Institute of chemical sciences and technologies “G. Natta” (SCITEC), Lab. NMR, CNR, v. Corti
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1
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ABSTRACT
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NMR based metabolic profiling was used to investigate metabolic changes in ciders fermented by
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yeast species as valuable tool for the identification of metabolites responsible for yeast activities. 1H
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NMR spectra of apple ciders produced by Saccharomyces cerevisiae EC1118, Torulaspora
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delbrueckii TD291, Starmerella bacillaris YR21, Hanseniaspora osmophila HO16, Hanseniaspora
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uvarum Y4M, and Saccharomyces uvarum SU3, revealed to be dominated by glycerol and organic
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acids signals. The aromatic region showed the presence of low intense signals referred to
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epicatechin, catechin, chlorogenate, xanthine, uracil, tyrosol, fumarate, histamine, and histidine.
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The anomeric region showed signals related to xylose, arabinose, glucose, fructose and sucrose.
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Finally, in the aliphatic region, signals due to amino acids like alanine, aspartate, asparagine,
25
isoleucine and threonine, organic acids like acetate, lactate, malate, pyruvate, quinate, succinate,
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and alcohols like 2,3 butanediol, ethanol, and glycerol and sterol were detected. Principal
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component analysis performed considering NMR data from either all spectral regions and only
28
aromatic region revealed the potentiality to discriminate the yeasts action. Signals due to glucose,
29
fructose, glycerol, malate, fumarate, tyrosol, histidine, and histamine resulted discriminant for
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sample differentiation. This study, the first one by NMR, provides preliminary insights on the
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metabolic differences among yeast species involved in cider production.
32 33
Keywords: apple cider, NMR, yeast, fermentation, metabolite profiling
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2
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1. Introduction
36
Cider is a slightly alcoholic beverage produced from the fermentation of apple juice that is often
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performed by selected Saccharomyces cerevisiae strains following an oenological protocol. This
38
procedure, generally utilized by the industry, assures a controlled and standardized cidermaking
39
process (Cusano, Simonato, & Consonni, 2018). Recently, a positive impact on the aromatic
40
compounds of cider has assessed by the fermentation with different Saccharomyces and non-
41
Saccharomyces strains, both in co-inoculation with S. cerevisiae (Xu, Zhao, & Wang, 2006; Aung,
42
Lee, Yu, & Liu, 2015; Liu, Aung, Lee, & Yu, 2016; Ye, Yue, & Yuan, 2014) and as single cultures
43
(Lorenzini, Simonato, Slaghenaufi, Ugliano, & Zapparoli, 2019; Wei, Wang, Zhang, Yuan, & Yue,
44
2019; Gutierrez, Boekhout, Gojkovic, & Katz, 2018). The increasing interest of this practice is
45
related to their unusual properties to produce specific metabolites and/or reduce the ethanol yield,
46
together with the production of aromatic compounds (Dashko et al., 2015). Only few data are
47
available on the impact of different yeast strains on the cider non-volatile compounds (Laaksonen,
48
Kuldjärv, Paalme, Virkki, & Yang, 2017).
49
A performing analytical technique to analyze the soluble metabolites characterizing the cider is the
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Nuclear Magnetic Resonance (NMR) spectroscopy (Del Campo, Berregi, Iturriza & Santos, 2006;
51
Monakhova, Kuballa & Lachenmeier, 2012; Zuriarrain, Zuriarrain, Puertas, Dueñas, & Berregi,
52
2015; Cusano et al., 2018). As a matter of fact, NMR is a valuable tool for the identification of
53
metabolites such as valine, pyruvate, succinate, proline responsible for differentiation of yeast
54
strains involved on the alcoholic fermentation (Son et al., 2009). These molecules being products of
55
cellular metabolism can have a key role for monitoring the fermentation process and relating yeasts
56
to final product quality. Indeed, due to the advantages of this technique, such as the simplicity of
57
sample preparation, measurement rapidity and the possibility to investigate a wide spectrum range
58
of chemical compounds present in different amount with a single experiment, it is easily possible to
59
assess important details of metabolites profiles related to a certain species or strains.
3
60
Despite the high potentiality of this technique to investigate the metabolomics of yeasts involved on
61
the production of the fermented beverages, 1H NMR investigations on this issue are still scarce.
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The aim of this study was the investigation of the metabolic content of ciders obtained by apple
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juice fermentation carried out by six different Saccharomyces and non-Saccharomyces yeast strains.
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The NMR approach allowed to determine the effect of different yeast strains on the metabolite
65
content of cider evolution.
66 67
2. Materials and Methods
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2.1. Yeast strains and fermentation process
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In this study six different yeast strains were tested. Saccharomyces cerevisiae EC1118 (Lallemand,
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Montreal, Canada) and Torulaspora delbrueckii TD291 (BIODIVATM, Lallemand) were
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commercial. Starmerella bacillaris YR21, Hanseniaspora osmophila HO16, Hanseniaspora
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uvarum Y4M, and Saccharomyces uvarum SU3 were strains isolated from apple juice and wine and
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belong to the collection of Dipartimento di Biotecnologie of University of Verona, Italy. Yeasts
74
were maintained on YPD agar (1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose and 1.5%
75
w/v agar) prior to use and also kept at −80°C with 25% w/v glycerol.
76
Golden Delicious apples were squeezed to obtain the juice characterized by 9.9 °Brix as total
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soluble solid, 3.4 g/L total acidity expressed as malic acid equivalents and pH 3.43. The apple juice
78
was pasteurized for 5 min at 70°C before fermentation. Each yeast strain was incubated in 10 mL
79
YPD medium at 28°C on a rotary shaker, and after two days cells were separated by centrifugation
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(5000 g, 4°C, 5 min) and washed with sterile 0.9% w/v NaCl. Each strain was suspended in 10 mL
81
of apple juice and finally added to 600 mL of pasteurized apple juice to a final concentration of 106
82
CFU/mL. The fermentations were performed at 20°C in duplicate and supervised following the
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amount of produced CO2 by measuring the weight loss until reaching a stable weight. At the end of
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fermentation the produced ciders were stored at 4°C for 72 h to favour the sedimentation of
85
suspended materials and then racked. 4
86 87
2.1 Sample preparation for NMR analysis
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A total of 12 ciders obtained by the apple juice fermentation carried out by each of six yeast strains
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investigated in a double biological replicates (two ciders for each yeast). About 100 mg of
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lyophilized sample was dissolved in 600 µL of deuterated methanol (CH3OH-d4 Eurisotop, 99.80
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atom % D, France) and 120 µL of deuterated water (H2O Sigma-Aldrich, 99.96 atom % D, Italy);
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the solutions were stirred for 3 min at room temperature and used for NMR analysis.
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2.2 NMR data acquisition
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Spectra were recorded with a Bruker DRX 600 spectrometer (Bruker Biospin GmbH Rheinstetten,
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Karlsruhe, Germany) operating at 14.1 T, equipped with a 5-mm inverse probe with a z-gradient, at
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room temperature (300 K). Spectra were acquired with a spectral with of 7184 Hz, over 32 K data
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points. The residual water suppression was achieved by applying a presaturation scheme with a
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low-power radiofrequency irradiation for 1.2 s.
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2.2.1 NMR data processing and analysis
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A resolution enhancement function with an exponential multiplication of 0.3 Hz for the line
103
broadening was applied before Fourier transformation. All spectra were carefully phased and
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baseline-adjusted with TOPSPIN 3.0 software (Bruker Biospin GmbH Rheinstetten, Karlsruhe,
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Germany) and referenced to the methanol signal at 3.31 ppm and 49.9 ppm for 1H and
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respectively. Successively spectra were imported to ACD Spectrus processor (ACD Labs 2012,
107
Toronto, Canada). Intelligent bucketing was applied to all spectral region (the range of 0.00 and
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9.75 ppm) in order to compensate possible small signals shift due to different pH. The spectral
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region including the solvent signal (3.29 and 3.33 ppm) was set to zero constant value before the
110
bucket normalization performed with respect to the total integral value. Alternatively, only the
111
aromatic region was considered (the range of 5.52 and 9.75 ppm) and a new bucket normalization
13
C
5
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was performed. These normalizations allowed to evaluate the relative abundance of the identified
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metabolites. Table of buckets was imported into SIMCA-P 13 (Umetrics, Umea, Sweden) and
114
Principal Component Analysis (PCA) was performed, with mean centering as data pretreatment.
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3. Results and Discussion
117
1
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shown in Fig. S1 and Fig. 1A-C. The metabolites assignment was achieved by recording
119
bidimensional homo and heteronuclear NMR experiments and literature data (Cusano et al., 2018).
120
The relative abundance of all the identified metabolites is reported in Table 1 and figure S2 for all
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the investigated strains; their values are expressed as percentage value respect to the integral of all
122
resonances in the complete spectral region. In this way they reflect their relative abundance over the
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entire metabolite content. The 1H NMR spectra (Fig. S1) were dominated by glycerol organic acids
124
signals and, in some samples, by the saccharides content. The amount of glycerol and organic acids,
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produced during the alcoholic fermentation, can significantly be affected by the yeasts (Vileda et
126
al., 2019). As a matter of fact, glycerol, pyruvate, malate, and succinate were differentially
127
produced by S. cerevisiae strains in wine, previously analysed by 1H NMR (Son et al., 2009).
128
Moreover, glycerol, succinate, and α-glucose signals were found largely different between Fiano
129
wines produced by autochthonous and commercial S. cerevisiae strains (Mazzei, Spaccini,
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Francesca, Moschetti, & Piccolo, 2013).
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On the basis of the quantification values in Table 1, fructose (min. 0.61±0.02, max 7.59±1.20) and
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glucose (min. 0.81±0,05, max. 5.53±0.81) are largely present among saccharides, aspartate (min.
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0.51±0.03, max. 1.25±0.04), asparagine (min. 0.45±0.03, max. 0.54±0.06) and threonine (min
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0.27±0.90 max 0.591±0.005) among amino acids, malate (min. 3.72±0.25, max. 7.02±0.25) and
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succinate (min. 1.26±0.21, max. 3.00±0.02) among organic acids, while glycerol (min. 9.82±0.83,
H NMR spectra of apple ciders fermented with Saccharomyces and non-Saccharomyces yeasts are
6
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max 18.27±0.18) and 2,3-butanediol (min. 0.20±0.02, max. 1.39±0.14) resulted the largely
137
abundant among other compounds.
138
By considering the aliphatic region (Fig. 1A) of the 1H NMR spectrum, signals due to amino acids
139
like alanine, asparagine, aspartate, isoleucine, and threonine, organic acids like acetate, lactate,
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malate, succinate, pyruvate, and quinate, alcohols like ethanol and 2,3-butanediol, and sterol were
141
detected. Since molecules such as acetate, lactate, malate, and 2,3-butanediol have an important
142
impact on aroma of fermented beverages and their production can be related to yeast metabolism
143
(Romano, Brandolini, Ansaloni, & Menziani, 1998; Saayman & Viljoen-Bloom, 2006), their
144
identification may provide useful information on possible role of the fermenting yeast on
145
characterizing the volatile profile of cider. As regards to acetate signal, its presence in large amount
146
in ciders obtained by Hanseniaspora uvarum Y4M (0.15±0.04) and Hanseniaspora osmophila
147
HO16 (0.118±0.004) was expected since these species are generally high acetate producers
148
(Romano, Suzzi, Comi, & Zironi, 1992). Conversely, less obvious was the high acetate content in
149
Starmerella bacillaris YR21 sample (0.23±0.02), since this species generally produces low amount
150
of acetate during the alcoholic fermentation (Magyar & Tóth, 2011; Rantsiou et al., 2017). The high
151
content of malate (min. 3.72±0.25, max. 7.02±0.25) in ciders was expected being this organic acid
152
abundantly present in apple juice. Moreover data showed that this acid was most abundant in cider
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fermented by Saccharomyces cerevisiae EC1118 (7.02±0.25); this could be due to an inefficiently
154
use of malate by this yeast as reported in a previous study (Casal, Paiva, Queirόs, & Soares-Silva,
155
2008). Furthermore lactate was largely present in cider fermented by Saccharomyces uvarum SU3
156
strain (1.92±0.08), thus suggesting higher production of this acid through glyoxase pathway
157
(Martins, Cordeiro, & Ponces Freire, 2001) for this strain compared with the other yeasts. The
158
detection of amino acids can evidence interesting metabolic differences among the yeasts that may
159
impact on fermentation kinetics of apple juices as well as on sensory properties of cider since amino
160
acids are precursors of higher alcohols (Mangas, Cabranes, Moreno, & Gomis, 1994). Different 7
161
signals of amino acids, particularly aspartate, alanine and threonine, among cider profiles, are
162
congruent with different consumption patterns for amino acids by yeasts fermenting apple juice
163
(Suárez Valles, Palacios García, Rodríguez Madrera, & Picinelli Lobo, 2005; Eleutério Dos Santos
164
et al. 2015). The variability of amino acid profiles due to yeasts affects the formation of fusel
165
alcohols and esters in cider (Eleutério Dos Santos et al. 2015). The anomeric region of 1H NMR
166
spectra (Fig. 1B) showed signals related to xylose and arabinose in all samples; sucrose was largely
167
present in ciders fermented by Hanseniaspora osmophila HO16 (0.63±0.02), while in fermented
168
ciders by Torulaspora delbrueckii TD291 only in very low amount (0.0073±0.0001). In our
169
previous investigation (Cusano et al., 2018) the NMR signals of xylose and arabinose were
170
observed in cider from apple juice fermented by the same strain of Saccharomyces cerevisiae
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EC1118 used in the present study and were highly correlated in natural or sparkling ciders (Gomis,
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Tamayo, Valles, & Mangas Alonso, 2004). The large presence of sucrose in cider obtained by
173
Hanseniaspora osmophila HO16 (0.63±0.02) is probably due to its inability to ferment and
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assimilate this disaccharide. Interestingly, López (López, Mateo, & Maicas, 2016) reported strain-
175
specific differences on the sucrose assimilation among different species Hanseniaspora spp.,
176
including Hanseniaspora osmophila HO16 and Hanseniaspora uvarum Y4M. Glucose, present in
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both α and β isoforms in all samples, appeared dominant only in Starmerella bacillaris YR21
178
(5.53±0.81), while in lower amount in ciders fermented with all the other yeasts (min 0.81±0.05,
179
max 0.93±0.09). Fructose was largely present only in Torulaspora delbrueckii TD291 (7.59±1.20)
180
and Hanseniaspora uvarum Y4M (3.78±0.26) while lower amount was present in ciders fermented
181
with Hanseniaspora osmophila HO16 (1.83±0.15), and only in a few amount in Starmerella
182
bacillaris YR21 (0.61±0.02), Saccharomyces uvarum SU3 (0.73±0.03), and Saccharomyces
183
cerevisiae EC1118 (0.73±0.04). These results suggest differences in consumption rate on glucose
184
and fructose among these yeasts at the fermentation conditions used in the present study. In fact,
185
preferences of yeast for one or the other saccharidic moiety can determine an unbalance ratio of 8
186
these two sugars at the end of fermentation. Specifically, the large presence of glucose and the low
187
presence of fructose in cider fermented by Starmerella bacillaris YR21 (5.53±0.81 and 0.61±0.02,
188
respectively) is congruent with its fructophilic behavior (Magyar & Toth, 2011). Moreover, signals
189
of these two saccharides in cider produced by Hanseniaspora osmophila HO16 (0.81±0.05 and
190
1.83±0.15, respectively) and Hanseniaspora uvarum Y4M (0.84±0.14 and 3.78±0.26, respectively)
191
were consistent to their glucophilic character in agreement with previous determination (Granchi,
192
Granuci, Messini, & Vincenzini, 2002) for the former yeast, but in contrast with other results (Ciani
193
& Fatichenti, 1999) for the latter yeast. This last incongruence could be related to phenotypic
194
diversity among strains of the same species and deserve further investigation.
195
Conversely, the large presence of fructose on cider produced by Torulaspora delbrueckii TD291
196
(7.59±1.20) can be attributable to preference of this yeast on glucose. However, it is interesting how
197
different is the amount of this saccharide with respect to Saccharomyces cerevisiae EC1118
198
(0.73±0.04) which are glucophilic too. The patterns of saccharide utilization by these two yeasts
199
can be similar or different according to the fermentation conditions (Santos et al., 2008).
200
Specifically, in anaerobic conditions, fructose consumption rate of Torulaspora delbrueckii TD291
201
is much lower than Saccharomyces cerevisiae (Santos et al., 2008), and this rate, together with the
202
production of other metabolites, can change according to the oxygen availability during the
203
fermentation process (Ramírez & Velázquez, 2018). Further investigations are required to fully
204
assess whether sucrose and fructose can potentially be key metabolites for Hanseniaspora
205
osmophila HO16 and Torulaspora delbrueckii TD291, respectively, involved in cider production.
206
The aromatic region of 1H NMR spectra (Fig. 1C) showed only low intense signals. Specifically,
207
they referred to epicatechin, catechin, and chlorogenate commonly present in all samples (Tables 1,
208
Fig. S2), xanthine is present in low amount in all fermented ciders (lower than 0.0081±0.0001), as
209
well as uracil (lower than 0.0102±0.0009), and tyrosol (lower than 0.042±0.001) observed mainly in
210
ciders fermented by Saccharomyces cerevisiae EC1118 and Saccharomyces uvarum SU3, while in 9
211
lower amount in all other fermented ciders. Table 2 and figure S3 summarizes the relative amount
212
of the identified aromatic compounds, normalized to the total aromatic content, for a clearer
213
evaluation. The different amount of xanthine observed among all the fermented ciders (min
214
0.24±0.05, max 1.41±0.02) could suggest metabolic differences among yeast species in the purine
215
degradation. Similarly, the different content of uracil (min 0.26±0.11, max 1.78±0.18) could be
216
explained by different capacity to uracil release into the growth medium by the yeasts. Obviously,
217
the explanation of our results through possible different regulation of these pathways among yeasts
218
is merely speculative since elucidations on pathways involved in the metabolism of xanthine and
219
uracil have been carried out only on Saccharomyces cerevisiae (Séron, Blondel, Haguenauer-Tsapis
220
& Volland 1999; Rolfes, 2006). On the other hand, the presence of tyrosol, an aromatic alcohol that
221
can positively contribute to sensory properties of fermented beverages, observed among yeasts is
222
consistent with data reported by González (González et al., 2018). The Ehrlich pathway involved in
223
the production of this alcohol, together to tryptophan and 2-phenylethanol, in Torulaspora
224
delbrueckii TD291 and Starmerella bacillaris YR21 seems to be differentially regulated with
225
respect to that in Saccharomyces cerevisiae. In addition, the aromatic region of the 1H NMR
226
spectrum, showed that ciders obtained by Saccharomyces cerevisiae EC1118 and Saccharomyces
227
uvarum SU3 contained low levels of histamine (0.45±0.02 and 0.50±0.11,
228
histidine (0.57±0.05 and 0.52±0.14, respectively) with respect to all the other samples (min
229
1.59±0.02, max 6.26±1.07 for histamine, and min 3.07±0.20, max 7.88±1.88 for histidine).
230
Different levels of biogenic amines including histamine in wine or in other foodstuff produced by
231
various yeasts species or strains have been attributed to their diverse amino acid decarboxylase
232
activities involved on their production (Tristezza et al., 2013). Moreover, the variability on release
233
of biogenic amines among yeasts depends to medium composition and fermentation condition
234
(Restuccia, Loizzo, & Spizzirri, 2018). At the experimental condition used in the present study the
235
very low content of histamine and its precursor histidine only in cider produced by Saccharomyces
respectively) and
10
236
yeasts suggests that the histidine degradation could be carried out by aromatic amino acid
237
aminotransferases involved in a pathway alternative with respect to that produces histamine. Since
238
differences on the regulation of this pathway have been observed between Saccharomyces
239
cerevisiae and Candida glabrata (Brunke et al., 2014), we could not exclude that the higher
240
presence of histamine in cider obtained by non-Saccharomyces yeasts is due to metabolic
241
differences among yeasts to use histidine as a potential nitrogen source. Finally, fumarate resulted
242
present in all cider samples and with a high amount in Hanseniaspora osmophila HO16 and
243
Starmerella bacillaris YR21 (12.88±0.52 and 15.27±0.80, respectively). Cabranes (Cabranes,
244
Mangas, & Blanco, 1997) reported great intra-specific variations on the amount of this acid
245
produced during alcoholic fermentation of apple juices by Saccharomyces cerevisiae and
246
Hanseniaspora uvarum (synonym Kloeckera apiculata) strains. Further analyses, using various
247
strains of each yeast species, should be necessary to ascertain possible inter-specific differences on
248
the production of fumarate. This organic acid could inhibit the malolactic fermentation that can
249
naturally occur or be induced in cider (Herrero, Gracía, & Díaz, 2003). Nevertheless, the amount of
250
fumarate in ciders at the end of alcoholic fermentation is generally far below the level that is
251
considered inhibitory against malolactic bacteria (Herrero, Cuesta, García, & Díaz, 1999; Herrero et
252
al., 2003). To highlight possible differences in the metabolite content of all the investigated
253
samples, PCA analysis was initially performed considering NMR data from all spectral width
254
adopting “mean centering” as data pretreatment. The model yielded two components that explained
255
92.4% of the total variance. The score plot reported in figure 2A showed the first PC negatively
256
affecting Torulaspora delbrueckii TD291 and Starmerella bacillaris YR21 fermented samples,
257
while conversely it positively affected the other clustered ciders. In particular these latter samples
258
were belonging to the Saccharomyces and Hanseniaspora genus, thus showing similar metabolite
259
content. As expected due to the scaling adopted, in the corresponding loading plot (Fig. 2B, S4) the
260
more intense signals due to saccharides, glycerol and organic acids, resulted discriminant for
261
samples differentiation. As a matter of fact α and β glucose (buckets at 5.11 ppm, and 3.15, 3.32, 11
262
3.38, 4.49 ppm respectively) were the characterizing metabolites for Starmerella bacillaris YR21
263
samples, fructose (buckets at 3.47, 3.62, 3.74, 3.76, 3.84, and 3.99 ppm) for Torulaspora
264
delbrueckii TD291 samples, while fermented ciders obtained by using yeast belonging to
265
Saccharomyces and Hanseniaspora genus showed a higher content in glycerol (buckets at 3.50 and
266
3.58 ppm), malate (buckets at 2.58, 2.76, and 4.33 ppm), and succinate (bucket at 2.55 ppm). These
267
data confirmed the glucophilic and fructophilic beahaviour of Torulaspora delbrueckii TD291 and
268
Starmerella bacillaris YR21 respectively, respect to Hanseniaspora and Saccharomyces genus.
269
Successively, in order to investigate the possible samples discrimination according to the non-
270
volatile aroma compounds induced by the action of different yeasts, a new PCA considering only
271
the aromatic NMR spectral regions was performed. This model resulted in five components
272
explaining the 98.2% of the total variance. The score plot reported in figure 3A showed that
273
according to the first PC, a clear-cut separation could be achieved among Saccharomyces cerevisiae
274
EC1118 and Saccharomyces uvarum SU3, and all the other samples grouped in the opposite space
275
of the plot, resulting the former essentially characterized by tyrosol content (buckets at 6.71 and
276
7.01 ppm, Fig. 3B, S5). The latter group of samples could be better characterized by the use of the
277
second PC. As a matter of fact, this PC highlighted histidine (buckets at 6.98 and 7.70 ppm) content
278
negatively affecting Hanseniaspora uvarum Y4M and Torulaspora delbrueckii TD291 samples
279
while histamine (buckets at 7.10 and 7.77 ppm) and fumarate (bucket at 6.64 ppm) content
280
positively affected Starmerella bacillaris YR21 and Hanseniaspora osmophila HO16 (Fig. 3B, S5).
281
The statistical evaluation of aromatic region of 1H NMR spectra of yeast fermented apple juices,
282
highlighted a higher content of biogenic amines for all yeast with the exception of Saccharomyces
283
genus. Furthermore, Starmerella bacillaris YR21 and Hanseniaspora osmophila H016, showing a
284
higher histamine content, suggesting a
285
Finally, the higher content of tyrosol observed for Saccharomyces cerevisiae EC1118 and
286
Saccharomyces uvarum SU3 suggested a more performing Ehrlich metabolic pathway.
larger aromatic amino acid aminotransferase activity.
287 12
288
4. Conclusions
289
The NMR analysis of ciders evidenced the metabolic differences among yeast species involved in
290
apple juice fermentation. The discrimination of ciders according to NMR spectra was linked to
291
metabolic profiles of yeasts that were characterized by signals of different molecules mostly
292
belonging to the primary metabolism. In particular, metabolisms of saccharides, purines and
293
pyrimidines, and amino acids seem to be important in characterizing yeasts. However, the lack of
294
knowledges for most of species used in this study on molecular mechanisms involved on these
295
pathways did not allow a deeper understanding of NMR fingerprinting generation for each yeast at
296
the fermentation conditions here reported. Although the data here presented constitute a preliminary
297
report in the view of finding potential species-specific key compounds (biomarkers) related to the
298
apple juice fermentation, insights gained by the present work, as the first on NMR-based metabolic
299
profiling of yeasts fermenting apple juice, are useful as starting-points for further investigations.
300 301
Acknowledgments
302
We thank Lorenzini Marilinda for technical assistance.
303
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428
Figure captions
429
Fig. 1. Aliphatic (A), anomeric (B), and aromatic (C) expansions of 1H NMR spectra of
430
methanol/D2O extract of apple ciders samples analyzed showing principal metabolite assignments.
431
a) Hanseniaspora uvarum Y4M, b) Torulaspora delbrueckii TD291, c) Saccharomyces cerevisiae
432
EC1118, d) Saccharomyces uvarum SU3, e) Starmerella bacillaris YR21, f) Hanseniaspora
433
osmophila HO16.
434
Fig. 2. Score (A) and loading (B) plot obtained by performing PCA on NMR data considering all
435
spectral regions. 2PC, R2X= 92.4% and Q2=86.4%. EC1118: Saccharomyces cerevisiae EC1118,
436
TD291: Torulaspora delbrueckii TD291, YR21: Starmerella bacillaris YR21, HO16:
437
Hanseniaspora osmophila HO16, Y4M: Hanseniaspora uvarum Y4M, and SU3: Saccharomyces
438
uvarum SU3,
439
Fig. 3. Score (A) and loading (B) plot obtained by performing PCA on NMR data considering only
440
the aromatic spectral regions. 5PC, R2X= 98.2% and Q2=81.2%. EC1118: Saccharomyces
441
cerevisiae EC1118, TD291: Torulaspora delbrueckii TD291, YR21: Starmerella bacillaris YR21,
442
HO16: Hanseniaspora osmophila HO16, Y4M: Hanseniaspora uvarum Y4M, and SU3:
443
Saccharomyces uvarum SU3,
444
19
445
Fig. 1A
446 447
448
449
450
451
452
453
20
454
Fig. 1B
455
456
457
458
459
460
461
462
463
21
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Fig. 1C
465
466
467
468
469
470
471
472
473
474
475
22
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Fig. 2A
477 478
479
480
481
482
483
484
485 23
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Fig. 2B
487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502
24
503
Fig. 3A
504 505 506
25
507
Fig. 3B
508
509 510
26
511
Tables.
512
Table 1. Relative amount of selective integrals of the identified compounds expresses as
513
percentage values respect to the total values of all integrals over the entire integrated spectral
514
region. Standard deviation is included for all values. Discriminating metabolites from PCA analysis
515
are reported in bold.
RELATIVE AMOUNT FOR SINGLE STRAIN COMPOUNDS
c
YR21
HO16
SU3
EC1118
TD291
Y4M
0.0627±0.0237
0.0338±0.0004
0.0268±0.0035
0.0464±0.0035
0.0311±0.0031
0.0281±0.0007
0.6078±0.0249
1.8288±0.1457
0.7323±0.0273
0.7293±0.0431
7.5851±1.1968
3.7807±0.2577
5.5342±0.811
0.8078±0.0496
0.8687±0.0848
0.8860±0.0176
0.9265±0.091
0.8400±0.1442
Saccharides Arabinose Fructose Glucose
a
b
Sucrose
0.0206±0.0010
0.6308±0.0198
0.0110±0.0023
0.0092±0.0008
0.0073±0.0001
0.0134±0.0003
b
Xylose
0.3594±0.0432
0.3862±0.0137
0.5428±0.0366
0.5997±0.0096
0.3777±0.0381
0.3513±0.0192
Alanine
0.0651±0.0041
0.1120±0.0015
0.1199±0.0064
0.0629±0.0152
0.1117±0.0214
0.1348±0.0439
Aspartate
0.5103±0.0275
0.7106±0.0378
1.2487±0.0447
1.0487±0.0351
0.5396±0.0361
0.6392±0.0128
Asparagine
0.4856±0.0203
0.4463±0.0324
0.4802±0.0547
0.4618±0.0269
0.5039±0.0498
0.5426±0.0597
Histidine
0.0238±0.0030
0.0170±0.0010
0.0038±0.0008
0.0032±0.0003
0.0258±0.0059
0.0344±0.0099
Isoleucine
0.1664±0.0521
0.2670±0.0379
0.4024±0.0096
0.3459±0.0149
0.1989±0.0573
0.1778±0.0042
Threonine
0.2653±0.9010
0.4787±0.0729
0.5546±0.0935
0.5912±0.0046
0.4165±0.0670
0.4204±0.0135
Acetate
0.2291±0.0166
0.1179±0.0044
0.0895±0.0007
0.0733±0.0003
0.0595±0.0087
0.1527±0.0383
Chlorogenate
0.0080±0.0009
0.0167±0.0002
0.0171±0.0004
0.0156±0.0011
0.0084±0.0009
0.0125±0.0005
Fumarate
0.0606±0.0093
0.0712±0.0032
0.0570±0.0016
0.0455±0.0027
0.0160±0.0009
0.0214±0.0021
Amino acids
Organic acids
Lactate
0.1529±0.0307
0.1931±0.0076
1.9183±0.0773
0.5074±0.0010
0.2382±0.0823
0.2490±0.0549
Malate
3.7200±0.2538
5.8234±0.7499
6.6235±0.8637
7.0220±0.2456
4.9397±0.5423
5.4840±0.0679
Pyruvate
0.1023±0.0212
0.1848±0.0128
0.0847±0.0078
0.0939±0.0017
0.0550±0.0012
0.1035±0.0068
Quinate
0.1650±0.0250
0.3179±0.0121
0.3967±0.0174
0.2984±0.0149
0.2124±0.0316
0.2152±0.0064
Succinate
1.2646±0.2139
2.9780±0.0421
2.2667±0.0889
2.9984±0.0216
1.8441±0.3766
2.1159±0.1415
Catechine
0.0039±0.0008
0.0091±0.0001
0.0094±0.0001
0.0096±0.0001
0.0055±0.0009
0.0064±0.0001
Epicatechine
0.0016±0.0005
0.0033±0.0002
0.0042±0.0001
0.0052±0.0001
0.0021±0.0001
0.0027±0.0001
Histamine
0.0246±0.0017
0.0088±0.0001
0.0037±0.0007
0.0026±0.0001
0.0102±0.0002
0.0145±0.0035
Tyrosol
0.0096±0.0087
0.0083±0.0001
0.0418±0.0013
0.0227±0.0056
0.0075±0.0036
0.0086±0.0001
Xantine
0.0009±0.0001
0.0031±0.0001
0.0047±0.0003
0.0081±0.0001
0.0014±0.0005
0.0021±0.0001
Uracil
0.0013±0.0002
0.0017±0.0002
0.0035±0.0001
0.0102±0.0009
0.0012±0.0001
0.0011±0.0004
2.3-butanediol
0.5353±0.0359
0.2010±0.0161
0.8599±0.1034
1.3866±0.1389
0.4578±0.1660
0.5990±0.0535
Sterol
0.0053±0.0037
0.0080±0.0065
0.0036±0.0004
0.0062±0.0003
0.0097±0.0021
0.0087±0.0001
Glycerol
13.4829±1.9407 15.5139±0.2248 17.3432±0.6179 18.2692±0.1801
9.8221±0.8283
16.9777±0.7331
Others
516 27
517
a
The most abundant isoform of fructose (β-FP).
518
b
α and β isoforms.
519
c
EC1118: Saccharomyces cerevisiae EC1118; TD291: Torulaspora delbrueckii TD291; YR21,
520
Starmerella bacillaris YR21; HO16: Hanseniaspora osmophila HO16; Y4M: Hanseniaspora
521
uvarum Y4M, and SU3: Saccharomyces uvarum SU3.
522
28
523
Table 2. Relative amount of selective integral expresses as percentage values respect to the total
524
values of all integrals over the aromatic integrated spectral region. Discriminating metabolites from
525
PCA analysis are reported in bold.
526 RELATIVE AMOUNT FOR SINGLE STRAIN
a
AROMATIC COMPOUNDS Xanthine
YR21
HO16
SU3
EC1118
TD291
Y4M
0.2409±0.0467
0.5548±0.0224
0.6434±0.0633
1.4108±0.0227
0.3886±0.1521
0.4887±0.05162
Histamine
6.2599±1.0742
1.5868±0.0178
0.4971±0.1137
0.4477±0.0246
2.9327±0.1068
3.3332±0.6437
Histidine
6.0783±1.3902
3.0679±0.2034
0.5242±0.1361
0.5670±0.0540
7.4485±1.8463
7.8834±1.8786
Tyrosol
5.0113±2.7231
3.0025±0.0087
11.3319±0.2017
7.9470±1.0521
4.3458±1.1452
3.9709±0.4115
Fumarate
15.2684±0.8018 12.8764±0.5160
7.7147±0.0320
7.9460±0.5350
4.6124±0.1906
4.9439±0.7443
Chlorogenate
1.2949±0.1916
2.3828±0.1047
1.4477±0.0461
2.2112±0.1261
1.5386±0.2303
2.1293±0.0628
Epicatechin
0.3924±0.1036
0.5899±0.0418
0.5653±0.0239
0.9166±0.0099
0.5970±0.0199
0.6203±0.03763
Catechin
0.9883±0.0999
1.6430±0.0233
1.2745±0.0330
1.6805±0.0023
1.5861±0.2437
1.4849±0.0973
Uracil
0.3191±0.0258
0.3106±0.0354
0.4727±0.0067
1.7805±0.1824
0.3415±0.0237
0.2599±0.1099
527 528
a
529
Starmerella bacillaris YR21; HO16: Hanseniaspora osmophila HO16; Y4M: Hanseniaspora
530
uvarum Y4M, and SU3: Saccharomyces uvarum SU3.
EC1118: Saccharomyces cerevisiae EC1118; TD291: Torulaspora delbrueckii TD291; YR21,
531
29
532
Supplementary data
533 534
Figure S1. 1H NMR spectra of methanol/D2O extract of apple ciders samples analyzed. a)
535
Hanseniaspora Y4M, b) Torulaspora delbrueckii TD291, c) Saccharomyces cerevisiae EC1118, d)
536
Saccharomyces uvarum SU3, e) Starmerella bacillaris YR21, f) Hanseniaspora osmophila HO16.
537 538 539 540 541 542 543 544 545 546 547 548 549 550
30
551
Figure S2. Heatmap based on the relative amount of selective integrals expresses as percentage
552
values respect to the total values of all integrals over the entire spectral region. Color key indicates
553
the relative abundance of the identified metabolites; (red: highest, yellow: medium, green: lowest).
554
Rows represent the identified metabolites (in bold discriminating metabolites from PCA analysis)
555
and columns represent the yeast strains. EC1118: Saccharomyces cerevisiae EC1118; TD291:
556
Torulaspora delbrueckii TD291; YR21, Starmerella bacillaris YR21; HO16: Hanseniaspora
557
osmophila HO16; Y4M: Hanseniaspora uvarum Y4M, and SU3: Saccharomyces uvarum SU3.
558
559
arabinose fructose glucose sucrose xylose alanine aspartate asparagine histidine isoleucine threonine acetate chlorogenate fumarate lactate malate pyruvate quinate succinate catechine epicatechine histamine tyrosol xantine uracil 2,3-butanediol sterol glycerol
YR21 0.0627 0.6078 5.5342 0.0206 0.3594 0.0651 0.5103 0.4856 0.0238 0.1664 0.2653 0.2291 0.0080 0.0606 0.1529 3.7200 0.1023 0.1650 1.2646 0.0039 0.0016 0.0246 0.0096 0.0009 0.0013 0.5353 0.0053 13.4829
HO16 0.0338 1.8288 0.8078 0.6308 0.3862 0.1120 0.7106 0.4463 0.0170 0.2670 0.4787 0.1179 0.0167 0.0712 0.1931 5.8234 0.1848 0.3179 2.9780 0.0091 0.0033 0.0088 0.0083 0.0031 0.0017 0.2010 0.0080 15.5139
SU3 0.0268 0.7323 0.8687 0.0110 0.5428 0.1199 1.2487 0.4802 0.0038 0.4024 0.5546 0.0895 0.0171 0.0570 1.9183 6.6235 0.0847 0.3967 2.2667 0.0094 0.0042 0.0037 0.0418 0.0047 0.0035 0.8599 0.0036 17.3432
EC1118 0.0464 0.7293 0.8860 0.0092 0.5997 0.0629 1.0487 0.4618 0.0032 0.3459 0.5912 0.0733 0.0156 0.0455 0.5074 7.0220 0.0939 0.2984 2.9984 0.0096 0.0052 0.0026 0.0227 0.0081 0.0102 1.3866 0.0062 18.2692
TD291 0.0311 7.5851 0.9265 0.0073 0.3777 0.1117 0.5396 0.5039 0.0258 0.1989 0.4165 0.0595 0.0084 0.0160 0.2382 4.9397 0.0550 0.2124 1.8441 0.0055 0.0021 0.0102 0.0075 0.0014 0.0012 0.4578 0.0097 9.8221
Y4M 0.0281 3.7807 0.8400 0.0134 0.3513 0.1348 0.6392 0.5426 0.0344 0.1778 0.4204 0.1527 0.0125 0.0214 0.2490 5.4840 0.1035 0.2152 2.1159 0.0064 0.0027 0.0145 0.0086 0.0021 0.0011 0.5990 0.0087 16.9777 31
560
Figure S3. Heatmap based on the relative amount of selective integrals expresses as percentage
561
values respect to the total values of all integrals over the aromatic spectral region. Color key
562
indicates the relative abundance of the identified metabolites; (red: highest, yellow: medium, green:
563
lowest). Rows represent the identified metabolites (in bold discriminating metabolites from PCA
564
analysis) and columns represent the yeast strains. EC1118: Saccharomyces cerevisiae EC1118;
565
TD291: Torulaspora delbrueckii TD291; YR21, Starmerella bacillaris YR21; HO16:
566
Hanseniaspora osmophila HO16; Y4M: Hanseniaspora uvarum Y4M, and SU3: Saccharomyces
567
uvarum SU3.
568
569
xanthine histamine histidine tyrosol fumarate chlorogenate epicatechin catechin uracil
YR22 0.2409 6.2599 6.0783 5.0113 15.2684 1.2949 0.3924 0.9883 0.3191
HO16 0.5548 1.5868 3.0679 3.0025 12.8764 2.3828 0.5899 1.6430 0.3106
SU4 0.6434 0.4971 0.5242 11.3319 7.7147 1.4477 0.5653 1.2745 0.4727
EC1119 1.4108 0.4477 0.5670 7.9470 7.9460 2.2112 0.9166 1.6805 1.7805
TD292 0.3886 2.9327 7.4485 4.3458 4.6124 1.5386 0.5970 1.5861 0.3415
Y4M 0.4887 3.3332 7.8834 3.9709 4.9439 2.1293 0.6203 1.4849 0.2599
570
571
572
573
574
575
576 32
577
Figure S4. Loading plot obtained of PCA performed on NMR data considering all spectral regions.
578
Buckets values are reported.
579 580
581 582
33
583
Figure S5 Loading plot obtained of PCA performed on NMR data considering aromatic spectral
584
regions. Buckets values are reported.
585
586 587
34
Highlights •
Apple juice fermented by 6 yeast species
•
NMR characterization of metabolite profiles of fermented apple juice
•
Specie-specific biomarkers related to apple juice fermentation
Consiglio Nazionale delle Ricerche
Conflict of Interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. On behalf of all the co-authors,
R. Consonni
Milan 17th June 2019 Partita IVA 02118311006 - C.F. 80054330586 Sede Legale: Via E. Bassini 15, 20133 Milano. Ingresso presso: Via A. Corti 12, 20133, Milano Altre Sedi: 16149 Genova - Via de Marini, 6 - Tel. 010.64751 – 13900 Biella - C.so G. Pella, 16 - Tel. 015.8493043 Web: www.ismac.cnr.it
S0023-6438(19)31113-2
DOI:
https://doi.org/10.1016/j.lwt.2019.108771
Reference:
YFSTL 108771
To appear in:
LWT - Food Science and Technology
Received Date: 19 June 2019 Revised Date:
11 October 2019
Accepted Date: 20 October 2019
Please cite this article as: Cusano, E., Cagliani, L.R., Consonni, R., Simonato, B., Zapparoli, G., NMRbased metabolic profiling of different yeast fermented apple juices, LWT - Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.108771. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
NMR-based metabolic profiling of different yeast fermented apple juices
1 2 3 4
Erica Cusanoa, Laura Ruth Cagliania, Roberto Consonnia*, Barbara Simonatob, Giacomo Zapparolib
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a
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12, 20133 Milan, Italy
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b
Department of Biotechnology, Università degli Studi di Verona, 37134 Verona, Italy
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*
Corresponding author
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Email address: [email protected] (R. Consonni)
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Tel.: +39-02-23699578
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Fax: +39-02-23699620
Institute of chemical sciences and technologies “G. Natta” (SCITEC), Lab. NMR, CNR, v. Corti
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1
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ABSTRACT
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NMR based metabolic profiling was used to investigate metabolic changes in ciders fermented by
17
yeast species as valuable tool for the identification of metabolites responsible for yeast activities. 1H
18
NMR spectra of apple ciders produced by Saccharomyces cerevisiae EC1118, Torulaspora
19
delbrueckii TD291, Starmerella bacillaris YR21, Hanseniaspora osmophila HO16, Hanseniaspora
20
uvarum Y4M, and Saccharomyces uvarum SU3, revealed to be dominated by glycerol and organic
21
acids signals. The aromatic region showed the presence of low intense signals referred to
22
epicatechin, catechin, chlorogenate, xanthine, uracil, tyrosol, fumarate, histamine, and histidine.
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The anomeric region showed signals related to xylose, arabinose, glucose, fructose and sucrose.
24
Finally, in the aliphatic region, signals due to amino acids like alanine, aspartate, asparagine,
25
isoleucine and threonine, organic acids like acetate, lactate, malate, pyruvate, quinate, succinate,
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and alcohols like 2,3 butanediol, ethanol, and glycerol and sterol were detected. Principal
27
component analysis performed considering NMR data from either all spectral regions and only
28
aromatic region revealed the potentiality to discriminate the yeasts action. Signals due to glucose,
29
fructose, glycerol, malate, fumarate, tyrosol, histidine, and histamine resulted discriminant for
30
sample differentiation. This study, the first one by NMR, provides preliminary insights on the
31
metabolic differences among yeast species involved in cider production.
32 33
Keywords: apple cider, NMR, yeast, fermentation, metabolite profiling
34
2
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1. Introduction
36
Cider is a slightly alcoholic beverage produced from the fermentation of apple juice that is often
37
performed by selected Saccharomyces cerevisiae strains following an oenological protocol. This
38
procedure, generally utilized by the industry, assures a controlled and standardized cidermaking
39
process (Cusano, Simonato, & Consonni, 2018). Recently, a positive impact on the aromatic
40
compounds of cider has assessed by the fermentation with different Saccharomyces and non-
41
Saccharomyces strains, both in co-inoculation with S. cerevisiae (Xu, Zhao, & Wang, 2006; Aung,
42
Lee, Yu, & Liu, 2015; Liu, Aung, Lee, & Yu, 2016; Ye, Yue, & Yuan, 2014) and as single cultures
43
(Lorenzini, Simonato, Slaghenaufi, Ugliano, & Zapparoli, 2019; Wei, Wang, Zhang, Yuan, & Yue,
44
2019; Gutierrez, Boekhout, Gojkovic, & Katz, 2018). The increasing interest of this practice is
45
related to their unusual properties to produce specific metabolites and/or reduce the ethanol yield,
46
together with the production of aromatic compounds (Dashko et al., 2015). Only few data are
47
available on the impact of different yeast strains on the cider non-volatile compounds (Laaksonen,
48
Kuldjärv, Paalme, Virkki, & Yang, 2017).
49
A performing analytical technique to analyze the soluble metabolites characterizing the cider is the
50
Nuclear Magnetic Resonance (NMR) spectroscopy (Del Campo, Berregi, Iturriza & Santos, 2006;
51
Monakhova, Kuballa & Lachenmeier, 2012; Zuriarrain, Zuriarrain, Puertas, Dueñas, & Berregi,
52
2015; Cusano et al., 2018). As a matter of fact, NMR is a valuable tool for the identification of
53
metabolites such as valine, pyruvate, succinate, proline responsible for differentiation of yeast
54
strains involved on the alcoholic fermentation (Son et al., 2009). These molecules being products of
55
cellular metabolism can have a key role for monitoring the fermentation process and relating yeasts
56
to final product quality. Indeed, due to the advantages of this technique, such as the simplicity of
57
sample preparation, measurement rapidity and the possibility to investigate a wide spectrum range
58
of chemical compounds present in different amount with a single experiment, it is easily possible to
59
assess important details of metabolites profiles related to a certain species or strains.
3
60
Despite the high potentiality of this technique to investigate the metabolomics of yeasts involved on
61
the production of the fermented beverages, 1H NMR investigations on this issue are still scarce.
62
The aim of this study was the investigation of the metabolic content of ciders obtained by apple
63
juice fermentation carried out by six different Saccharomyces and non-Saccharomyces yeast strains.
64
The NMR approach allowed to determine the effect of different yeast strains on the metabolite
65
content of cider evolution.
66 67
2. Materials and Methods
68
2.1. Yeast strains and fermentation process
69
In this study six different yeast strains were tested. Saccharomyces cerevisiae EC1118 (Lallemand,
70
Montreal, Canada) and Torulaspora delbrueckii TD291 (BIODIVATM, Lallemand) were
71
commercial. Starmerella bacillaris YR21, Hanseniaspora osmophila HO16, Hanseniaspora
72
uvarum Y4M, and Saccharomyces uvarum SU3 were strains isolated from apple juice and wine and
73
belong to the collection of Dipartimento di Biotecnologie of University of Verona, Italy. Yeasts
74
were maintained on YPD agar (1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose and 1.5%
75
w/v agar) prior to use and also kept at −80°C with 25% w/v glycerol.
76
Golden Delicious apples were squeezed to obtain the juice characterized by 9.9 °Brix as total
77
soluble solid, 3.4 g/L total acidity expressed as malic acid equivalents and pH 3.43. The apple juice
78
was pasteurized for 5 min at 70°C before fermentation. Each yeast strain was incubated in 10 mL
79
YPD medium at 28°C on a rotary shaker, and after two days cells were separated by centrifugation
80
(5000 g, 4°C, 5 min) and washed with sterile 0.9% w/v NaCl. Each strain was suspended in 10 mL
81
of apple juice and finally added to 600 mL of pasteurized apple juice to a final concentration of 106
82
CFU/mL. The fermentations were performed at 20°C in duplicate and supervised following the
83
amount of produced CO2 by measuring the weight loss until reaching a stable weight. At the end of
84
fermentation the produced ciders were stored at 4°C for 72 h to favour the sedimentation of
85
suspended materials and then racked. 4
86 87
2.1 Sample preparation for NMR analysis
88
A total of 12 ciders obtained by the apple juice fermentation carried out by each of six yeast strains
89
investigated in a double biological replicates (two ciders for each yeast). About 100 mg of
90
lyophilized sample was dissolved in 600 µL of deuterated methanol (CH3OH-d4 Eurisotop, 99.80
91
atom % D, France) and 120 µL of deuterated water (H2O Sigma-Aldrich, 99.96 atom % D, Italy);
92
the solutions were stirred for 3 min at room temperature and used for NMR analysis.
93 94
2.2 NMR data acquisition
95
Spectra were recorded with a Bruker DRX 600 spectrometer (Bruker Biospin GmbH Rheinstetten,
96
Karlsruhe, Germany) operating at 14.1 T, equipped with a 5-mm inverse probe with a z-gradient, at
97
room temperature (300 K). Spectra were acquired with a spectral with of 7184 Hz, over 32 K data
98
points. The residual water suppression was achieved by applying a presaturation scheme with a
99
low-power radiofrequency irradiation for 1.2 s.
100 101
2.2.1 NMR data processing and analysis
102
A resolution enhancement function with an exponential multiplication of 0.3 Hz for the line
103
broadening was applied before Fourier transformation. All spectra were carefully phased and
104
baseline-adjusted with TOPSPIN 3.0 software (Bruker Biospin GmbH Rheinstetten, Karlsruhe,
105
Germany) and referenced to the methanol signal at 3.31 ppm and 49.9 ppm for 1H and
106
respectively. Successively spectra were imported to ACD Spectrus processor (ACD Labs 2012,
107
Toronto, Canada). Intelligent bucketing was applied to all spectral region (the range of 0.00 and
108
9.75 ppm) in order to compensate possible small signals shift due to different pH. The spectral
109
region including the solvent signal (3.29 and 3.33 ppm) was set to zero constant value before the
110
bucket normalization performed with respect to the total integral value. Alternatively, only the
111
aromatic region was considered (the range of 5.52 and 9.75 ppm) and a new bucket normalization
13
C
5
112
was performed. These normalizations allowed to evaluate the relative abundance of the identified
113
metabolites. Table of buckets was imported into SIMCA-P 13 (Umetrics, Umea, Sweden) and
114
Principal Component Analysis (PCA) was performed, with mean centering as data pretreatment.
115 116
3. Results and Discussion
117
1
118
shown in Fig. S1 and Fig. 1A-C. The metabolites assignment was achieved by recording
119
bidimensional homo and heteronuclear NMR experiments and literature data (Cusano et al., 2018).
120
The relative abundance of all the identified metabolites is reported in Table 1 and figure S2 for all
121
the investigated strains; their values are expressed as percentage value respect to the integral of all
122
resonances in the complete spectral region. In this way they reflect their relative abundance over the
123
entire metabolite content. The 1H NMR spectra (Fig. S1) were dominated by glycerol organic acids
124
signals and, in some samples, by the saccharides content. The amount of glycerol and organic acids,
125
produced during the alcoholic fermentation, can significantly be affected by the yeasts (Vileda et
126
al., 2019). As a matter of fact, glycerol, pyruvate, malate, and succinate were differentially
127
produced by S. cerevisiae strains in wine, previously analysed by 1H NMR (Son et al., 2009).
128
Moreover, glycerol, succinate, and α-glucose signals were found largely different between Fiano
129
wines produced by autochthonous and commercial S. cerevisiae strains (Mazzei, Spaccini,
130
Francesca, Moschetti, & Piccolo, 2013).
131
On the basis of the quantification values in Table 1, fructose (min. 0.61±0.02, max 7.59±1.20) and
132
glucose (min. 0.81±0,05, max. 5.53±0.81) are largely present among saccharides, aspartate (min.
133
0.51±0.03, max. 1.25±0.04), asparagine (min. 0.45±0.03, max. 0.54±0.06) and threonine (min
134
0.27±0.90 max 0.591±0.005) among amino acids, malate (min. 3.72±0.25, max. 7.02±0.25) and
135
succinate (min. 1.26±0.21, max. 3.00±0.02) among organic acids, while glycerol (min. 9.82±0.83,
H NMR spectra of apple ciders fermented with Saccharomyces and non-Saccharomyces yeasts are
6
136
max 18.27±0.18) and 2,3-butanediol (min. 0.20±0.02, max. 1.39±0.14) resulted the largely
137
abundant among other compounds.
138
By considering the aliphatic region (Fig. 1A) of the 1H NMR spectrum, signals due to amino acids
139
like alanine, asparagine, aspartate, isoleucine, and threonine, organic acids like acetate, lactate,
140
malate, succinate, pyruvate, and quinate, alcohols like ethanol and 2,3-butanediol, and sterol were
141
detected. Since molecules such as acetate, lactate, malate, and 2,3-butanediol have an important
142
impact on aroma of fermented beverages and their production can be related to yeast metabolism
143
(Romano, Brandolini, Ansaloni, & Menziani, 1998; Saayman & Viljoen-Bloom, 2006), their
144
identification may provide useful information on possible role of the fermenting yeast on
145
characterizing the volatile profile of cider. As regards to acetate signal, its presence in large amount
146
in ciders obtained by Hanseniaspora uvarum Y4M (0.15±0.04) and Hanseniaspora osmophila
147
HO16 (0.118±0.004) was expected since these species are generally high acetate producers
148
(Romano, Suzzi, Comi, & Zironi, 1992). Conversely, less obvious was the high acetate content in
149
Starmerella bacillaris YR21 sample (0.23±0.02), since this species generally produces low amount
150
of acetate during the alcoholic fermentation (Magyar & Tóth, 2011; Rantsiou et al., 2017). The high
151
content of malate (min. 3.72±0.25, max. 7.02±0.25) in ciders was expected being this organic acid
152
abundantly present in apple juice. Moreover data showed that this acid was most abundant in cider
153
fermented by Saccharomyces cerevisiae EC1118 (7.02±0.25); this could be due to an inefficiently
154
use of malate by this yeast as reported in a previous study (Casal, Paiva, Queirόs, & Soares-Silva,
155
2008). Furthermore lactate was largely present in cider fermented by Saccharomyces uvarum SU3
156
strain (1.92±0.08), thus suggesting higher production of this acid through glyoxase pathway
157
(Martins, Cordeiro, & Ponces Freire, 2001) for this strain compared with the other yeasts. The
158
detection of amino acids can evidence interesting metabolic differences among the yeasts that may
159
impact on fermentation kinetics of apple juices as well as on sensory properties of cider since amino
160
acids are precursors of higher alcohols (Mangas, Cabranes, Moreno, & Gomis, 1994). Different 7
161
signals of amino acids, particularly aspartate, alanine and threonine, among cider profiles, are
162
congruent with different consumption patterns for amino acids by yeasts fermenting apple juice
163
(Suárez Valles, Palacios García, Rodríguez Madrera, & Picinelli Lobo, 2005; Eleutério Dos Santos
164
et al. 2015). The variability of amino acid profiles due to yeasts affects the formation of fusel
165
alcohols and esters in cider (Eleutério Dos Santos et al. 2015). The anomeric region of 1H NMR
166
spectra (Fig. 1B) showed signals related to xylose and arabinose in all samples; sucrose was largely
167
present in ciders fermented by Hanseniaspora osmophila HO16 (0.63±0.02), while in fermented
168
ciders by Torulaspora delbrueckii TD291 only in very low amount (0.0073±0.0001). In our
169
previous investigation (Cusano et al., 2018) the NMR signals of xylose and arabinose were
170
observed in cider from apple juice fermented by the same strain of Saccharomyces cerevisiae
171
EC1118 used in the present study and were highly correlated in natural or sparkling ciders (Gomis,
172
Tamayo, Valles, & Mangas Alonso, 2004). The large presence of sucrose in cider obtained by
173
Hanseniaspora osmophila HO16 (0.63±0.02) is probably due to its inability to ferment and
174
assimilate this disaccharide. Interestingly, López (López, Mateo, & Maicas, 2016) reported strain-
175
specific differences on the sucrose assimilation among different species Hanseniaspora spp.,
176
including Hanseniaspora osmophila HO16 and Hanseniaspora uvarum Y4M. Glucose, present in
177
both α and β isoforms in all samples, appeared dominant only in Starmerella bacillaris YR21
178
(5.53±0.81), while in lower amount in ciders fermented with all the other yeasts (min 0.81±0.05,
179
max 0.93±0.09). Fructose was largely present only in Torulaspora delbrueckii TD291 (7.59±1.20)
180
and Hanseniaspora uvarum Y4M (3.78±0.26) while lower amount was present in ciders fermented
181
with Hanseniaspora osmophila HO16 (1.83±0.15), and only in a few amount in Starmerella
182
bacillaris YR21 (0.61±0.02), Saccharomyces uvarum SU3 (0.73±0.03), and Saccharomyces
183
cerevisiae EC1118 (0.73±0.04). These results suggest differences in consumption rate on glucose
184
and fructose among these yeasts at the fermentation conditions used in the present study. In fact,
185
preferences of yeast for one or the other saccharidic moiety can determine an unbalance ratio of 8
186
these two sugars at the end of fermentation. Specifically, the large presence of glucose and the low
187
presence of fructose in cider fermented by Starmerella bacillaris YR21 (5.53±0.81 and 0.61±0.02,
188
respectively) is congruent with its fructophilic behavior (Magyar & Toth, 2011). Moreover, signals
189
of these two saccharides in cider produced by Hanseniaspora osmophila HO16 (0.81±0.05 and
190
1.83±0.15, respectively) and Hanseniaspora uvarum Y4M (0.84±0.14 and 3.78±0.26, respectively)
191
were consistent to their glucophilic character in agreement with previous determination (Granchi,
192
Granuci, Messini, & Vincenzini, 2002) for the former yeast, but in contrast with other results (Ciani
193
& Fatichenti, 1999) for the latter yeast. This last incongruence could be related to phenotypic
194
diversity among strains of the same species and deserve further investigation.
195
Conversely, the large presence of fructose on cider produced by Torulaspora delbrueckii TD291
196
(7.59±1.20) can be attributable to preference of this yeast on glucose. However, it is interesting how
197
different is the amount of this saccharide with respect to Saccharomyces cerevisiae EC1118
198
(0.73±0.04) which are glucophilic too. The patterns of saccharide utilization by these two yeasts
199
can be similar or different according to the fermentation conditions (Santos et al., 2008).
200
Specifically, in anaerobic conditions, fructose consumption rate of Torulaspora delbrueckii TD291
201
is much lower than Saccharomyces cerevisiae (Santos et al., 2008), and this rate, together with the
202
production of other metabolites, can change according to the oxygen availability during the
203
fermentation process (Ramírez & Velázquez, 2018). Further investigations are required to fully
204
assess whether sucrose and fructose can potentially be key metabolites for Hanseniaspora
205
osmophila HO16 and Torulaspora delbrueckii TD291, respectively, involved in cider production.
206
The aromatic region of 1H NMR spectra (Fig. 1C) showed only low intense signals. Specifically,
207
they referred to epicatechin, catechin, and chlorogenate commonly present in all samples (Tables 1,
208
Fig. S2), xanthine is present in low amount in all fermented ciders (lower than 0.0081±0.0001), as
209
well as uracil (lower than 0.0102±0.0009), and tyrosol (lower than 0.042±0.001) observed mainly in
210
ciders fermented by Saccharomyces cerevisiae EC1118 and Saccharomyces uvarum SU3, while in 9
211
lower amount in all other fermented ciders. Table 2 and figure S3 summarizes the relative amount
212
of the identified aromatic compounds, normalized to the total aromatic content, for a clearer
213
evaluation. The different amount of xanthine observed among all the fermented ciders (min
214
0.24±0.05, max 1.41±0.02) could suggest metabolic differences among yeast species in the purine
215
degradation. Similarly, the different content of uracil (min 0.26±0.11, max 1.78±0.18) could be
216
explained by different capacity to uracil release into the growth medium by the yeasts. Obviously,
217
the explanation of our results through possible different regulation of these pathways among yeasts
218
is merely speculative since elucidations on pathways involved in the metabolism of xanthine and
219
uracil have been carried out only on Saccharomyces cerevisiae (Séron, Blondel, Haguenauer-Tsapis
220
& Volland 1999; Rolfes, 2006). On the other hand, the presence of tyrosol, an aromatic alcohol that
221
can positively contribute to sensory properties of fermented beverages, observed among yeasts is
222
consistent with data reported by González (González et al., 2018). The Ehrlich pathway involved in
223
the production of this alcohol, together to tryptophan and 2-phenylethanol, in Torulaspora
224
delbrueckii TD291 and Starmerella bacillaris YR21 seems to be differentially regulated with
225
respect to that in Saccharomyces cerevisiae. In addition, the aromatic region of the 1H NMR
226
spectrum, showed that ciders obtained by Saccharomyces cerevisiae EC1118 and Saccharomyces
227
uvarum SU3 contained low levels of histamine (0.45±0.02 and 0.50±0.11,
228
histidine (0.57±0.05 and 0.52±0.14, respectively) with respect to all the other samples (min
229
1.59±0.02, max 6.26±1.07 for histamine, and min 3.07±0.20, max 7.88±1.88 for histidine).
230
Different levels of biogenic amines including histamine in wine or in other foodstuff produced by
231
various yeasts species or strains have been attributed to their diverse amino acid decarboxylase
232
activities involved on their production (Tristezza et al., 2013). Moreover, the variability on release
233
of biogenic amines among yeasts depends to medium composition and fermentation condition
234
(Restuccia, Loizzo, & Spizzirri, 2018). At the experimental condition used in the present study the
235
very low content of histamine and its precursor histidine only in cider produced by Saccharomyces
respectively) and
10
236
yeasts suggests that the histidine degradation could be carried out by aromatic amino acid
237
aminotransferases involved in a pathway alternative with respect to that produces histamine. Since
238
differences on the regulation of this pathway have been observed between Saccharomyces
239
cerevisiae and Candida glabrata (Brunke et al., 2014), we could not exclude that the higher
240
presence of histamine in cider obtained by non-Saccharomyces yeasts is due to metabolic
241
differences among yeasts to use histidine as a potential nitrogen source. Finally, fumarate resulted
242
present in all cider samples and with a high amount in Hanseniaspora osmophila HO16 and
243
Starmerella bacillaris YR21 (12.88±0.52 and 15.27±0.80, respectively). Cabranes (Cabranes,
244
Mangas, & Blanco, 1997) reported great intra-specific variations on the amount of this acid
245
produced during alcoholic fermentation of apple juices by Saccharomyces cerevisiae and
246
Hanseniaspora uvarum (synonym Kloeckera apiculata) strains. Further analyses, using various
247
strains of each yeast species, should be necessary to ascertain possible inter-specific differences on
248
the production of fumarate. This organic acid could inhibit the malolactic fermentation that can
249
naturally occur or be induced in cider (Herrero, Gracía, & Díaz, 2003). Nevertheless, the amount of
250
fumarate in ciders at the end of alcoholic fermentation is generally far below the level that is
251
considered inhibitory against malolactic bacteria (Herrero, Cuesta, García, & Díaz, 1999; Herrero et
252
al., 2003). To highlight possible differences in the metabolite content of all the investigated
253
samples, PCA analysis was initially performed considering NMR data from all spectral width
254
adopting “mean centering” as data pretreatment. The model yielded two components that explained
255
92.4% of the total variance. The score plot reported in figure 2A showed the first PC negatively
256
affecting Torulaspora delbrueckii TD291 and Starmerella bacillaris YR21 fermented samples,
257
while conversely it positively affected the other clustered ciders. In particular these latter samples
258
were belonging to the Saccharomyces and Hanseniaspora genus, thus showing similar metabolite
259
content. As expected due to the scaling adopted, in the corresponding loading plot (Fig. 2B, S4) the
260
more intense signals due to saccharides, glycerol and organic acids, resulted discriminant for
261
samples differentiation. As a matter of fact α and β glucose (buckets at 5.11 ppm, and 3.15, 3.32, 11
262
3.38, 4.49 ppm respectively) were the characterizing metabolites for Starmerella bacillaris YR21
263
samples, fructose (buckets at 3.47, 3.62, 3.74, 3.76, 3.84, and 3.99 ppm) for Torulaspora
264
delbrueckii TD291 samples, while fermented ciders obtained by using yeast belonging to
265
Saccharomyces and Hanseniaspora genus showed a higher content in glycerol (buckets at 3.50 and
266
3.58 ppm), malate (buckets at 2.58, 2.76, and 4.33 ppm), and succinate (bucket at 2.55 ppm). These
267
data confirmed the glucophilic and fructophilic beahaviour of Torulaspora delbrueckii TD291 and
268
Starmerella bacillaris YR21 respectively, respect to Hanseniaspora and Saccharomyces genus.
269
Successively, in order to investigate the possible samples discrimination according to the non-
270
volatile aroma compounds induced by the action of different yeasts, a new PCA considering only
271
the aromatic NMR spectral regions was performed. This model resulted in five components
272
explaining the 98.2% of the total variance. The score plot reported in figure 3A showed that
273
according to the first PC, a clear-cut separation could be achieved among Saccharomyces cerevisiae
274
EC1118 and Saccharomyces uvarum SU3, and all the other samples grouped in the opposite space
275
of the plot, resulting the former essentially characterized by tyrosol content (buckets at 6.71 and
276
7.01 ppm, Fig. 3B, S5). The latter group of samples could be better characterized by the use of the
277
second PC. As a matter of fact, this PC highlighted histidine (buckets at 6.98 and 7.70 ppm) content
278
negatively affecting Hanseniaspora uvarum Y4M and Torulaspora delbrueckii TD291 samples
279
while histamine (buckets at 7.10 and 7.77 ppm) and fumarate (bucket at 6.64 ppm) content
280
positively affected Starmerella bacillaris YR21 and Hanseniaspora osmophila HO16 (Fig. 3B, S5).
281
The statistical evaluation of aromatic region of 1H NMR spectra of yeast fermented apple juices,
282
highlighted a higher content of biogenic amines for all yeast with the exception of Saccharomyces
283
genus. Furthermore, Starmerella bacillaris YR21 and Hanseniaspora osmophila H016, showing a
284
higher histamine content, suggesting a
285
Finally, the higher content of tyrosol observed for Saccharomyces cerevisiae EC1118 and
286
Saccharomyces uvarum SU3 suggested a more performing Ehrlich metabolic pathway.
larger aromatic amino acid aminotransferase activity.
287 12
288
4. Conclusions
289
The NMR analysis of ciders evidenced the metabolic differences among yeast species involved in
290
apple juice fermentation. The discrimination of ciders according to NMR spectra was linked to
291
metabolic profiles of yeasts that were characterized by signals of different molecules mostly
292
belonging to the primary metabolism. In particular, metabolisms of saccharides, purines and
293
pyrimidines, and amino acids seem to be important in characterizing yeasts. However, the lack of
294
knowledges for most of species used in this study on molecular mechanisms involved on these
295
pathways did not allow a deeper understanding of NMR fingerprinting generation for each yeast at
296
the fermentation conditions here reported. Although the data here presented constitute a preliminary
297
report in the view of finding potential species-specific key compounds (biomarkers) related to the
298
apple juice fermentation, insights gained by the present work, as the first on NMR-based metabolic
299
profiling of yeasts fermenting apple juice, are useful as starting-points for further investigations.
300 301
Acknowledgments
302
We thank Lorenzini Marilinda for technical assistance.
303
13
304
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Figure captions
429
Fig. 1. Aliphatic (A), anomeric (B), and aromatic (C) expansions of 1H NMR spectra of
430
methanol/D2O extract of apple ciders samples analyzed showing principal metabolite assignments.
431
a) Hanseniaspora uvarum Y4M, b) Torulaspora delbrueckii TD291, c) Saccharomyces cerevisiae
432
EC1118, d) Saccharomyces uvarum SU3, e) Starmerella bacillaris YR21, f) Hanseniaspora
433
osmophila HO16.
434
Fig. 2. Score (A) and loading (B) plot obtained by performing PCA on NMR data considering all
435
spectral regions. 2PC, R2X= 92.4% and Q2=86.4%. EC1118: Saccharomyces cerevisiae EC1118,
436
TD291: Torulaspora delbrueckii TD291, YR21: Starmerella bacillaris YR21, HO16:
437
Hanseniaspora osmophila HO16, Y4M: Hanseniaspora uvarum Y4M, and SU3: Saccharomyces
438
uvarum SU3,
439
Fig. 3. Score (A) and loading (B) plot obtained by performing PCA on NMR data considering only
440
the aromatic spectral regions. 5PC, R2X= 98.2% and Q2=81.2%. EC1118: Saccharomyces
441
cerevisiae EC1118, TD291: Torulaspora delbrueckii TD291, YR21: Starmerella bacillaris YR21,
442
HO16: Hanseniaspora osmophila HO16, Y4M: Hanseniaspora uvarum Y4M, and SU3:
443
Saccharomyces uvarum SU3,
444
19
445
Fig. 1A
446 447
448
449
450
451
452
453
20
454
Fig. 1B
455
456
457
458
459
460
461
462
463
21
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Fig. 1C
465
466
467
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469
470
471
472
473
474
475
22
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Fig. 2A
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479
480
481
482
483
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Fig. 2B
487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502
24
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Fig. 3A
504 505 506
25
507
Fig. 3B
508
509 510
26
511
Tables.
512
Table 1. Relative amount of selective integrals of the identified compounds expresses as
513
percentage values respect to the total values of all integrals over the entire integrated spectral
514
region. Standard deviation is included for all values. Discriminating metabolites from PCA analysis
515
are reported in bold.
RELATIVE AMOUNT FOR SINGLE STRAIN COMPOUNDS
c
YR21
HO16
SU3
EC1118
TD291
Y4M
0.0627±0.0237
0.0338±0.0004
0.0268±0.0035
0.0464±0.0035
0.0311±0.0031
0.0281±0.0007
0.6078±0.0249
1.8288±0.1457
0.7323±0.0273
0.7293±0.0431
7.5851±1.1968
3.7807±0.2577
5.5342±0.811
0.8078±0.0496
0.8687±0.0848
0.8860±0.0176
0.9265±0.091
0.8400±0.1442
Saccharides Arabinose Fructose Glucose
a
b
Sucrose
0.0206±0.0010
0.6308±0.0198
0.0110±0.0023
0.0092±0.0008
0.0073±0.0001
0.0134±0.0003
b
Xylose
0.3594±0.0432
0.3862±0.0137
0.5428±0.0366
0.5997±0.0096
0.3777±0.0381
0.3513±0.0192
Alanine
0.0651±0.0041
0.1120±0.0015
0.1199±0.0064
0.0629±0.0152
0.1117±0.0214
0.1348±0.0439
Aspartate
0.5103±0.0275
0.7106±0.0378
1.2487±0.0447
1.0487±0.0351
0.5396±0.0361
0.6392±0.0128
Asparagine
0.4856±0.0203
0.4463±0.0324
0.4802±0.0547
0.4618±0.0269
0.5039±0.0498
0.5426±0.0597
Histidine
0.0238±0.0030
0.0170±0.0010
0.0038±0.0008
0.0032±0.0003
0.0258±0.0059
0.0344±0.0099
Isoleucine
0.1664±0.0521
0.2670±0.0379
0.4024±0.0096
0.3459±0.0149
0.1989±0.0573
0.1778±0.0042
Threonine
0.2653±0.9010
0.4787±0.0729
0.5546±0.0935
0.5912±0.0046
0.4165±0.0670
0.4204±0.0135
Acetate
0.2291±0.0166
0.1179±0.0044
0.0895±0.0007
0.0733±0.0003
0.0595±0.0087
0.1527±0.0383
Chlorogenate
0.0080±0.0009
0.0167±0.0002
0.0171±0.0004
0.0156±0.0011
0.0084±0.0009
0.0125±0.0005
Fumarate
0.0606±0.0093
0.0712±0.0032
0.0570±0.0016
0.0455±0.0027
0.0160±0.0009
0.0214±0.0021
Amino acids
Organic acids
Lactate
0.1529±0.0307
0.1931±0.0076
1.9183±0.0773
0.5074±0.0010
0.2382±0.0823
0.2490±0.0549
Malate
3.7200±0.2538
5.8234±0.7499
6.6235±0.8637
7.0220±0.2456
4.9397±0.5423
5.4840±0.0679
Pyruvate
0.1023±0.0212
0.1848±0.0128
0.0847±0.0078
0.0939±0.0017
0.0550±0.0012
0.1035±0.0068
Quinate
0.1650±0.0250
0.3179±0.0121
0.3967±0.0174
0.2984±0.0149
0.2124±0.0316
0.2152±0.0064
Succinate
1.2646±0.2139
2.9780±0.0421
2.2667±0.0889
2.9984±0.0216
1.8441±0.3766
2.1159±0.1415
Catechine
0.0039±0.0008
0.0091±0.0001
0.0094±0.0001
0.0096±0.0001
0.0055±0.0009
0.0064±0.0001
Epicatechine
0.0016±0.0005
0.0033±0.0002
0.0042±0.0001
0.0052±0.0001
0.0021±0.0001
0.0027±0.0001
Histamine
0.0246±0.0017
0.0088±0.0001
0.0037±0.0007
0.0026±0.0001
0.0102±0.0002
0.0145±0.0035
Tyrosol
0.0096±0.0087
0.0083±0.0001
0.0418±0.0013
0.0227±0.0056
0.0075±0.0036
0.0086±0.0001
Xantine
0.0009±0.0001
0.0031±0.0001
0.0047±0.0003
0.0081±0.0001
0.0014±0.0005
0.0021±0.0001
Uracil
0.0013±0.0002
0.0017±0.0002
0.0035±0.0001
0.0102±0.0009
0.0012±0.0001
0.0011±0.0004
2.3-butanediol
0.5353±0.0359
0.2010±0.0161
0.8599±0.1034
1.3866±0.1389
0.4578±0.1660
0.5990±0.0535
Sterol
0.0053±0.0037
0.0080±0.0065
0.0036±0.0004
0.0062±0.0003
0.0097±0.0021
0.0087±0.0001
Glycerol
13.4829±1.9407 15.5139±0.2248 17.3432±0.6179 18.2692±0.1801
9.8221±0.8283
16.9777±0.7331
Others
516 27
517
a
The most abundant isoform of fructose (β-FP).
518
b
α and β isoforms.
519
c
EC1118: Saccharomyces cerevisiae EC1118; TD291: Torulaspora delbrueckii TD291; YR21,
520
Starmerella bacillaris YR21; HO16: Hanseniaspora osmophila HO16; Y4M: Hanseniaspora
521
uvarum Y4M, and SU3: Saccharomyces uvarum SU3.
522
28
523
Table 2. Relative amount of selective integral expresses as percentage values respect to the total
524
values of all integrals over the aromatic integrated spectral region. Discriminating metabolites from
525
PCA analysis are reported in bold.
526 RELATIVE AMOUNT FOR SINGLE STRAIN
a
AROMATIC COMPOUNDS Xanthine
YR21
HO16
SU3
EC1118
TD291
Y4M
0.2409±0.0467
0.5548±0.0224
0.6434±0.0633
1.4108±0.0227
0.3886±0.1521
0.4887±0.05162
Histamine
6.2599±1.0742
1.5868±0.0178
0.4971±0.1137
0.4477±0.0246
2.9327±0.1068
3.3332±0.6437
Histidine
6.0783±1.3902
3.0679±0.2034
0.5242±0.1361
0.5670±0.0540
7.4485±1.8463
7.8834±1.8786
Tyrosol
5.0113±2.7231
3.0025±0.0087
11.3319±0.2017
7.9470±1.0521
4.3458±1.1452
3.9709±0.4115
Fumarate
15.2684±0.8018 12.8764±0.5160
7.7147±0.0320
7.9460±0.5350
4.6124±0.1906
4.9439±0.7443
Chlorogenate
1.2949±0.1916
2.3828±0.1047
1.4477±0.0461
2.2112±0.1261
1.5386±0.2303
2.1293±0.0628
Epicatechin
0.3924±0.1036
0.5899±0.0418
0.5653±0.0239
0.9166±0.0099
0.5970±0.0199
0.6203±0.03763
Catechin
0.9883±0.0999
1.6430±0.0233
1.2745±0.0330
1.6805±0.0023
1.5861±0.2437
1.4849±0.0973
Uracil
0.3191±0.0258
0.3106±0.0354
0.4727±0.0067
1.7805±0.1824
0.3415±0.0237
0.2599±0.1099
527 528
a
529
Starmerella bacillaris YR21; HO16: Hanseniaspora osmophila HO16; Y4M: Hanseniaspora
530
uvarum Y4M, and SU3: Saccharomyces uvarum SU3.
EC1118: Saccharomyces cerevisiae EC1118; TD291: Torulaspora delbrueckii TD291; YR21,
531
29
532
Supplementary data
533 534
Figure S1. 1H NMR spectra of methanol/D2O extract of apple ciders samples analyzed. a)
535
Hanseniaspora Y4M, b) Torulaspora delbrueckii TD291, c) Saccharomyces cerevisiae EC1118, d)
536
Saccharomyces uvarum SU3, e) Starmerella bacillaris YR21, f) Hanseniaspora osmophila HO16.
537 538 539 540 541 542 543 544 545 546 547 548 549 550
30
551
Figure S2. Heatmap based on the relative amount of selective integrals expresses as percentage
552
values respect to the total values of all integrals over the entire spectral region. Color key indicates
553
the relative abundance of the identified metabolites; (red: highest, yellow: medium, green: lowest).
554
Rows represent the identified metabolites (in bold discriminating metabolites from PCA analysis)
555
and columns represent the yeast strains. EC1118: Saccharomyces cerevisiae EC1118; TD291:
556
Torulaspora delbrueckii TD291; YR21, Starmerella bacillaris YR21; HO16: Hanseniaspora
557
osmophila HO16; Y4M: Hanseniaspora uvarum Y4M, and SU3: Saccharomyces uvarum SU3.
558
559
arabinose fructose glucose sucrose xylose alanine aspartate asparagine histidine isoleucine threonine acetate chlorogenate fumarate lactate malate pyruvate quinate succinate catechine epicatechine histamine tyrosol xantine uracil 2,3-butanediol sterol glycerol
YR21 0.0627 0.6078 5.5342 0.0206 0.3594 0.0651 0.5103 0.4856 0.0238 0.1664 0.2653 0.2291 0.0080 0.0606 0.1529 3.7200 0.1023 0.1650 1.2646 0.0039 0.0016 0.0246 0.0096 0.0009 0.0013 0.5353 0.0053 13.4829
HO16 0.0338 1.8288 0.8078 0.6308 0.3862 0.1120 0.7106 0.4463 0.0170 0.2670 0.4787 0.1179 0.0167 0.0712 0.1931 5.8234 0.1848 0.3179 2.9780 0.0091 0.0033 0.0088 0.0083 0.0031 0.0017 0.2010 0.0080 15.5139
SU3 0.0268 0.7323 0.8687 0.0110 0.5428 0.1199 1.2487 0.4802 0.0038 0.4024 0.5546 0.0895 0.0171 0.0570 1.9183 6.6235 0.0847 0.3967 2.2667 0.0094 0.0042 0.0037 0.0418 0.0047 0.0035 0.8599 0.0036 17.3432
EC1118 0.0464 0.7293 0.8860 0.0092 0.5997 0.0629 1.0487 0.4618 0.0032 0.3459 0.5912 0.0733 0.0156 0.0455 0.5074 7.0220 0.0939 0.2984 2.9984 0.0096 0.0052 0.0026 0.0227 0.0081 0.0102 1.3866 0.0062 18.2692
TD291 0.0311 7.5851 0.9265 0.0073 0.3777 0.1117 0.5396 0.5039 0.0258 0.1989 0.4165 0.0595 0.0084 0.0160 0.2382 4.9397 0.0550 0.2124 1.8441 0.0055 0.0021 0.0102 0.0075 0.0014 0.0012 0.4578 0.0097 9.8221
Y4M 0.0281 3.7807 0.8400 0.0134 0.3513 0.1348 0.6392 0.5426 0.0344 0.1778 0.4204 0.1527 0.0125 0.0214 0.2490 5.4840 0.1035 0.2152 2.1159 0.0064 0.0027 0.0145 0.0086 0.0021 0.0011 0.5990 0.0087 16.9777 31
560
Figure S3. Heatmap based on the relative amount of selective integrals expresses as percentage
561
values respect to the total values of all integrals over the aromatic spectral region. Color key
562
indicates the relative abundance of the identified metabolites; (red: highest, yellow: medium, green:
563
lowest). Rows represent the identified metabolites (in bold discriminating metabolites from PCA
564
analysis) and columns represent the yeast strains. EC1118: Saccharomyces cerevisiae EC1118;
565
TD291: Torulaspora delbrueckii TD291; YR21, Starmerella bacillaris YR21; HO16:
566
Hanseniaspora osmophila HO16; Y4M: Hanseniaspora uvarum Y4M, and SU3: Saccharomyces
567
uvarum SU3.
568
569
xanthine histamine histidine tyrosol fumarate chlorogenate epicatechin catechin uracil
YR22 0.2409 6.2599 6.0783 5.0113 15.2684 1.2949 0.3924 0.9883 0.3191
HO16 0.5548 1.5868 3.0679 3.0025 12.8764 2.3828 0.5899 1.6430 0.3106
SU4 0.6434 0.4971 0.5242 11.3319 7.7147 1.4477 0.5653 1.2745 0.4727
EC1119 1.4108 0.4477 0.5670 7.9470 7.9460 2.2112 0.9166 1.6805 1.7805
TD292 0.3886 2.9327 7.4485 4.3458 4.6124 1.5386 0.5970 1.5861 0.3415
Y4M 0.4887 3.3332 7.8834 3.9709 4.9439 2.1293 0.6203 1.4849 0.2599
570
571
572
573
574
575
576 32
577
Figure S4. Loading plot obtained of PCA performed on NMR data considering all spectral regions.
578
Buckets values are reported.
579 580
581 582
33
583
Figure S5 Loading plot obtained of PCA performed on NMR data considering aromatic spectral
584
regions. Buckets values are reported.
585
586 587
34
Highlights •
Apple juice fermented by 6 yeast species
•
NMR characterization of metabolite profiles of fermented apple juice
•
Specie-specific biomarkers related to apple juice fermentation
Consiglio Nazionale delle Ricerche
Conflict of Interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. On behalf of all the co-authors,
R. Consonni
Milan 17th June 2019 Partita IVA 02118311006 - C.F. 80054330586 Sede Legale: Via E. Bassini 15, 20133 Milano. Ingresso presso: Via A. Corti 12, 20133, Milano Altre Sedi: 16149 Genova - Via de Marini, 6 - Tel. 010.64751 – 13900 Biella - C.so G. Pella, 16 - Tel. 015.8493043 Web: www.ismac.cnr.it