Food Chemistry 233 (2017) 29–37

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Impact of apple cultivar, ripening stage, fermentation type and yeast

strain on phenolic composition of apple ciders
Oskar Laaksonen a,⇑, Rain Kuldjärv b,c, Toomas Paalme c, Mira Virkki a, Baoru Yang a
a
Food Chemistry and Food Development, Department of Biochemistry, University of Turku, FI-20014 Turku, Finland
b
Center of Food and Fermentation Technologies, Akadeemia tee 15A, Tallinn 12618, Estonia
c
Department of Chemistry and Biotechnology, Tallinn University of Technology, Ehitajate tee 5, Tallinn 12618, Estonia

a r t i c l e i n f o a b s t r a c t

Article history: Hydroxycinnamic acids and flavonoids in apple juices and ciders were studied using liquid chromatogra-
Received 12 December 2016 phy. Samples were produced from four different Estonian apple cultivars using unripe, ripe and overripe
Received in revised form 10 April 2017 apples, and six different commercial yeasts including Saccharomyces cerevisiae, Saccharomyces bayanus,
Accepted 11 April 2017
and Torulaspora delbrueckii strains. Part of the samples was additionally inoculated with malolactic bac-
Available online 12 April 2017
teria, Oenococcus oeni. The most notable difference among the samples was the appearance of phloretin in
malolactic ciders in comparison to conventional ciders and the juices. Furthermore, the apple cultivars
Keywords:
were significantly different in their phenolic contents and compositions. Additionally, ciders and juices
Apple cider
Cultivar
made from unripe apples contained more phenolic compounds than the ripe or overripe, but the effect
Flavonoids was dependent on cultivar. The commercial yeast strains differed in the release of free HCAs, especially
Hydroxycinnamic acids p-coumaric acid, during the yeast fermentation. In ciders inoculated with S. bayanus, the content was
Ripening stage higher than in ciders fermented with S. cerevisiae.
Yeasts Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Apples are known for their health benefits. The adage ‘‘an apple
a day keeps the doctor away” is well known among the consumers
and results of scientific research support it by showing presence of
various bioactive molecules, such as various phenolic compounds.
Health benefits associated with polyphenols in apples are cancer
risk reduction, high antioxidative power, anti-inflammatory and
anti-tumor properties and inhibition of carcinogenesis in skin,
mammary, and colon etc. (Biedrzycka & Amarowicz, 2008;
Francini & Sebastiani, 2013).
Cider is generally regarded as a beverage made from apples. In
North-America, the term ‘‘cider” generally refers to cloudy unpas-
teurized apple juice, and the term ‘‘hard cider” is used for a fer-
mented product. In Europe, however, the term ‘‘cider” refers to
the fermented product, an alcoholic beverage with alcohol content
between 1.2 and 8.5% v/v (Lea & Piggott, 2003). In this study, the
term ‘‘cider” refers to a fermented alcoholic beverage. Apple cider
production starts with pressed apple juice. During the fruit crush-
ing, pressing and juice extraction only a fraction of phenolic com-
⇑ Corresponding author.
E-mail addresses: oskar.laaksonen@utu.fi (O. Laaksonen), rain@tftak.eu
(R. Kuldjärv), toomas.paalme@ttu.ee (T. Paalme), mira.j.virkki@utu.fi (M. Virkki),
baoru.yang@utu.fi (B. Yang).
pounds is extracted (Van der Sluis, Dekker, & van Boekel, 2005).
This is mainly due to the discard of the peel and seeds which are
rich in different phenolic compounds (Francini, 2013; Wolfe, Wu,
& Liu, 2003). The polyphenolic content of apple juice depends on
preparation method, such as possible utilisation of pectinolytic
enzymes in order to release phenolic compounds from polysaccha-
ride structures, and following treatments, such as pasteurisation,
or fermentation parameters in cider production (Ye, Yue, & Yuan,
2014). Endogenous enzymes, such as polyphenol oxidase, oxidise
phenolic compounds in the apple juice and affect the sensory qual-
ity of the product (Renard et al., 2011). Each apple cultivar has its
own polyphenolic profile that is also dependent on harvest year,
climatic variables, cultivation and storage conditions (Mattila,
Hellström, & Törrönen, 2006; Mikulic-Petkovsek, Slatnar,
Stampar, & Veberic, 2010; Thompson-Witrick et al., 2014; Wolfe
et al., 2003; Łata & Tomala, 2007). Therefore, in order to compare
different cultivars, apples used in the current study shall come
from the same orchard and are grown under the same conditions.
The phenolic contents and profile have important effects on the
sensory properties of apple ciders, mainly on colour, bitterness,
and astringency (Ye et al., 2014). High molecular weight procyani-
dins in ciders are known to contribute to astringency, whereas the
smaller compounds contribute to bitter taste (Lea & Arnold, 1978;
Symoneaux, Baron, Marnet, Bauduin, & Chollet, 2014; Symoneaux,
Chollet, Bauduin, Le Quéré, & Baron, 2014). Simultaneously, they

http://dx.doi.org/10.1016/j.foodchem.2017.04.067
0308-8146/Ó 2017 Elsevier Ltd. All rights reserved.
30 O. Laaksonen et al. / Food Chemistry 233 (2017) 29–37
influence the sweetness and sourness, thus further highlighting
their importance in the overall flavour development (Symoneaux,
Baron et al., 2014). In addition to the non-volatile phenolic com-
pounds, the volatile phenolics mainly formed by enzymatic decar-
boxylation during fermentation contribute to aroma (Vanbeneden,
Van Roey, Willems, Delvaux, & Delvaux, 2008).
The selection of yeast and fermentation conditions is an impor-
tant factor influencing the development of sensory properties in
ciders. The availability of different yeast strains is numerous;
therefore, monitoring their influence on apple cider content and
profile of phenolics is in the interest of both yeast manufacturers
and apple cider producers. Malolactic fermentation (conversion
of L-malic acid to L-lactic acid and carbon dioxide) used in cider-
making process mainly to reduce the sourness of the product.
The aim of the study was to investigate phenolic profiles of apple
juices with those of ciders after the yeast fermentation as well as
malolactic fermentation by using multivariate statistical models.
Moreover, we compared the contributions of apple cultivar, with
an emphasis on Estonian cultivars, and ripening stage to the phe-
nolic composition of ciders and juices. Special focus was also on
the impact of various commercial yeast strains on the composi-
tional profiles of the fermented ciders.
2. Materials and methods
2.1. Preparation of ciders
Four autumn or winter apple cultivars, ‘Antei’, ‘Kulikovskoye’,
‘Melba’, and ‘Orlovski sinap’ grown in South Estonia, at a private
orchard in Valgjärve (58°80 N, 26°660 E) were used in the study.
Samples were subsequently taken at three different stages of
ripening: unripe, ripe, and overripe. Ripening time and thus the
harvesting time varies in each season due to varying weather con-
ditions. Therefore the estimation of ripening stage was based on
the assessment of the experienced farmer and additionally esti-
mated using the iodine starch test under a laboratory setting
(Travers, Jacquet, Brisset, & Maite, 2002). All apples were first har-
vested at the ‘unripe’ stage (0 weeks) of their maturity and left to
ripen at +4 °C. This is a common practice in Northern-Europe
region, because some of the autumn and the most winter cultivars
do not reach their maturity before the first frost. The ripe samples
were collected after 2–8 (depending on the cultivar: ‘Melba’
2 weeks, ‘Kulikovskoye’ 3 weeks, ‘Antei’ 6 weeks and ’Orlovski
sinap’ 8 weeks) weeks in storage at +4 °C and the overripe after
6–12 weeks in storage (approx. one month after the ripe stage: 6,
8, 10 and 12 weeks, respectively). Unripe apples had approximated
starch index 1–2, ripe apples starch index 3–4 and overripe apples
starch index 5 (Travers et al., 2002).
Apples were washed with tap water and drained. Apples with
obvious signs of biological contamination (rotting, molding, etc.)
were excluded; however, no further selection was made according
to the size or appearance of the apple. The apple juice was pre-
pared from 20 kg apples of each cultivar in each ripening stage
using a centrifugal juice press (Vita Pro-Active JE810; Kenwood).
The resulting juice batches were first combined and then dis-
tributed immediately into 1 L bottles for fermentation (cider sam-
ples) or frozen (juice samples). To achieve a stable fermentation,
each bottle was inoculated with 1 g of a chosen starter culture.
The commercial starter cultures used in this study were provided
by Lallemand, Inc., including Biodiva (Torulaspora delbrueckii),
C1108 (Saccharomyces bayanus), EC1118 (Saccharomyces
cerevisiae), OKAY (Saccharomyces cerevisiae), OPALE (Saccharomyces
cerevisiae), and QA23 (Saccharomyces bayanus) and instructions in
the products were followed. Prior to inoculation, weighed amounts
of the starter cultures were rehydrated using small amounts of the
apple juice to be fermented. All juices and ciders were prepared in
food-grade conditions.
The bottles were sealed with a rubber stopper equipped with an
airlock. The bottles were left to ferment at temperature of
+21 ± 1 °C until no signs of further fermentation could be observed.
Fermentations with Biodiva yeast culture lasted for 2–3 days. After
this, an additional inoculation with S. cerevisiae (specifically,
EC1118) was performed because non-Saccharomyces yeasts are
typically used in a sequential inoculation. 500 mL of ciders fer-
mented with Biodiva, EC1118, OKAY and QA23 yeast cultures
was additionally inoculated with malolactic bacteria (Oenococcus
oeni, VP41). Prior to inoculation, each bacterial culture weighed
0.01 g with the addition of water for rehydration. After inoculation,
0.1 g of nutrients (Opti ML Blanc; Scott Laboratories) was added.
Inoculated bottles were sealed once again with rubber stoppers
equipped with an airlock. Malolactic fermentation lasted three
weeks at +21 ± 1 °C. Samples without malolactic fermentation
(n = 4 cultivars  3 ripening stages  6 yeasts  2 replicates = 144)
are referred in this study as ‘ciders’ whereas samples with both fer-
mentations are referred as ‘malolactic ciders’
(n = 4  3  4  2 = 96). The number of apple juice samples was
24 (4  3  1  2). A representative sample was taken of each juice
and cider and stored at 20 °C in 10 mL plastic tubes.
2.2. Analysis of phenolic compounds
The phenolic compounds were extracted from the samples and
analysed using a liquid chromatographic method described in
Mäkilä et al. (2016). A Phenomenex Aeris peptide XB-C18
(3.6 lm, 150  4.60 mm, Torrance, CA) column was used. The
high-performance liquid chromatography-diode-array detection
instrument consisted of a Shimadzu (Shimadzu Corporation, Kyoto,
Japan) SIL-30AC auto sampler, a sample cooler, two LC-30AD
pumps, a CTO-20AC column oven, an SPD-M20A diode array detec-
tor and a CBM-20A central unit. Quantifications were carried out
using selected external standards for each compound group:
chlorogenic acid (5-O-caffeoylquinic acid; Sigma-Aldrich, St. Louis,
MO) for caffeic acid derivatives (monitored at UV–vis wavelength
320 nm, peak maxima around 320 nm), p-coumaric acid (Sigma-
Aldrich) for p-coumaric acid derivatives (monitored at 320 nm,
peak maxima around 310 nm), phloridzin (Sigma-Aldrich) for
dihydrochalcones (280 nm) and quercetin-3-O-glucopyranoside
(Extrasynthese, Genay, France) for flavonol glucosides (monitored
at 360 nm, peak maxima around 350 nm) and aglycons (monitored
at 360 nm, peak maxima around 370 nm). A B-type procyanidin
dimer was prepared by the Department of Chemistry, University
of Turku, and it was used as an external standard for proantho-
cyanidins (280 nm).
Phenolic compounds were identified with HPLC-DAD–ESI-MS/
MS method described in Mäkilä et al. (2016). The analyses were
performed using a Waters Acquity Ultra Performance LC system
in combination with a Waters 2996 DAD detector and a Waters
Quattro Premier mass spectrometer (Waters Corp., Milford, MA)
equipped with an ionspray interface. Mass spectra were obtained
by scanning between m/z 130 and 1200. The identifications of
compounds were based on retention times, reference compounds,
UV spectra, mass spectral characteristics and literature references
focusing on apples, apple juices and/or ciders.
2.3. Statistical analyses
All the samples were analyzed in duplicate. A one-way analysis
of variance (ANOVA) and student’s t-test were performed to com-
pare the compositional differences between cultivars, ripening
stages, and yeasts. Statistical univariate analyses were performed
by using IBM SPSS Statistics 22 (SPSS Inc., Chicago, IL). Principal
 O. Laaksonen et al. / Food Chemistry 233 (2017) 29–37 31

component analysis (PCA) and partial least squares regression dis-
crimination analysis (PLS-DA) were applied for standardised data
to study the differences and classifications among the apple culti-
vars (n = 4), ripening stages (n = 3), fermentation type (n = 3; juice,
fermentation and malolactic fermentation) and yeast strains
(n = 4–6) using the phenolic variables as X-data and aforemen-
tioned classes as Y-data. Full cross- validation was used to estimate
the number of factors for a statistically reliable model. Multivariate
models were performed by using Unscrambler X, version 10.4
(CAMO Software, Oslo, Norway).
3. Results and discussion
3.1. Identification of the phenolic compounds
Identification of the analytes extracted from ciders and juices
were based on the HPLC retention behaviour, UV–vis spectra,
ESI-MS(-MS2) spectra, reference compounds, and literature com-
parisons (Chagné et al., 2012; Clifford, Johnston, Knight, &
Kuhnert, 2003; Diñeiro García, Valles, & Picinelli Lobo, 2009;
Malec et al., 2014; Marks, Mullen, & Crozier, 2007; Picinelli Lobo,
García, Sánchez, Madrera, & Valles, 2009; Ramirez-Ambrosi et al.,
2013; Sanoner, Guyot, Marnet, Molle, & Drilleau, 1999;
Thompson-Witrick et al., 2014; Verdu et al., 2013, 2014; Çam &
Aaby, 2010). Representative samples from each cultivar, ripening
stage, fermentation type, and yeast strain were used to identify
the compounds in the samples.
The compounds were classified into four groups of phenolic
compounds observed in the samples (Table 1; Supplementary
Fig. 1) (Guyot, Marnet, Laraba, Sanoner, & Drilleau, 1998). The
malolactic ciders showed less peaks in the chromatograms proba-
bly due to the fact that these samples were twice more diluted
than the juice and cider samples. For this reason the malolactic
ciders were not included in Table 2. The most abundant class in
the chromatograms was hydroxycinnamic acid derivatives
(phenylpropanoid structures) followed by three different groups
of flavonoids: flavan-3-ols and proanthocyanidins, flavonols, and
dihydrochalcones. The apple cultivars used in this study contain
red pigments in their skins derived from anthocyanins, but these
compounds were not examined in this study.

Table 1
Identification of phenolic compounds in cider samples.

Peak Rt k max [MH] Other ions in [M+H]+ (m/z) Other ions in Tentative identification Abbreviationc
No. (min)a (nm)b (m/z) negative mode positive mode
(m/z) (m/z)
Hydroxycinnamic acids
1 10.25 353 191 – 3-O-caffeoylquinic acid (neochlorogenic acid) nChlor*
2 10.42 310 337 339 147 3-O-p-coumaroylquinic acid 3pC-qa
3 11.08 325 353 191 355 163 5-O-caffeoylquinic acid (chlorogenic acid) Chlor*
4 11.70 327 353 173 355 163 4-O-caffeoylquinic acid 4Ca-qa*
5 12.37 324 179 181 Caffeic acid Ca*
6 12.80 313 337 173 339 5-O-p-coumaroylquinic acid 5pC-qa
7 13.05 353 191 355 163 Caffeoylquinic acid Ca-qa
8 13.85 313 337 173 339 147 4-O-p-coumaroylquinic acid 4pC-qa
9 15.32 367 369 Ferulic acid hexoside Fe-hex I
10 15.78 310 163 165 p-coumaric acid pC*
11 16.20 367 369 Ferulic acid hexoside Fe-hex II
12 17.18 323 193 195 Ferulic acid Fe*
13 17.70 367 369 Ferulic acid hexoside Fe-hex III
Flavan-3-ols
14 9.97 280 577 579 PC dimer Di I
15 11.15 279 291 Flavan-3-ol monomer (catechin) Cat
16 11.78 284 865 867 PC trimer Tri I
17 12.82 280 577 579 PC dimer Di II
18 13.00 279 865 867 PC trimer Tri II
19 13.65 279 279 291 Flavan-3-ol monomer (epicatechin) Ecat
20 15.08 282 865 867 PC trimer Tri III
21 15.52 280 577 579 PC dimer Di III
22 16.00 280 1153 865 1155 867 PC tetramer Tet
23 18.75 280 577 579 PC dimer Di IV
Flavonols
24 18.48 348 463 465 303 Quercetin-3-O-galactoside Qu-gal
25 18.95 342 463 465 303 Quercetin-3-O-glucoside Qu-glc*
26 20.30 342 433 435 303 Quercetin-3-O-xyloside Qu-xyl
27 21.23 433 435 303 Quercetin pentoside Qu-pen
28 21.55 477 479 317 Isorhamnetin-3-O-glucoside Is-glc*
29 22.57 348 433 435 303 Quercetin-3-O-arabinoside Qu-ara
30 23.20 344 447 449 303 Quercetin-3-O-rhamnoside Qu-rha
31 30.90 461 463 317 Isorhamnetin-3-O-rhamnoside Is-rha
32 33.70 301 303 Quercetin Qu*
Dihydrochalcones
33 18.38 282 583 585 291, 453 Hydroxyphloretin diglycoside HP-diglc
34 21.73 283 451 289 453 291 Hydroxyphloretin monoglycoside HP-gly
35 22.78 285 567 569 275, 437 Phloretin-20 -O-xyloglucoside Ph-xylglc I
36 23.82 283 567 569 275, 437 Phloretin xyloglucoside Ph-xylglc II
37 28.20 285 435 273 437 275 Phloretin-20 -O-glucoside (phloridzin) Ph-glc*
38 34.55 283 273 275 Phloretin Ph
a
Retention time in the LC-MS analysis (Supplementary Figure).
b
Maximum in the UV–vis spectrum; in the cases of missing values the peak overlaps with another more abundant peak.
c
Abbreviated names are used in Figs. 1–3.
32 O. Laaksonen et al. / Food Chemistry 233 (2017) 29–37

Table 2
Contents of major classes of phenolic compounds (mg/100 mL) in samples before and after the yeast fermentationa.

Hydroxycinnamic acids Flavonols Procyanidins Dihydrochalcones Total sum

PC sum
HCA Free HCAs Flavonol Free Dihydrochal Free phloretin
derivatives glycosides quercetin derivatives
Comparison of juices and ciders
All cultivars Juice (n = 23) 7.37 ± 3.1 0.24 ± 0.4 1.64 ± 0.7 0.12 ± 0.3 b 17.3 ± 12 3.61 ± 1.4 0.01 ± 0.01 b 30.2 ± 15
Cider (n = 140) 7.53 ± 2.3 0.32 ± 0.1 1.31 ± 0.6 0.18 ± 0.1 a 20.8 ± 9.5 3.46 ± 1.1 0.28 ± 0.1 a 33.6 ± 12
Comparison of apple cultivars in juices and ciders
‘Antei’ (n = 42) 10.0 ± 2.3 a 0.23 ± 0.1 b 0.74 ± 0.2 d 0.13 ± 0.1 23.7 ± 13 a 3.17 ± 0.7 b 0.26 ± 0.2 38.0 ± 15 a
‘Kulikovskoye’ (n = 40) 7.49 ± 1.3 b 0.36 ± 0.1 a 1.91 ± 0.3 a 0.17 ± 0.1 20.5 ± 9.6 ab 2.74 ± 0.7 b 0.27 ± 0.2 33.1 ± 11 ab
‘Melba’ (n = 42) 6.33 ± 1.8 c 0.34 ± 0.1 ab 1.58 ± 0.5 b 0.20 ± 0.1 21.4 ± 7.3 a 4.24 ± 1.3 a 0.26 ± 0.1 34.1 ± 11 a
’Orlovski sinap’ (n = 39) 6.08 ± 1.6 c 0.29 ± 0.3 ab 1.21 ± 0.6 c 0.18 ± 0.2 15.3 ± 5.9 b 3.76 ± 1.0 a 0.22 ± 0.1 26.8 ± 8.4 b
Comparison of juices and ciders within cultivars
‘Antei’ Juice (n = 6) 10.5 ± 2.7 0.18 ± 0.03 b 1.05 ± 0.4 0.05 ± 0.05 21.9 ± 12 3.68 ± 0.9 0.09 ± 0.09 b 37.4 ± 16
Cider (n = 36) 9.93 ± 2.2 0.24 ± 0.1 a 0.69 ± 0.2 0.14 ± 0.1 24.0 ± 14 3.08 ± 0.6 0.29 ± 0.1 a 38.1 ± 16
‘Kulikovskoye’ Juice (n = 6) 8.26 ± 1.3 0.15 ± 0.01 b 2.25 ± 0.2 a 0.05 ± 0.04 b 19.9 ± 16 2.94 ± 0.6 0.05 ± 0.07 b 33.6 ± 16
Cider (n = 34) 7.36 ± 1.3 0.40 ± 0.1 a 1.86 ± 0.3 b 0.19 ± 0.06 a 20.6 ± 8 2.70 ± 0.7 0.31 ± 0.1 a 33.1 ± 9.8
‘Melba’ Juice (n = 6) 6.02 ± 2.5 0.15 ± 0.05 b 2.00 ± 0.8 0.08 ± 0.04 b 18.7 ± 11 4.79 ± 2.1 0.19 ± 0.1 b 31.8 ± 16
Cider (n = 36) 6.4 ± 1.7 0.37 ± 0.1 a 1.51 ± 0.4 0.22 ± 0.1 a 21.8 ± 6.6 4.15 ± 1.1 0.27 ± 0.1 a 34.5 ± 9.5
‘Orlovski sinap’ Juice (n = 5) 4.13 ± 1.2 0.53 ± 0.09 1.17 ± 0.9 0.34 ± 0.6 6.66 ± 0.3 b 2.91 ± 0.4 b 0.04 ± 0.03 b 15.5 ± 1.7 b
Cider (n = 34) 6.37 ± 1.5 0.26 ± 0.09 1.22 ± 0.6 0.15 ± 0.07 16.6 ± 5.2 a 3.89 ± 1.1 a 0.25 ± 0.1 a 28.5 ± 7.7 a
Comparison samples in different apple ripening stages
All cultivars Unripe (n = 54) 8.45 ± 2.4 a 0.31 ± 0.1 1.68 ± 0.6 a 0.22 ± 0.1 a 25.4 ± 13 a 4.03 ± 1.4 a 0.23 ± 0.1 b 40.1 ± 15 a
Ripe (n = 55) 7.05 ± 2.6 b 0.34 ± 0.3 1.27 ± 0.6 b 0.15 ± 0.2 b 18.0 ± 5.3 b 3.09 ± 0.9 b 0.21 ± 0.1 b 29.8 ± 7.8 b
Overripe (n = 54) 7.03 ± 2.4 b 0.28 ± 0.2 1.13 ± 0.5 b 0.13 ± 0.05 b 17.6 ± 8.4 b 3.34 ± 0.8 b 0.32 ± 0.2 a 29.5 ± 9.8 b
‘Antei’ Unripe (n = 14) 11.2 ± 2.4 a 0.25 ± 0.1 a 0.90 ± 0.3 a 0.17 ± 0.2 32.8 ± 20 a 3.34 ± 0.9 a 0.20 ± 0.1 b 48.7 ± 22 a
Ripe (n = 14) 10.8 ± 1.4 a 0.32 ± 0.1 a 0.63 ± 0.1 b 0.10 ± 0.05 21.0 ± 3.4 b 3.48 ± 0.5 a 0.26 ± 0.1 ab 37.1 ± 5.0 ab
Overripe (n = 14) 8.00 ± 1.2 b 0.12 ± 0.03 b 0.70 ± 0.1 ab 0.11 ± 0.03 16.4 ± 3.5 b 2.69 ± 0.4 b 0.34 ± 0.2 a 28.1 ± 5.0 b
‘Kulikovskoye’ Unripe (n = 14) 7.54 ± 1.1 ab 0.32 ± 0.1 ab 2.12 ± 0.4 a 0.21 ± 0.09 a 22.1 ± 10 2.28 ± 0.4 ab 0.22 ± 0.1 b 35.0 ± 11 a
Ripe (n = 12) 6.79 ± 0.9 b 0.31 ± 0.1 b 1.88 ± 0.3 ab 0.12 ± 0.05 b 18.1 ± 3.7 2.24 ± 0.6 b 0.20 ± 0.1 b 29.4 ± 4.2 ab
Overripe (n = 14) 8.16 ± 1.5 a 0.45 ± 0.2 a 1.78 ± 0.3 b 0.18 ± 0.07 ab 21.5 ± 13 3.19 ± 0.7 a 0.39 ± 0.2 a 35.3 ± 14 b
‘Melba’ Unripe (n = 14) 8.45 ± 0.6 a 0.39 ± 0.1 2.07 ± 0.5 a 0.31 ± 0.1 a 27.1 ± 5.4 a 5.51 ± 1.1 a 0.32 ± 0.1 a 43.7 ± 7.2 a
Ripe (n = 14) 5.41 ± 1.2 b 0.33 ± 0.1 1.40 ± 0.5 b 0.19 ± 0.09 b 17.3 ± 6.7 b 3.64 ± 0.9 b 0.21 ± 0.08 b 28.2 ± 8.9 ab
Overripe (n = 14) 5.15 ± 1.2 b 0.30 ± 0.1 1.28 ± 0.3 b 0.10 ± 0.04 c 19.9 ± 6.1 b 3.57 ± 0.8 b 0.25 ± 0.1 ab 30.3 ± 8.1 b
‘Orlovski sinap’ Unripe (n = 14) 6.45 ± 1.7 a 0.26 ± 0.09 1.70 ± 1.2 a 0.20 ± 0.1 19.2 ± 6.7 a 4.31 ± 1.0 a 0.20 ± 0.1 ab 32.1 ± 9.7 a
Ripe (n = 13) 5.04 ± 1.5 b 0.39 ± 0.5 1.16 ± 0.6 b 0.20 ± 0.4 14.6 ± 4.4 ab 2.98 ± 0.9 b 0.17 ± 0.1 b 24.2 ± 6.6 ab
Overripe (n = 12) 6.78 ± 1.0 a 0.23 ± 0.1 0.71 ± 0.2 c 0.13 ± 0.04 11.6 ± 3.4 b 4.00 ± 0.9 a 0.30 ± 0.2 a 23.5 ± 5.4 b
a
Results are shown as means ± standard deviations. Table do not include malolactic cider samples. Statistical differences between four cultivars or three ripening stages are
based on oneway ANOVA with Tukey’s posthoc test and between juices and ciders is based on student’s t-test; significant differences are shown with letters a–d (p < 0.05).

The hydroxycinnamic acid derivatives detected in the samples
were quinic acid derivatives of caffeic and p-coumaric acid with
traces of corresponding ferulic acid derivatives or free hydroxycin-
namic acids (Table 1). Tentative groupings to either p-coumaric
acid derivatives or caffeic and ferulic acid derivatives were first
made according to the UV–vis spectra, and the identifications were
followed by the MS spectra and the literature. Total of 13
hydroxycinnamic acids were detected: three isomers of p-
coumaroylquinic acid, four isomers of caffeoylquinic acid, three
isomers of feruloylquinic acid (ferulic acid hexosides) and free caf-
feic, p-coumaric and ferulic acid. The most abundant peak (peak 3)
in every chromatogram was identified as chlorogenic acid accord-
ing to MS spectra and commercial standard (5-O-caffeoyl quinic
acid). The mass detected on positive mode was m/z 355 and 353
on the negative mode with similar MS fragments as shown in liter-
ature (Clifford et al., 2003; Malec et al., 2014; Ramirez-Ambrosi
et al., 2013). Confusingly, the major peak, chlorogenic acid, has
been reported in the literature as either 3-O-caffeoylquinic acid
(Thompson-Witrick et al., 2014) or 5-O-caffeoylquinic acid
(Clifford, 2000; Clifford et al., 2003; Ramirez-Ambrosi et al.,
2013). Three other similar peaks (similar MS spectra) were
detected in the chromatograms (peaks 1, 4 and 7). The commercial
standards for neochlorogenic acid (3-O-caffeoylquinic acid) and 4-
O-caffeoylquinic acid (Sigma-Aldrich) were used to identify peaks
1 and 4, respectively. Three p-coumaroylquinic acids were detected
(peaks 2, 6 and 8) with the m/z 339 in positive and 337 in the neg-
ative modes. They were identified according to a similar fragmen-
tation pattern as 3-, 5- and 4-O-p-coumaroylquinic acids,
respectively. The 4-O-p-coumaroylquinic acid was the second most
abundant peak in the chromatogram. A similar pattern may be con-
sidered for the three notably less abundant ferulic acid hexosides
which were tentatively identified as feruloylquinic acids (peaks 9,
11 and 13). In positive and negative modes, m/z 181 or 179, respec-
tively, indicates caffeic acid (peak 5), m/z 165 or 163 p-coumaric acid
(peak 10) and m/z 195 or 193 (ferulic acid (peak 12).
Concerning proanthocyanidins, apple ciders contain mainly
procyanidins which are composed of two diastereoisomeric
monomeric configurations of flavan-3-ols, (+)-catechin and
()-epicatechin (Hellström, Törrönen, & Mattila, 2009). Contents
of procyanidins are generally lower in ciders than in apples
(Hellström et al., 2009). In this study, two procyanidin monomers,
four dimers, three trimers, and a tetramer were detected (Table 1)
based on MS spectra and literature (Malec et al., 2014; Marks et al.,
2007; Ramirez-Ambrosi et al., 2013; Verdu et al., 2013). Based on
the literature, peaks 14 and 17 are tentatively dimers B1 and B2,
respectively, whereas peak 23 is tentatively dimer B5. Both
flavan-3-ol monomers were detected (peaks 15 and 19) with
(+)-catechin co-eluting with chlorogenic acid (peak 3). Addition-
ally, four dimers were detected indicating four possible variations
of catechin and epicatechin dimers. A procyanidin compound co-
eluted together with the caffeic acid (peak 5) at around 12.3 min.
The compound was not further identified, but the overlapping
may have interfered with the further quantifications and, thus,
affected the results concerning the caffeic acid.
 O. Laaksonen et al. / Food Chemistry 233 (2017) 29–37 33

Dihydrochalcones and flavonols eluted generally in the latter
part of the chromatograms (Table 1; Supplementary Fig. 1). Six
dihydrochalcones were detected in this study. The most abundant
peaks were 20 -O-xyloglucoside (peak 35) and 20 -O-glucoside (peak
37) of phloretin (Marks et al., 2007; Ramirez-Ambrosi et al., 2013;
Sanoner et al., 1999). Two of the less abundant compounds were
tentatively identified as glycosides of hydroxyphloretin based on
literature (Alonso-Salces et al., 2005; Ramirez-Ambrosi et al.,
2013) whereas one showed exactly same MS fragments as phlore-
tin 20 -O-xyloglucoside (Ramirez-Ambrosi et al., 2013). Nine flavo-
nols were detected (Table 1) including eight glycosides
(glucosides and rhamnosides of quercetin and isorhamnetin) and
quercetin aglycon (Malec et al., 2014; Marks et al., 2007;
Ramirez-Ambrosi et al., 2013). Flavonols were the least abundant
group of the compounds investigated in this study. However, the
compounds are known to have very low thresholds for astringency
(Hufnagel & Hofmann, 2008); thus, the compounds may have an
important role in the sensory quality of the ciders.
3.2. Contribution of fermentation type to phenolic composition
Principal component analysis was first used to study the poten-
tial hidden structures in the data and, thus, the main factors con-
tributing to dispersion in the data of all samples using only the
selected most abundant phenolic variables (n = 24) found in juices,
ciders and malolactic ciders. The key factor on the first PC (Supple-
mentary Fig. 2) was the difference between malolactic ciders and
others, whereas the second PC separated samples of cultivar ‘Antei’
from the other cultivars. Supervised classification method, partial
least squares discriminant analysis (PLS-DA) was further used to
study the impact of the cider process type (juice, cider and malo-
lactic cider as Y-data; n = 3) on the phenolic composition (X-data,
n = 24). Three processes are classified with a model of five vali-
dated factors (R2 = 0.745; validated R2 = 0.657). The most impor-
tant variation in the data among the three classes is the
difference between malolactic ciders with three validated factors
(R2 = 0.815; validated R2 = 0.776) and others shown already on
the first factor in Fig. 1. Malolactic fermented ciders are located
on the right with strong positive correlation with free phloretin
(variable P) and negative correlation with glycosylated form the
compound, phloridzin (P-glc). The juice (R2 = 0.680; validated
R2 = 0.500) and cider samples (R2 = 0.723; validated R2 = 0.680)
are not equally well classified in the model.
Sum variables of the main phenolic compound classes are in
Table 2, as well as the corresponding free aglycons potentially
released during fermentations. The major significant differences
between all juices and ciders and malolactic samples were the
higher contents of quercetin and phloretin in the fermented sam-
ples (Table 2). Processing of apple juices resulted in breaking of
glycosidic bonds. The phloretin is almost non-existing
(0.01 ± 0.01 mg/100 mL) in apple juices whereas the content is
notable higher in ciders (0.28 ± 0.01) and finally significantly the

Scores
5

3
Factor-2 (6%, 2 6%)

-1

-2

-3

-4
-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6
Factor-1 (22%, 38%)

Correlation Loadings (X and Y)

1

0.8
Juice
0.6 nChlor
Ca
Factor-2 (6%, 26%)

0.4 Qu-ara
Is-glc
Fe-hexQu-glc
I Qu-gal Qu-xyl 5pC-qa Malolactic cider
0.2 Di IV QuDi II Ph
0 Ph-glc 4Ca-qaTri IIIEcat Di I
Fe-hex II
-0.2 ChlorPh-xylglc I 4pC-qa pC

3pC-qa
-0.4
Cider Fe
-0.6

-0.8

-1
-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1
Factor-1 (22%, 38%)

Fig. 1. PLS-DA model for juice, cider and malolactic cider samples (n = 259) classified using the selected abundant phenolic compounds as X-data (n = 24) process method as
Y-data (n = 3; juice, squares; cider, circles; malolactic cider, triangles). Abbreviations of compounds refer to Table 1.
34 O. Laaksonen et al. / Food Chemistry 233 (2017) 29–37

highest in the malolactic ciders (0.74 ± 0.6). Oenococcus oeni possi- correlation with chlorogenic acid. ‘Melba’, on the other hand, is
bly produces enzymes with glycosidase activity especially suitable profiled only in the third and following factors (not shown). If
for dihydrochalcone 2-O-glycosides as the content of free quercetin the impact of malolactic ciders is excluded, ‘Antei’ samples had
from the quercetin 3-O-glycosides decreased in malolactic ciders the highest contents of HCAs among the cultivars (Table 2). ‘Kuli-
(from 0.18 mg/100 mL in ciders to 0.13 mg/100 mL in malolactic kovskoye’, on the other hand, had the highest contents of flavonol
ciders). Phloretin has been shown to activate bitter taste receptors glycosides whereas ‘Melba’ and ‘Orlovski sinap’ contained highest
in in vitro-tests (Roland et al., 2013) thus the increased content amounts of dihydrochalcones.
may potentially result in increased bitterness. In order to examine the impact of cider processing on the phe-
nolic composition of the sample, juices were compared to ciders in
3.3. Contribution of apple cultivar to the phenolic composition each cultivar and malolactic cider samples were excluded from the
comparison (Table 2). The impact of the process was mainly
The impact of apple cultivars on the phenolic profiles is shown detected in the release of free HCAs from caffeoyl-, coumaroyl-
in Fig. 2 and Table 2. Classification of the same 259 samples (as in or feruloylquinic acids, quercetin from corresponding 3-O-
Fig. 1) with PLS-DA was carried out using the selected major indi- glycosides or especially phloretin from glycosides of dihydrochal-
vidual phenolic compounds as X-data (n = 24; same as in Fig. 1) cones. The increase of phloretin in ciders was significant in all
and four cultivars (‘Antei’, ‘Kulikovskoye’, ‘Melba’ and ‘Orlovski cultivars whereas increases of HCAs were detected only in three
sinap’) as Y-data. All cultivars were successfully classified in the out of four cultivars and quercetin in only cultivars ‘Kulikovskoye’
model: ‘Antei’ with four validated factors (R2 = 0.788; validated and ‘Melba’. Our findings are in accordance with Alberti et al.
R2 = 0.772), ‘Kulikovskoye’ with seven (R2 = 0.826; validated (2016) that impact of fermentation process (i.e. comparison of
R2 = 0.739), ‘Melba’ with seven (R2 = 0.734; validated R2 = 0.666) juices and ciders) on the phenolic contents is dependent on apple
and ‘Orlovski sinap’ with eight (R2 = 0.857; validated R2 = 0.797). cultivar.
In the Fig.2 showing the first two validated factors, ‘Antei’ is
located on the left side of the plot with some of the p-coumaroyl 3.4. Impact of apple ripening stage
and caffeoylquinic acids whereas ‘Orlovski sinap’ is located on
the right side of the plot with certain quercetin glycosides. In general with all juice and cider samples included, the ones
‘Kulikovskoye’ is located on the bottom of the plots with negative from unripe contained statistically significantly more HCAs,

Scores
4

2
Factor-2 (13% . 24%)

-1

-2

-3

-4

-5
-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4
Factor-1 (14%, 26%)

Correlation Loadings (X and Y)

1

0.8
Fe-hex I Orlovski Sinap
0.6
Chlor
Ph-xylglc I
Factor-2 (13% ,24%)

0.4 nChlor Qu-glc

Antei Fe-hex IICa Ph-glc
0.2 3pC-qa
4Ca-qa Ecat Melba Qu-ara
0 Tri III
-0.2 4pC-qa Di I
pC Qu-xyl Qu-gal
Di II
-0.4 Di IV Ph
Fe Is-glc
5pC-qa
-0.6
Kulikovskoye
-0.8

-1
-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1
Factor-1 (14%, 26%)

Fig. 2. PLS-DA model for juice, cider and malolactic cider samples (n = 259) classified using the selected abundant phenolic compounds as X-data (n = 24) and cultivars as Y-
data (n = 4; ‘Antei’, squares; ‘Kulikovskoye’, circles; ‘Melba’, triangles; ‘Orlovski sinap’, diamonds) Abbreviations of the phenolic variables in loadings-plots refer to Table 1.
 O. Laaksonen et al. / Food Chemistry 233 (2017) 29–37 35

Correlation Loadings (X and Y) Correlation Loadings (X and Y)

A C 1
1

0.8 Unripe 0.8

Qu-gal Ca Ripe
0.6 0.6
Qu-glc FlaSum Qu nChlor

Factor-2 (6%, 30%)

0.4
Factor-2 (13%, 45%)

0.4 Di II Qu-rha
Is-glc
Fe-hex I
Fe-hexCa-qa
II 3pC-qa
3pC-qa Ecat Di IIIQu-xyl UnripeQu-ara
Qu 4pC-qa
0.2 Overripe Fe-hex II pC-der Qu-araPCSumTotalSum 0.2 HCAQQu-xyl
Is-glc4Ca-qa
HP-diglc
Chlor Chlor
FlaSum
P-glc Di II
HP-diglc Qu-rha
P-xylglc I 5pC-qa Di IV
Ph-xylglc I 0 HchalSum
Qu-galQu-glc
0
Ph
Ca
HCADerSum HP-gly pC-derFreeHCASum
pC
Ph
-0.2 Fe
nChlor HP-glyHchalSum
4pC-qa -0.2 TotalSum
Ecat Fe
5pC-qaDi IV Ph-glc PCSum Di III
Tri III pC
-0.4 -0.4 Di I
Fe-hex I Free HCAsum Tri III
-0.6 Ca-qa -0.6 Overripe
Di I 4Ca-qa
-0.8 Ripe -0,8

-1 -1
-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1
Factor-1 (37%, 27%) Factor-1 (49%, 29%)

Correlation Loadings (X and Y) Correlation Loadings (X and Y)

B 1 D 1 HCADerSum
4pCo-qa HchalSum
4Ca-qa Ph-glc Ph-xylglc I
Chlor
0.8 0.8
Ca-qa HP-gly
Di II Fe TotalSum
0.6 0.6 Overripe Fe-hex II
Di I Ph Qu-glcEcatIs-glc
Di IV Ripe PCSumQu-gal
Tri III
Factor-2 (19%, 24%)

Factor-2 (25%, 25%)

0.4 Overripe 0.4 nChlor Qu-rha
Fe Ca
Fe-hex I FlaSum
0.2 pC Ca-qa 0.2 Di IV HP-diglc Unripe Di III
Free HCA sum 3pC-qa
P Fe-hex II
0 0 Qu
Di II Di I FreeHCASumQu-xyl
HP-diglc pC-der 5pC-qa
-0.2 HP-gly Ecat -0.2 pC pC-der
PCSum Tri III Qu-glc
P-glc P-xylglcQu
I Qu-gal
-0.4 HchalSum 4pCo-qa TotalSum 3pC-qa
nChlor
-0.4 Qu-ara
HCADerSum4Ca-qa 5pC-qa Ca
Chlor Qu-ara Di III Fe-hex I Ripe
-0.6 Qu-xyl Is-glc Unripe -0.6
Qu-rha FlaSum
-0.8 -0.8

-1 -1
-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1
Factor-1 (21%, 29%) Factor-1 (28%, 40%)

Fig. 3. PLS-DA models for the impact of apple ripening stage (Y-data, n = 3) on the phenolic composition (X-data, n = 37) in juice and cider samples within cultivars. A. ‘Antei’;
B. ‘Kulikovskoye’; C. ‘Melba’; D. ‘Orlovski sinap’. Abbreviations of the phenolic variables in loadings-plots refer to Table 1 and sum variables to Table 2.

flavonols, procyanidins and dihydrochalcones than the ripe or ples had the highest contents of HCAs and dihydrochalcones
overripe samples (Table 2). Only the content of phloretin was the (Table 2). The differences between ripening stages in each cultivar
highest in overripe samples. In accordance with a previous study can also be seen from Fig. 3A–D with the sum variables and indi-
(Alberti et al., 2016), the impact of the ripening stage on the chem- vidual compounds found in juice and cider samples (X-data,
ical composition was not equally significant as the impact of apple n = 37). PLS-DA models were created in order to use phenolic vari-
cultivar. The degree of polymerisation (DP) and the content of pro- ables as X-data to explained difference between three ripening
cyanidins with high DP, which were not investigated in this study, stages (Y-data) within each cultivar (malolactic ciders excluded).
decreases during apple ripening (Alonso-Salces et al., 2005). In all cultivars, the multivariate models were able to discriminate
The impact of ripening was dependent on apple cultivar. For the three classes based on their phenolic composition (‘Antei’:
example, in cultivar ‘Antei’ the juices and ciders produced from R2 = 0.919, validated R2 = 0.865, with five factors; ‘Kulikovskoye’:
the unripe apples contained more HCAs and procyanidins than R2 = 0.753, validated R2 = 0.573, with four factors; ‘Melba’:
the overripe samples, whereas in ‘Kulikovskoye’ the overripe sam- R2 = 0.919, validated R2 = 0.744, with six factors; ‘Orlovski sinap’:

Table 3
Impact of different yeasts on the free hydroxycinnamic acids in ciders and malolactic ciders (mg/100 mL)*.

Yeast Sum of free HCAs Caffeic acid p-Coumaric acid Ferulic acid
Cider samples
S. bayanus (n = 45) 0.36 ± 0.1 a 0.04 ± 0.01 0.25 ± 0.1 a 0.06 ± 0.03 a
S. cerevisiae (n = 72) 0.31 ± 0.1 b 0.05 ± 0.02 0.21 ± 0.1 b 0.05 ± 0.02 b
Biodiva (n = 23) 0.28 ± 0.1 bc 0.05 ± 0.01 0.18 ± 0.1 bc 0.05 ± 0.03 bc
+EC1118
C1108 (n = 22) 0.41 ± 0.1 a 0.04 ± 0.01 0.30 ± 0.1 a 0.07 ± 0.02 a
EC1118 (n = 24) 0.21 ± 0.09 c 0.04 ± 0.02 0.13 ± 0.07 c 0.04 ± 0.02 c
OPALE (n = 24) 0.34 ± 0.1 ab 0.04 ± 0.01 0.23 ± 0.1 ab 0.06 ± 0.02 ab
OKAY (n = 24) 0.37 ± 0.1 ab 0.05 ± 0.03 0.26 ± 0.1 ab 0.06 ± 0.02 ab
QA23 (n = 23) 0.31 ± 0.1 bc 0.04 ± 0.02 0.21 ± 0.1 bc 0.06 ± 0.03 ab
Malolactic cider samples
Biodiva (n = 24) 0.30 ± 0.1 0.02 ± 0.02* 0.22 ± 0.09 b 0.06 ± 0.02
+EC1118
* * *
EC1118 (n = 24) 0.28 ± 0.07 0.02 ± 0.02 0.21 ± 0.06 b 0.06 ± 0.02*
OKAY (n = 24) 0.37 ± 0.07 0.01 ± 0.02* 0.29 ± 0.07 a* 0.07 ± 0.02
QA23 (n = 24) 0.31 ± 0.09 0.02 ± 0.02* 0.23 ± 0.06 b 0.06 ± 0.02

Results are shown as means ± standard deviations. Statistical differences between six yeasts are based on student’s t-test or oneway ANOVA with Tukey’s posthoc test
(separate yeast products); significant differences are shown with letters a–c (p < 0.05); an asterisk shows the significant differences between ciders and malolactic ciders.
36 O. Laaksonen et al. / Food Chemistry 233 (2017) 29–37
R2 = 0.904, validated R2 = 0.772, with five factors). In general, the
unripe (validated R2-values 0.897, 0.605, 0.897 and 0.757, respec-
tively) and overripe (0.803, 0.611, 0.755 and 0.908, respectively)
were better classified along the first factors of the models in com-
parison to the ripe (0.885, 0.502, 0.581 and 0.652, respectively)
within each model as the unripe correlated with the majority of
the phenolic compounds and compound sums. The correlation
was the strongest in ‘Antei’ and ‘Melba’ (Fig. 3A and C) However,
the overripe in ‘Kulikovskoye’ correlated with the free HCA vari-
ables and phloretin. Additionally, many of the other compounds,
including the most abundant compounds, the chlorogenic acid
and 4-O-p-coumaroylquinic acid, in the cultivar correlated with
both the unripe and overripe (Fig. 3B).
3.5. Impact of the yeast strain
The selection of various yeast strains of S. bayanus, S. cerevisiae
or T. delbrueckii origin had significantly less impact on the phenolic
composition than the processing in general or the apple cultivar or
its ripening stage. Nevertheless, Table 3 shows the observed differ-
ences between strains. Fermentation with S. bayanus yeasts
resulted in higher contents of free HCAs compared to S. cerevisiae
indicating more enzymes were produced to break the ester bonds
between the hydroxycinnamic acids and quinic acid. The T. del-
brueckii (Biodiva) was used together with S. cerevisiae (EC1118).
These samples were not significantly different from the samples
produced with only EC1118, suggesting low corresponding activity
of the enzymes in T. delbrueckii. In the comparison between ciders
and malolactic ciders (Table 3) only EC1118 fermentation
increased the contents of free HCAs in ciders. O. oeni used in the
malolactic fermentation do not have a similar impact on the ester
bonds. On the other hand, the significant increase of free phloretin
in malolactic ciders discussed earlier was not dependent on the
yeasts used for fermentation.
4. Conclusions
The impact of four different factors, fermentation type, apple
cultivar, apple ripening stage and yeast strain, on the phenolic
composition was studied using multivariate statistical models.
The major difference was between ciders inoculated with addi-
tional malolactic bacteria in comparison to juices or ciders pro-
duced only with yeast fermentation. Malolactic fermentation
which is typically used to ‘‘soften” the sour taste of wines and
ciders by converting malic acid to lactic acid was shown in this
study to result in increased contents of free phloretin in the corre-
sponding cider samples.
On the other hand, the apple cultivar had a very significant role
in phenolic composition highlighting and verifying the previously
known importance of cultivar selection in the cider production.
This study included local Estonian apple cultivars with potential
to be used in ciders. The ripening stage of the apple had less signif-
icant, but yet important impact on the phenolic composition. The
unripe apples contain generally higher contents of the phenolic
compounds although the effect is dependent on cultivar. Typically,
ripe or overripe apples are used in cider processing due to softened
structures thus resulting in higher juice yields and due to increase
in sugar content during apple ripening. Based on the results of this
study, the less ripe apples can be used to produce ciders with sig-
nificantly high contents of phenolics. The less ripe apples can also
be used in blends with other cultivars to increase the content of
phenolic compounds in the resulting cider product. The commer-
cial yeast products used in this study had only a small impact on
the phenolic profiles by differing in the release of free HCAs from
the corresponding quinic acid esters. However, this effect may also
result in altered flavour profile due to the potential release of vola-
tiles bound with ester bonds. All in all, the significant differences in
phenolic compositions of the juices and ciders may also contribute
to notable differences in sensory quality, such as astringency or
bitterness.
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgements
This research was supported by the Enterprise Estonia project
EU48667. Authors would also like to thank OÜ Siidrikoda for pro-
viding the apples and Lallemand Inc. for providing the yeast cul-
tures and malolactic strain. Additionally, Jukka-Pekka Suomela,
Heta Haikonen and Matilda Lintunen are thanked for their contri-
butions to the LC-DAD and LC-MS analyses.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2017.
04.067.
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