LWT - Food Science and Technology 126 (2020) 109316

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

A new approach to the use of apple pomace in cider making for the recovery T
of phenolic compounds
Débora Gonçalves Bortolinia, Laís Benvenuttib, Ivo Mottin Demiatea, Alessandro Nogueiraa,
Aline Albertia, Acácio Antonio Ferreira Zielinskib,∗
a
Graduate Program in Food Science and Technology, State University of Ponta Grossa, Av. Carlos Cavalcanti 4748, CEP 84.030-900, Ponta Grossa, PR, Brazil
b
Department of Chemical Engineering and Food Engineering, Federal University of Santa Catarina (UFSC), Florianópolis, SC, C.P. 476, 88040-900, Brazil

ARTICLE INFO ABSTRACT

Keywords: The aim of this study was to validate the process of recovery of phenolic compounds from apple pomace by
Natural phenolic extraction immobilization of the pomace during the alcoholic fermentation of ciders. The apple must (cv. Fuji) was divided
Malus domestica into two groups: Cider I (control); and Cider II (with added dried apple pomace, 47.3 g/L). The latter were
By-product fermented and monitored for 15 days in terms of physicochemical, phenolic, antioxidant, colorimetric and
Alcoholic fermentation
sensory analysis. During fermentation, Cider II produced increased levels of phenolic compounds and, conse-
Antioxidant activity
quently, increased antioxidant activity. Cider II was lighter than Cider I based on sensory and color analysis.
Sensory analysis revealed that Cider II had lower levels of sourness and higher levels of bitterness, whereas the
astringency and odor quality were similar for both ciders. Therefore, this study revealed that apple pomace
shows great promise as a source of phenolic compounds to be re-incorporated into cider.

1. Introduction
Apples (Malus domestica Borkh) are one of the main fruits cultivated
worldwide, with production around 89 million tonnes in 2016
(FAOSTAT, 2018). Approximately 35% of the total production of apples
is processed and juice and cider are the main products (Malec et al.,
2014). Cider is an alcoholic beverage (< 8.5 °GL) obtained from the
fermentation of apple must (Symoneaux, Chollet, Bauduin, Le Quéré, &
Baron, 2014).
Cider contains phenolic compounds, which confers antioxidant ac-
tivity to this beverage. Phenolic compounds have been shown to have
health benefits, reducing the risk of chronic diseases such as diabetes
(Yassin, Alberti, Zielinski, Oliveira-Emilio, & Nogueira, 2018), cancer
(Link, Balaguer, & Goel, 2010), and cardiovascular (Blumberg, Vita, &
Chen, 2015). Furthermore, phenolic compounds may modify the sen-
sory characteristics of cider (Symoneaux et al., 2014).
Cider processing generates a large quantity (about 30%) of apple
pomace. This by-product is composed of parts of the apple such as the
endocarp, mesocarp, epicarp, calyx, stem and seeds (Ferrentino,
Morozova, Mosibo, Ramezani, & Scampicchio, 2018). Based on the
phenolic composition of apples, flavanols (monomeric forms and pro-
cyanidins) represent around 60% of the phenolics found in the epicarp
and mesocarp (Alberti et al., 2017), whereas flavonols are almost
exclusively found in the epicarp (Bat et al., 2018). Apple pomace is
generally underutilized; it is usually used in landfills as fertilizer or as
animal feed (Madrera, Bedriñana, & Valles, 2015). Thus, a large
number of bioactive compounds are currently wasted.
The recovery of waste, and sustainable production, are current
trends in the food and beverage industries (Benvenutti, Zielinski, &
Ferreira, 2019). A method to valorize the bioactive compounds in apple
pomace is to use them to improve the antioxidant activity of cider. This
study examined the possibility of immersing apple pomace in the apple
must during alcoholic fermentation, as is performed in the maceration
of grape peel in winemaking. Consequently, the aims of this study were
to validate the recovery of phenolic compounds from apple pomace by
immobilizing them during the alcoholic fermentation of cider, and to
evaluate their effects on the composition and sensorial quality of cider.
2. Materials and methods
2.1. Processing of ciders
Processing of apple must: Apples of the Fuji variety were obtained
commercially in the city of Ponta Grossa, Paraná, Brazil. Then, the
apples (22 kg) were selected, washed, sanitized and processed into
apple must as described by Braga et al. (2013). The apple pomace was

∗
Corresponding author.
E-mail addresses: acacio.zielinski@ufsc.br, aczielinski@gmail.com (A.A.F. Zielinski).

https://doi.org/10.1016/j.lwt.2020.109316
Received 23 October 2019; Received in revised form 20 March 2020; Accepted 21 March 2020
Available online 26 March 2020
0023-6438/ © 2020 Elsevier Ltd. All rights reserved.
D.G. Bortolini, et al.
recovered, placed on shelves and dried in an air circulation oven (MA-
035 model, Marconi, Piracicaba, Sao Paulo, Brazil) at 60 °C. The dried
apple pomace was milled (IKA, WERKE, M20, USA) and sieved
(0.42–0.84 mm) before being added to the apple must. The proximate
composition of the apple pomace used was: Moisture: 3.00 ± 0.04 g/
100 g; Glucose: 18.4 ± 0.7 g/100 g; Fructose: 19.3 ± 0.6 g/100 g;
Sucrose: 5.5 ± 0.2 g/100 g; Nitrogen: 0.34 ± 0.01 g/100 g; Crude
fat: 1.12 ± 0.05 g/100 g; Total dietary fibre: 47 ± 2 g/100 g; In-
soluble dietary fibre 18.6 ± 0.8 g/100 g; Soluble dietary fibre:
30 ± 1 g/100 g; Ash: 1.83 ± 0.02 g/100 g.
Processing of ciders: the cider fermentation was performed in 20 glass
fermenters, in accordance with Braga et al. (2013). The apple must was
added into the fermenters and inoculated with 1.7 × 106 cell/mL of
Saccharomyces cerevisiae (Fermol Reims Champagne, Ref. PD 2002 AEB
Group, San Polo, Brescia, Italy), which had been previously rehydrated
and counted in a Neubauer chamber (CB–K-25, SMIC, China) in ac-
cordance with Bonneu, Crouzet, Urdaci, and Aigle (1991). The fer-
menters were divided into two groups: Cider I, without added apple
pomace (control); and Cider II, with added apple pomace. The im-
mobilization of apple pomace (3% moisture) was carried out in sachets
made of voil (10 × 7 cm). Glass balls were also added in the sachets to
maintain solids immersed in the liquid, in the proportion of 47.3g/L.
This quantity of dried apple pomace is obtained by processing 1 L of
apple must. The sachet with dry apple pomace was immersed in the
apple must and was kept immersed throughout the whole period of
fermentation. After fermentation period, the apparatus was removed.
The immobilization apparatus was chosen to facilitate the removal of
the pomace from fermenters, in order to do not affect the extraction of
phenolic compounds or the fermentation process. Alcoholic fermenta-
tion was carried out for 15 days at 20 ± 1 °C (BOD Labor, SP – 500,
Brazil). On days 1, 4, 7, 11 and 15 the fermentation of both groups of
cider was stopped. The ciders were centrifuged at 10,200 g at 5 °C
(Hitachi Himac CR21GII centrifuge, Tokyo, Japan) for 20 min, and the
supernatants were frozen (−18 °C) separately until further analysis. All
the experiment was performed in triplicate.
2.2. Monitoring of fermentation and characterization of ciders
Due the release of carbon dioxide, the weight loss of the system, was
monitored for 15 days. The carbon dioxide production rate (dCO2/dt)
was used to calculate a fourth-order polynomial equation.
The total nitrogen levels of the samples were determined using the
micro-Kjeldahl method; the results were expressed as mg/L, and ti-
tratable acidity was carried out in accordance with the AOAC (2005).
The pH was measured using a portable pH meter (Tecnal, Tec-3MP,
Piracicaba, São Paulo, Brazil).
The determination of the sugar and ethanol contents were per-
formed in accordance with Zielinski et al. (2014) in a HPLC system
(Waters Alliance 2695, Milford, MA, USA) coupled with a refractive
index detector (Waters RI 2414, Milford MA, USA) and Sugar Pak™
column (300 × 6.5 mm i.d.). The areas of the peaks were obtained and
compared with the standard curves (sucrose, D-glucose, D-fructose,
sorbitol, and ethanol). The results were expressed as gram per 100 mL
of must or cider.
2.3. Color analysis
Color analysis were performed using a colorimeter (Minolta CM-5,
Minolta Co. Ltd., Osaka, Japan). Firstly, the instrument was calibrated
with water. Then, the samples were placed in glass cells, and the L*, a*
and b* values were obtained using Iluminant D65 with an observation
angle of 10°. Using CIELab parameters the chroma (C*) was calculated.
The latter was used to estimate the color saturation (Equation (1)); the
hue angle (h°), which gives the tonality of the color (Equations (2) and
(3)); and the total color difference (ΔE*) between the ciders and the
apple must, which measures how one sample differs from a determined
2
LWT - Food Science and Technology 126 (2020) 109316
standard (Equation (4)). All the parameters were calculated using the
following equations:
C = (a ) 2 + (b ) 2 (1)
b b
h° = tan 1 ; If >0
a a (2)
b b
h° = tan 1 + 180°; If <0
a a (3)
E = ( L )2 + ( a ) 2 + ( b )2 (4)
2.4. Phenolic composition
The apple musts and ciders were analyzed in terms of total phenolic
compounds (TPC) using the Folin-Ciocalteu method, as developed by
Singleton and Rossi (1965), with changes. The measurement was per-
formed at 765 nm the results were expressed as milligram of 5-caf-
feoylquinic acid equivalent per liter of product (mg CAE/L). The total
flavonoid content (TFC) was quantified as described by Zhishen,
Mengcheng, and Jianming (1999), with minor changes. the absorbance
was measured at 510 nm. The results were expressed as milligram of
catechin per liter of product (mg CAT/L). The flavanols were de-
termined by the vanillin method according to Broadhurst and Jones
(1978), with slight changes. The measurements were performed at
500 nm the results were expressed as milligram of catechin per liter of
cider (mg CAT/L). All the absorbances were determined in triplicate
using a spectrophotometer (Mini-UV 1240 model, Shimadzu, Japan).
The individual phenolic compounds were determined using HPLC
system (Waters Alliance 2695, Milford, MA, USA) coupled with a PDA
2998 photodiode array detector and Symmetry C18 (4.6 × 150 mm,
3.5 mm) column (Waters, Milford, USA) in accordance with Alberti
et al. (2014). The identification and quantification of the compounds
from the samples were subsequently performed by comparing the re-
tention times and the spectra of the standards. The results were ex-
pressed as milligram per liter of apple must or cider.
2.5. In vitro antioxidant activity
The measurement of the free-radical scavenging antioxidant by
ABTS assay for the apple must and ciders was performed according to
Re et al. (1999). The samples were mixed with ABTS reagent, reacted in
the absence of light for 30 min, and then read at 734 nm. The free-
radical scavenging was also measured by DPPH assay, as described by
Brand-Williams, Cuvelier, and Berset (1995). The samples and metha-
nolic solution of DPPH were mixed in tubes and reacted for 30 min in
darkness. The absorbance reading was performed at 517 nm. The cupric
reducing antioxidant capacity was assessed by the CUPRAC method
previously described by Apak et al. (2008). Neocuproine solution was
mixed with the previously diluted samples, kept in the dark for 30 min,
and the absorbance was read at 450 nm. The ferric reducing antioxidant
power was assessed by the FRAP method, in accordance with Benzie
and Strain (1996). The samples and the FRAP reagent mixtures reacted
in the dark for 30 min. The absorbance reading was performed at
593 nm. All the antioxidant activity assays were performed in a spec-
trophotometer (Mini-UV 1240 model, Shimadzu, Japan), in triplicate,
with minor modifications from the original methods. The results were
expressed as micromol of Trolox equivalent per liter of cider or apple
must (μmol TE/L).
2.6. Sensory analysis
The final products of the ciders (after day 15 of fermentation) were
evaluated by sensory analysis, which was performed according to
Madrera, Lobo, and Alonso (2010), after approval by the Research
D.G. Bortolini, et al. LWT - Food Science and Technology 126 (2020) 109316

Ethics Committee (COEP) of the State University of Ponta Grossa
(UEPG); project number 62047516.3.0000.0105. The panel was com-
posed by of eight judges, two male and six female, who were experts in
cider and aged 23 to 41. The evaluated attributes were color, bitterness,
sourness, astringency and odor quality. Each attribute was evaluated on
different days to avoid fatigue for the judges. The judges tasted the
ciders (randomly coded), and rated them from 1 (lowest rating) to 9
(highest rating). Only the attribute of odor quality was analyzed using
the affective method. In this case the judges smelled the tubes con-
taining the randomly encoded samples and evaluated the samples using
a hedonic scale from 1 = “I disliked extremely” to 9 = “I liked ex-
tremely”.
2.7. Statistical analysis
All the results were expressed as mean ± standard deviation. The
experimental data were submitted to one-way ANOVA analysis, fol-
lowed by Fisher's LSD test or Student's t-test, to estimate the differences
in the paired samples. Difference was considered to be significant when
the samples showed values p < 0.05. All the statistical tests were
performed using Statistica v. 13.3 software (TIBCO Software Inc., Palo
Alto, CA, USA).
3. Results and discussion
3.1. Influence of the addition of apple pomace on physicochemical
properties of ciders
Fructose was the main sugar found in the apple must, followed by
glucose and sucrose, totaling 13.38 g/100 mL (Table 1). At the begin-
ning of alcoholic fermentation, the sucrose was hydrolyzed into glucose
and fructose (Table 1) by exogenous enzymes released by S. cerevisiae
until day 4 of fermentation. On the first day of fermentation an increase
in the level of sugars was observed in Cider II, which was probably
related to the diffusion of superficial sugars in the apple pomace. Be-
tween day 1 and day 4 the fructose and glucose contents started to
decrease in Cider II as a result of alcoholic fermentation. Both cider
samples showed fructose residuals between 0.3 and 0.6 g/100 mL. The
ethanol content was 1.39 g/100 mL higher in Cider II than Cider I
(Table 1). The sugars were not totally used up to produce ethanol.
According to the manufacturers (AEB Group) strains of S. cerevisiae
Fermol Reims Champagne are able to produce esters and acetates.
Caliari, Panceri, Rosier, and Bordignon-Luiz (2015) used this yeast
strain to manufacture Moscato Giallo sparkling wines and found 24
different volatile compounds. Part of the sugars contained in apple
pomace may be consumed in the production of aromas during alcoholic
fermentation.
On day 1 of alcoholic fermentation the ciders showed lower con-
centrations of sorbitol than the apple must. This may have been related
to sorbitol dehydrogenase, the enzyme that converts sorbitol in fructose
(Jia, Wong, Sweetman, Bruning, & Ford, 2015). The levels of sorbitol
were stable in Cider I; however, Cider II showed increasing sorbitol
levels after day 4 of alcoholic fermentation. According to Persic,
Mikulic-Petkovsek, Slatnar, and Veberic (2017), a portion of sorbitol
from the fruit can be retained in apple pomace. Thus, the increase in
sorbitol in Cider II may have been related to the use of apple pomace
during the fermentation of the cider. However, both ciders showed
sorbitol contents close to those reported by Antón, Valles, Hevia, and
Lobo (2014), who analyzed nine ciders and found values ranging from
0.53 to 0.89 mg/L.
The high initial nitrogen content in apple must is quickly assimi-
lated by yeasts in the growth phase, as was observed in the present
study (Table 1). Ciders usually have a low quantity of residual ni-
trogenous substances (Villar et al., 2017). As for the other parameters,
Cider I showed lower levels of nitrogen than Cider II.
During cider fermentation, organic acids are produced, decreasing
the pH and increasing the titratable acidity. Cider II had lower pH and
higher acidity than Cider I.
The use of apple pomace during the alcoholic fermentation of Cider
II promoted the increase of some nutrients such as sugars and nitrogen,
as discussed previously. This factor affects the kinetic parameters of
alcoholic fermentation. Thus, the maximum fermentation rate (dCO2/
dt)max was higher for Cider II (22.9 g/L.day) than Cider I (22.3 g/
L.day). The fermentation time was 14.8 days for both ciders, whereas
the time of the maximum fermentation rate (tvmax) was 0.25 days
earlier in relation to Cider II. The levels of CO2 released were 71.3 g/L
and 80.3 g/L for Cider I and Cider II, respectively. The higher content of
nutrients from the pomace stimulates the yeast growth and hence the
fermentation rate which can influence on cider quality (Alberti, Vieira,
Drilleau, Wosiacki, & Nogueira, 2011; Santos et al., 2015). Therefore,
the addition of apple pomace to the apple must can be a tool to avoid
sluggish fermentations due to the lack of nutrients, such as amino acids
and phytosterols.
3.2. Phenolic composition
The TPC of Cider I decreased 40% after fermentation (Table 2). Ye,
Yue, and Yuan (2014) observed a variation in phenolic content during
the fermentation of ciders made from Fuji apples; in the final process,
the majority of compounds showed lower levels than apple must, as was
observed in the present study. Variations in phenolic content can be
related to the polymerization of procyanidins, and the bioconversion of
compounds performed by yeasts (Ye et al., 2014).
The quantity of flavonoids and their main sub-class, flavanols, also

Table 1
Physical and chemical characteristics of apple must and ciders.
Days Samples Sucrose (g/100 mL) Glucose (g/100 mL) Fructose (g/100 mL) Ethanol (g/100 mL) Sorbitol (g/100 mL) Nitrogen (mg/L) pH TA (g/100 mL)

0 Must 1.90 ± 0.1a 3.1 ± 0.1b 8.4 ± 0.1b – 0.68 ± 0.01de 175 ± 0.1a 3.8 0.25 ± 0.01f

1 Cider I 0.50 ± 0.08c 2.1 ± 0.1c 6.0 ± 0.1d 1.4 ± 0.4d 0.52 ± 0.01f 36.9 ± 7.8e 3.8 0.25 ± 0.01f
4 Cider I – 0.3 ± 0.1e 3.8 ± 0.2e 6.1 ± 0.1c 0.67 ± 0.01de 38.7 ± 3.5ef 3.7 0.34 ± 0.01cd
7 Cider I – – 0.6 ± 0.1f 6.6 ± 0.6bc 0.69 ± 0.06d 34.1 ± 2.5f 3.7 0.35 ± 0.01b
11 Cider I – – 0.3 ± 0.1g 5.9 ± 0.2bc 0.64 ± 0.02e 34.1 ± 2.5ef 3.7 0.32 ± 0.04de
15 Cider I – – 0.3 ± 0.1g 7.1 ± 0.1ab 0.75 ± 0.01c 38.7 ± 0.1ef 3.7 0.37 ± 0.01b

1 Cider II 1.03 ± 0.03b 3.9 ± 0.6a 9.4 ± 1.1a 1.8 ± 0.1d 0.85 ± 0.14ab 58.1 ± 3.9bcd 3.7 0.31 ± 0.01e
4 Cider II – 0.7 ± 0.2d 6.6 ± 0.7c 6.6 ± 0.1bc 0.81 ± 0.01b 52.5 ± 2.9d 3.6 0.45 ± 0.01a
7 Cider II – – 0.6 ± 0.1fg 8.2 ± 0.1a 0.89 ± 0.01a 58.9 ± 4.9bc 3.7 0.48 ± 0.01a
11 Cider II – – 0.5 ± 0.1fg 8.0 ± 0.1a 0.87 ± 0.01a 63.6 ± 3.9b 3.7 0.47 ± 0.01a
15 Cider II – – 0.6 ± 0.1fg 8.5 ± 0.5a 0.90 ± 0.04a 58.1 ± 3.9c 3.6 0.47 ± 0.01a

Note: Results are presented as mean ± standard deviation. Different letters in same column indicate statistical difference between the samples (p < 0.05). Cider I:
without added apple pomace (control). Cider II: with added apple pomace. TA: titratable acidity.

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D.G. Bortolini, et al. LWT - Food Science and Technology 126 (2020) 109316

Table 2
Phenolic composition and in vitro antioxidant activity of apple must and ciders.
Days Samples Phenolic composition In vitro antioxidant activity

TPC (mg CAE/L) Flavonoids (mg CAT/L) Flavanols (mg CAT/L) ABTS (μmol TE/L) CUPRAC (μmol TE/L) DPPH (μmol TE/L) FRAP (μmol TE/L)

b a a a a a
0 Apple must 485 ± 22 150.8 ± 5.8 134.3 ± 5.4 693 ± 31 4399 ± 108 706 ± 69 852 ± 13d

1 Cider I 427 ± 8cd 71.7 ± 4.6f 33.5 ± 2.2d 500 ± 41bc 2854 ± 71e 645 ± 9bcde 742 ± 18de
4 Cider I 358 ± 25f 76.6 ± 6.5f 31.6 ± 1.7de 479 ± 51bc 3142 ± 339c 659 ± 23bc 826 ± 79de
7 Cider I 334 ± 13f 84.9 ± 3.8e 31.7 ± 2.4de 483 ± 12bc 3067 ± 53de 659 ± 8bc 883 ± 54d
11 Cider I 281 ± 24g 71.3 ± 3.5f 27.8 ± 0.9f 557 ± 7b 4108 ± 117c 603 ± 24f 843 ± 12d
15 Cider I 292 ± 13g 82.1 ± 2.6e 28.6 ± 1.6ef 471 ± 10c 2988 ± 169d 637 ± 26cde 820 ± 35de

1 Cider II 554 ± 24a 104.6 ± 4.1cd 72.6 ± 4.3bc 663 ± 17a 3938 ± 133b 613 ± 27ef 845 ± 7d
4 Cider II 490 ± 32b 106.8 ± 3.7c 75.5 ± 3.4b 686 ± 53a 4135 ± 404a 672 ± 15b 1202 ± 81c
7 Cider II 447 ± 14c 111.9 ± 4.5b 76.5 ± 2.6b 710 ± 12a 4488 ± 132a 672 ± 18b 1259 ± 48a
11 Cider II 416 ± 13d 106.9 ± 4.2c 70.5 ± 2.5c 696 ± 12a 4440 ± 163a 654 ± 14bcd 1364 ± 83b
15 Cider II 385 ± 35e 100.1 ± 1.6d 65.1 ± 5.2e 680 ± 9a 4389 ± 165a 668 ± 18b 1270 ± 52b

Note: Results are presented as mean ± standard deviation. Different letters in same column indicate statistical difference between the samples (p < 0.05). Cider I:
without added apple pomace (control). Cider II: with added apple pomace. TPC: total phenolic content.

reduced (45.5% and 21.3%, respectively), during the fermentation of
Cider I (Table 2). These results are in agreement with those of Alberti
et al. (2016), who observed that flavonoids were the main phenolic
class that decreased with alcoholic fermentation, mainly in terms of
flavanols, in varietal ciders.
The reduction in phenolic compounds during the fermentation
process of cider cannot be avoided; however, the content of phenolic
compounds in cider can be increased by recovering phenolic com-
pounds from apple pomace. The present study represents a new attempt
to valorize phenols from apple pomace by adding this by-product to the
apple must in the process of manufacturing cider (Cider II). The ex-
traction of phenolic compounds from apple pomace occurs by two main
mechanisms: firstly by dissolution and then by diffusion, the same
principles that are utilized in winemaking. An extraction of total phe-
nolics from the apple pomace (Cider II) was observed from day 1 of
fermentation; this contained 14% more total phenolic compounds than
the apple must.
As was observed in Cider I, Cider II also showed a decrease in the
quantity of phenolic compounds during fermentation compared with
the apple must. However, on day 15 Cider II contained 32%, 18% and
56% higher total phenols, total flavonoids and flavanols, respectively,
than Cider I.
In relation to total phenolics, the addition of apple pomace also
increased the level of individual phenolic compounds in the ciders
(Table 3). Furthermore, according to the HPLC analysis, some com-
pounds increased during fermentation and other were modified by al-
coholic fermentation, in agreement with previous studies (Madrera,
Lobo, & Valles, 2006; Ye et al., 2014).
The phloretin-2′-β-D-glucoside (phloridzin) content increased in
both ciders; the content in Cider II was three times higher than in Cider
I, which showed the influence of the addition of apple pomace
(Table 3). Phloretin-2′-β-D-glucoside shows antioxidant activity, as well
as potentially reducing the risks of diabetes and obesity (Gosch,
Halbwirth, & Stich, 2010; Yassin et al., 2018).
According to the experimental data (Table 3), 5-caffeoylquinic acid
was found in the apple must and both ciders. However, between days 7
and 11 the quantity of 5-caffeoylquinic acid began to decrease in both
ciders, probably due to the cleavage reaction, which forms caffeic and
quinic acids (Madrera et al., 2006; Ye et al., 2014). Therefore, the
quantity of caffeic acid formed during the fermentation of the ciders
may have been derived from the 5-caffeoylquinic acid. Cider II had
higher phenolic acid content than Cider I (p < 0.05) because of the
extraction of 5-caffeoylquinic acid from the apple pomace, which oc-
curred until around day 7 of alcoholic fermentation.
p-Coumaric acid was not found in the apple must but was formed
during fermentation until day 4. According to Madrera et al. (2006), p-
coumaric acid may originate from p-coumaroylquinic acid hydrolysis.
The levels of p-coumaric acid decreased from day 7 of fermentation.
However, Cider II still had higher p-coumaric acid content than Cider I
at the end of fermentation.
Cider II showed higher levels of flavonols (quercetin glycosides)
than Cider I. Apple pomace is mainly composed of the epicarp, which
contains the majority of flavonols in apples (Bat et al., 2018). Thus, the
fermentation containing apple pomace promoted the extraction of these
compounds. The consumption of flavonols has been correlated to re-
ducing the risks of developing heart diseases, as well as anti-allergic,
anti-inflammatory, antimicrobial and anti-thrombotic effects (Sultana &
Anwar, 2008). Many biochemical reactions can occur during fermen-
tation with S. cerevisiae, including glucosylation and deglucosylation,
which modify the profile of flavonoids (Huynh, Van Camp, Smagghe, &
Raes, 2014). The levels of quercetin-3-rutinoside, quercetin-3-D-ga-
lactoside, quercetin-3-β-D-glucoside and quercetin-3-O-rhamnoside
were higher in both ciders than in the apple must, whereas, quercetin-3-
D-xyloside and quercetin-O-α-L-arabinofuranoside were exclusively
extracted from the apple pomace.
The phenolic compounds are the main bioactive compounds that
provide antioxidant potential in the ciders. In the present study, the free
radical scavenging (ABTS and DPPH assays) and the ion reducing power
(CUPRAC and FRAP assays) were the methods used to determine the in
vitro antioxidant activity. According to the antioxidant tests, a reduction
in the antioxidant activity of up to 32% was verified in Cider I in re-
lation to apple must. Sorption of phenolic compounds by yeast and
bioconversion of the must phenolics are mainly responsible for this
variation in antioxidant activity (Nguela, Sieczkowski, Roi, & Vernhet,
2015). Proanthocyanidins are recognized for their high antioxidant
activity (Guyot, Le Bourvellec, Marnet, & Drilleau, 2002; Tsao, Yang,
Xie, Sockovie, & Khanizadeh, 2005), however they can be adsorbed by
yeast which justifies the reduction of phenolic compounds of the Cider I
on the first day of fermentation and consequently their antioxidant
activity (Table 2). On the other hand, the conversion of 5-caffeoylquinic
acid to caffeic acid, by enzymes, released by yeasts during fermenta-
tion, increase the antioxidant activity of the medium (Sato et al., 2011;
Ye et al., 2014). While, the esterification of caffeic acid into chlorogenic
acid decreases the antioxidant activity (Cuvelier, Richard, & Berset,
1992).
The addition of apple pomace in the must had a positive influence
on antioxidant activity in the final cider (Table 2). Cider II showed
values for antioxidant activity up to 54.9% higher than Cider I. A sig-
nificant linear correlation (r > 0.65; p < 0.01) were observed be-
tween antioxidant assays with the total flavonoids and total flavanols,
respectively. The increase in the antioxidant activity were also corre-
lated (FRAP: r > 0.8; ABTS: r > 0.7 and CUPRAC: r > 0.64) with the

4
D.G. Bortolini, et al. LWT - Food Science and Technology 126 (2020) 109316

added apple pomace. nd: not detected. PLZ: phloretin-2′-β-D-glucoside. CQA: 5-caffeoylquinic acid. CFA: caffeic acid. CA: p-coumaric acid. QRU: quercetin-3-rutinoside. QGAL: quercetin-3-D-galactoside. QGLU: quercetin-
Note: Results are presented as mean ± standard deviation. Different letters in same column indicate statistical difference between the samples (p < 0.05). Cider I: without added apple pomace (control). Cider II: with
increase of individual flavonols that were extracted from pomace.
Therefore, the antioxidant activity of the phenolic compounds is asso-

0.03fg

0.06fg
0.01h

2.57 ± 0.01d

3.27 ± 0.01b
0.03g

3.37 ± 0.01a

2.87 ± 0.01c
0.01f
0.92 ± 0.01i
ciated to its chemical structure. Quercetin glycosides are compounds

±
±
±
±
±
with high antioxidant activity as were aforementioned, and the in-

1.45 ±
0.01e
QRA

1.19
1.16
1.18
1.13
1.18
novative fermentation with pomace made the beverage with superior
antioxidant activity.

0.88 ± 0.01d
0.99 ± 0.01b
a

0.85 ± 0.01e
3.3. Color

0.90 ± 0.01c

1.34 ± 0.01
The apple musts showed lower L* values, and higher a* (from
QAF

greenness to redness) and b* (from blueness to yellowness) values, than

nd

nd
nd
nd
nd
nd

the ciders (Table 4). A natural clarification was observed during fer-
d
mentation in the ciders, which may have been related to natural reac-

2.36 ± 0.03b
2.54 ± 0.01a

2.04 ± 0.01c
1.62 ± 0.02
tions connected to alcoholic fermentation, such as the consumption of
sugars and nitrogen, as well as the retention of phenolic compounds in
the cell wall of the yeasts. Thus, the lightness (L*) values increased, and
QXI

nd

nd
nd
nd
nd
nd

nd

the a* and b* values decreased during the fermentation of the ciders.

The addition of apple pomace in the cider (Cider II) provided a higher
impact on these characteristics than in the control cider (Cider I). The
0.02h

2.13 ± 0.01d

3.10 ± 0.01b
0.03g

0.02g
0.01e

2.86 ± 0.01c
0.01f
i
1.41 ± 0.01

3.09 ± 0.01

same behavior was confirmed by the saturation index (C*), which

showed a lower value in the final product for Cider II than Cider I.
±
±
±
±
±

3.45 ±
QGLU

During the processing of apple must, the oxidation of phenolic

0.02a
1.63
1.58
1.61
1.56
1.59

compounds occurs by PPO, which forms brown pigments at the be-

ginning of the process that are responsible for the color of the apple
must (Le Deun, Van der Werf, Le Bail, Le Quéré, & Guyot, 2015). In the
0.01h

7.09 ± 0.01d
7.92 ± 0.07b
0.08g
0.01g
0.01g

9.75 ± 0.01a
4.66 ± 0.01e

c
i

0.01f

7.46 ± 0.04
0.73 ± 001

present study the apple must showed h° of approximately 75° (orange

yellow), while there was an increase in the hue angle (~90° - yellow
±
±
±
±
±
QGAL

color) during the fermentation of the ciders.

1.06
0.79
0.80
0.77
0.73

Total color difference is a way to estimate the differences in color

between two samples (Persic et al., 2017). Thus, each cider sample was
3-β-D-glucoside. QXI: quercetin-3-D-xyloside. QAF: quercetin-O-α-L-arabinofuranoside. QRA: quercetin-3-O-rhamnoside.

compared with the apple must. There were significant differences

g

0.02ef

3.89 ± 0.01d

4.49 ± 0.24b
0.01g
0.04g
0.04g

a
0.01e

4.13 ± 0.04c
4.13 ± 0.21c
2.68 ± 0.01

4.65 ± 0.03

(p < 0.01) in terms of the ΔE* values between the ciders and the apple
must (Table 4), with a higher difference for Cider II. Overall, the ad-
±
±
±
±
±
QRU

2.83
2.73
2.70
2.70
2.69

dition of apple pomace in the cider (Cider II) provided a significant

difference in the measured color parameters.

3.4. Sensory analysis

1.45 ± 0.07fg
0.06h

1.25 ± 0.04h
0.01d

1.83 ± 0.10b
0.01g

1.50 ± 0.04e
0.03c

0.04i

2.03 ± 0.07
±
±
±
±
±

According to Olejar, Fedrizzi, and Kilmartin (2016), the maceration

1.41
1.68
1.74
1.31
1.16

process can modify the bitterness, astringency, color and aroma of

CA

nd

wines due to the excessive extraction of phenols. As was observed in

terms of sensory analysis (Fig. 1), ciders did not show significant dif-
0.05h

0.01d
0.08b
0.05g

3.87 ± 0.03a
1.83 ± 0.34e

2.50 ± 0.10c

ference between them for astringency (t-test = 1.11, p = 0.29). Cider II

f
0.01i

1.52 ± 0.01

(fermented with apple pomace) was bitter (t-test = 3.74, p = 0.003),

±
±
±
±
±

which was probably related to its phenolic composition (Tables 2 and

0.51
0.70
1.42
2.23
2.70
CFA

3). The flavonols were the main sub-class of flavonoids that increased
nd

nd

after fermentation in Cider II (Table 2), which may have influenced the
perception of increased bitterness. In their study, Symoneaux, Le Quéré,
Individual phenolic compounds (mg/L)

0.11h

0.50d
0.32b
0.51g

23.59 ± 0.29a
29.37 ± 0.19e

26.85 ± 0.04c
0.01i
j
18.96 ± 0.02

Baron, Bauduin, and Chollet (2015) concluded that polyphenols con-

centration in ciders are the main factor responsible to modify the bit-
±
±
±
±
±

26.17 ±

27.48 ±

terness and astringency. Moreover, the increase of quercetin derivatives

0.2 2f
24.31
24.11
24.01
22.60
20.62

0.01 j
CQA

in Cider II could increase the taste threshold limit (Olejar et al., 2016).
Although Cider II had higher levels of titratable acidity and lower
Individual phenolic composition of ciders.

pH, its sourness was less perceived by the judges, which could have
11.51 ± 0.03d
b

15.55 ± 0.09a
13.01 ± 0.43c

been influenced by its bitterness, which may have masked this attribute
14.50 ± 0.08
0.01h
0.01g

8.34 ± 0.02e
0.11f
0.04f
0.05f
i
2.72 ± 0.01

(Fig. 1). Thus, the addition of apple pomace did not influence the
±
±
±
±
±

sourness, being the results reported by judges for Cider II lower than
4.81
5.50
5.76
5.87
5.91

control cider (t-test = −4.13, p = 0.0014). As observed in the in-

PLZ

strumental color analysis, the color of Cider II evaluated by the judges

was lighter than Cider I (t-test = −3.15, p = 0.007).
Samples

The odor quality test showed no difference (t-test = 0.61, p = 0.53)

Cider II

Cider II

Cider II

Cider II
Cider II
I
I
I
I
I
Cider
Cider
Cider
Cider
Cider
Must

between the ciders; both were evaluated between six (liked slightly)
and seven (liked moderately). Thus, the apple pomace had no negative
influence on the aroma of the ciders. Madrera et al. (2010) also eval-
Table 3

uated the odor quality of cider spirits and observed a positively corre-
Days

11
15

11
15
0

1
4
7

lation with ethyl acetate content. According to authors, ethyl acetate

5
D.G. Bortolini, et al. LWT - Food Science and Technology 126 (2020) 109316

Table 4
Color parameters of apple must and ciders.
Days Samples L* a* b* ΔE* C* h°

h a a a i
0 Apple must 65.06 ± 0.04 20.27 ± 0.08 78.04 ± 0.12 – 80.63 ± 0.12 75.44 ± 0.05

g b
1 Cider I 87.01 ± 0.08 −1.93 ± 0.02 51.89 ± 0.17 b 40.73 ± 0.16 i
51.93 ± 0.17 b
92.13 ± 0.03 h
e ef
4 Cider I 90.43 ± 0.16 −2.60 ± 0.02 32.97 ± 0.19 c 56.55 ± 0.17 g
33.07 ± 0.19 c
94.50 ± 0.06 ef
e c
7 Cider I 90.41 ± 0.01 −2.35 ± 0.02 31.43 ± 0.48 d 57.68 ± 0.39 f
31.52 ± 0.47 d
94.27 ± 0.09 f
de cd
11 Cider I 90.65 ± 0.05 −2.41 ± 0.04 29.2 ± 0.46 e 59.61 ± 0.34 e
29.31 ± 0.46 e
94.71 ± 0.03 de
d de
15 Cider I 90.82 ± 0.45 −2.55 ± 0.13 29.48 ± 0.45 e 59.52 ± 0.40 e
29.59 ± 0.44 e
94.9 ± 0.42 d

f
1 Cider II 88.14 ± 0.13 −2.82 ± 0.03 gh 50.96 ± 0.14 b
42.42 ± 0.16 h
51.04 ± 0.14 b
93.17 ± 0.04 g

c
4 Cider II 92.25 ± 0.13 −2.95 ± 0.06 h 26.37 ± 0.22 f
62.84 ± 0.14 d
26.53 ± 0.22 f
96.38 ± 0.07 c

b
7 Cider II 92.97 ± 0.11 −2.64 ± 0.13 efg 22.84 ± 0.29 g
65.97 ± 0.23 c
22.99 ± 0.31 g
96.59 ± 0.25 c

a
11 Cider II 93.24 ± 0.16 −2.76 ± 0.014 fg 21.74 ± 0.34 h
67.04 ± 0.18 a
21.91 ± 0.35 h
97.23 ± 0.25 b

a
15 Cider II 93.40 ± 0.07 −3.31 ± 0.27 i 22.83 ± 0.85 g
66.39 ± 0.63 bc
23.07 ± 0.88 g
98.25 ± 0.36 a

Note: Results are presented as mean ± standard deviation. Different letters in same column indicate statistical difference between the samples (p < 0.05). Cider I:
without added apple pomace (control). Cider II: with added apple pomace. L: lightness. ΔE*: total color difference. C*: Chroma. h°: hue angle.

Investigation, Writing - original draft. Ivo Mottin Demiate:

Methodology, Investigation, Writing - review & editing. Alessandro
Nogueira: Conceptualization, Writing - review & editing, Project ad-
ministration. Aline Alberti: Conceptualization, Supervision, Writing -
review & editing. Acácio Antonio Ferreira Zielinski: Formal analysis,
Supervision, Project administration, Writing - review & editing.

Acknowledgments

The authors are grateful to CAPES for financial support.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://

doi.org/10.1016/j.lwt.2020.109316.

Fig. 1. Sensory analysis of ciders with and without added apple pomace.
Note: Cider I: without added apple pomace (control). Cider II: with added apple
pomace. From 1 (lowest rating) to 9 (highest rating). *from 1 = “I disliked
extremely” to 9 = “I liked extremely”.
shows glue-like aroma in high concentrations and fruity perception in
low concentrations. Although there was an increase of phenolic com-
pounds in the Cider II, aroma compounds show a complex synthesis
during the alcoholic fermentation. As reported by Santos et al. (2016),
amino acids are the main precursors of volatile compounds. However,
yeasts strains, organic acids, esters, and acetates also contribute to cider
flavor (Xu, Fan, & Qian, 2007).
4. Conclusion
The addition of apple pomace immobilized during cider fermentation
showed to be a low-cost alternative that resulted in increased levels of
phenolic antioxidants, as well as representing the potential valorization of
an agroindustrial by-product. In addition, the ciders fermented with apple
pomace were lighter than those that contained no apple pomace, as well as
showing lower levels of sourness and higher levels of bitterness than the
control samples. The astringency and odor quality were evaluated as being
similar for both ciders. Overall, the addition of apple pomace during cider
fermentation represents an excellent opportunity to recover phenolic
compounds from apple pomace, improving the phenolic composition,
antioxidant activity and sensorial parameters of cider.
CRediT authorship contribution statement
Débora Gonçalves Bortolini: Methodology, Investigation,
Visualization, Writing - original draft. Laís Benvenutti: Methodology,
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