Food Chemistry 300 (2019) 125130

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

Effect of reduced glutathione on the quality characteristics of apple wine during T

alcoholic fermentation
Junnan Xu, Yiman Qi, Jie Zhang, Miaomiao Liu, Xinyuan Wei, Mingtao Fan
College of Food Science and Engineering, Northwest A&F University, Yangling 712100, Shaanxi, China

ARTICLE INFO ABSTRACT

Keywords: To investigate the effect of reduced glutathione (GSH) on the quality characteristics of apple wine, 10, 20 and 30 mg/L of
Reduced glutathione GSH were added to apple juice before alcoholic fermentation. Meanwhile, apple wine fermented by Saccharomyces cerevisiae
Apple wine which had been pre-incubated with GSH (100 mg/L) was another experimental group. Mono-phenols, GSH and oxidized
Antioxidant glutathione (GSSG) were determined by HPLC. Aroma compounds were analysed by GC–MS. Further, E-nose was applied to
Phenolic compounds
monitor the odor. After fermentation, GSH content was the same in all of the samples. However, for the apple wine with GSH
Volatile aroma compounds
addition, GSSG content increased significantly. Notably, GSH could reduce the color index, protect chlorogenic acid and
phloretin, decrease the content of epicatechin and catechin as well as change the profile of aroma compounds (higher levels of
2-methyl-1-pro-panol, 3-methyl-1-butanol, ethyl benzoate, linalool, etc.). GSH may be used for flavor enhancement and
quality improvement of apple wine.

1. Introduction even replace SO2.

In recent years, apple wine consumption has been increasing and become
a worldwide popular drink, second only to grape wine among wines
consumed. Apple wine combines the advantages of wine and fruit juice, and it
has low alcohol content between 1.2 and 8.5% v/v as well as the mellow taste
(Laaksonen, Kuldjärv, Paalme, Virkki, & Yang, 2017). Besides, abundant
nutrients such as polyphenols, minerals, or-ganic acids and amino acids
(Picinelli Lobo, García, Sánchez, Madrera,
& Valles, 2009; Ye, Yue, & Yuan, 2014a,b) are also the chief reasons why it
is so popular. Because of the high yield and plentiful varieties of apples in the
world, apple wine production continues to be an im-portant and promising
segment of the fruit industry. However, although the apple wine brewing
technology has been relatively mature, there are still some problems to be
solved such as juice oxidation, aroma loss, browning and sulfur dioxide (SO 2)
residue. In order to solve these problems, winemakers have extensively
examined the process of fruit wine brewing. But up to now, the main
preservative utilized in wine to prevent oxidative spoilage and inhibit the
growth of miscellaneous bacteria is SO2 (Sonni, Clark, Prenzler, Riponi, &
Scollary, 2011). De-spite the fact that SO 2 has the remarkable effects and low
cost, its use in wine must be reduced because it has negative impacts on
people's health such as toxicity and allergy (Arapitsas, Guella, & Mattivi,
2018). Therefore, it is urgent to get an additive to reduce SO 2 consumption or
GSH is a nucleophilic, thiol-containing tripeptide composed of glutamic
acid, cysteine and glycine and it is naturally present in most eukaryotes and
many prokaryotic species at concentrations of 0.2–10 mM (Anderson, 1998).
Generally, glutathione exists in cells as reduced form of GSH and in oxidized
form as GSSG. Under natural conditions, more than 90% glutathione is
present in reduced form (Kritzinger, Bauer, & Du Toit, 2013). The difference
between GSH and GSSG is significant because only the reduced form
possesses physiolo-gical activity and the thiol group of GSH is the active site
responsible for its biochemical properties (Kritzinger et al., 2013).
GSH serves many crucial functions including antioxidant activity,
detoxification and influencing the metabolism of sulfur and nitrogen, etc
(Bachhawat & Kaur, 2017). Recently, the treatment of musts and wines with
GSH has been approved by the International Organization of Vine and Wine
(OIV), which provides a scientific guidance for the production of high quality
fruit wine (Antoce, Badea, & Cojocaru, 2016). Based on this, adding GSH to
apple wine may play a role in protecting phenols, inhibiting browning and
improving flavor owing to its oxidation resistance.
Polyphenols are one of the most important quality parameters of wine
because its type, structure and contents are closely related to color, taste and
aroma of wine (Ye, Yue, & Yuan, 2014b). Certain phenols, such as caffeic
acid, catechin and epicatechin, can lead to

Corresponding author.
E-mail address: fanmt@nwsuaf.edu.cn (M. Fan).
https://doi.org/10.1016/j.foodchem.2019.125130
Received 13 November 2018; Received in revised form 3 July 2019; Accepted 3 July 2019
Available online 04 July 2019
0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
J. Xu, et al. browning of wine through an enzymatic or non-enzymatic pathway
(Fernández-Zurbano et al., 1995; Singleton, Salgues, Zaya, & Trousdale,
1985). GSH is known to play a crucial role in the limitation of browning Food Chemistry 300 (2019) 125130
during winemaking as it can react with o-quinones resulting from the
oxidation of phenolic substances, generating 2-S-glutathionyl caftaric acid, At the end of fermentation, the physicochemical parameters of apple wine
also known as Grape Reaction Product (GRP) (Rodríguez-Bencomo, were shown in the supplementary table 2. All samples were centrifuged at 4
Andújar-Ortiz, Sánchez-Patán, Moreno-Arribas, & Pozo-Bayón, 2016). °C, 2795×g for 10 min and then stored at −20 °C until analysis. The study
Previous research has shown that during a three year bottling storage of dry was divided into five groups with three repeats.
white wine, the addition of 10 mg/L GSH sig-nificantly decreased the yellow
tint compared to the control wine (Ye, Yue, & Yuan, 2014a).
2.3. Quantification of GSH and GSSG
Higher alcohols, esters, fatty acids and carbonyls have been iden-tified as
important contributors to the aroma of apple wine and many of them are
GSH concentrations were monitored in apple wine at the end of alcoholic
mainly produced during the alcoholic fermentation as the primary or
fermentation. Analyses were carried out by High Performance Liquid
secondary metabolites of yeast (Ye et al., 2014a). GSH can affect the
Chromatography (HPLC; LC-20A, Shimadzu, Japan) described by Webber et
metabolism of yeast because of its detoxification and an es-sential role in the
al (Webber et al., 2017) with some modifications. The separation was
sulfur and nitrogen metabolism of cells (Wegmann-Herr, Ullrich, Schmarr, &
performed on a ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm, Agilent,
Durner, 2016). The effects of GSH on some aroma compounds may be
USA) with 0.05 mol/L sodium acetate buffer (pH = 5.6) as solvent A and
ascribed to either its free sulfhydryl (–SH) moiety, which confers unique
methanol as solvent B (A:B = 9:1). The qualitative and quantitative
redox and nucleophilic properties or the regulatory role in the metabolism of
characterization of GSH was based on the retention time and the standard
yeast (Roussis, Papadopoulou, & Sakarellos-Daitsiotis, 2010; Wegmann-Herr
curve, respectively. The determi-nation of GSSG was based on the analytical
et al., 2016). However, the exact mechanism of how GSH affects aroma
method of GSH. Briefly, 150 μL DL-Dithiothreitol (DTT, 25 mmol/L) was
compounds is yet to be confirmed.
added to 150 μL wine sample. Then, the mixed sample was stationary at 4 °C
for 5 min so that GSSG could be reduced to GSH. Finally, the qualitative and
Recently, studies about the effects of GSH addition are mainly re-lated to
quantitative method of GSH was implemented.
the red (white) grape wine during the storage period, but little attention has
been paid to the effects of GSH on apple wine. Therefore, the study aimed to
evaluate the influence of GSH addition and in-oculating S. cerevisiae pre-
incubated with GSH on apple wine char-acteristics during alcoholic
2.4. Determination of total phenolic compounds and color index
fermentation. As far as the writer knows, it is the first time to use E-nose
systems to monitor the effects of GSH on the odorant characteristics of apple
The total phenolic compounds were determined by the method of Folin-
wine. Additionally, the quality parameters including GSH content, color
Ciocalteu (Kwaw et al., 2017) with some modifications. Briefly, 0.5 mL apple
index, phenolics and volatile compounds profiles were also determined and
wine samples were appropriately diluted and added to the colorimetrical
assessed.
cylinder. Then, 2 mL distilled water, 0.5 mL Folin-Cio-calteu and 1.5 mL
7.5% sodium carbonate solution were added to the same colorimetrical
2. Materials and methods
cylinder in order. The mixture was kept at 20 °C for 2 h for reaction; the
absorbance of samples was measured at 765 nm. The concentrations of total
2.1. Experimental materials and reagents
phenolic compounds were calculated by the absorbance and the
corresponding standard curve and the results were expressed as milligrams of
Fuji apples were purchased from the market of Yangling (Shaanxi,
gallic acid (GAE) per liter of apple wine.
China). S. cerevisiae (WLP775) was preserved in the Lab of Food
The color index of samples was determined by the following method
Microbiology and Food Biotechnology of Northwest A&F University. All
(Webber et al., 2017) with some modifications. 3 mL apple wine sample and 6
chemicals and reagents used were HPLC grade and obtained from Solarbio
mL anhydrous ethanol were mixed. After centrifugation (2795×g, 4 °C, 10
Chemical Company, Ltd (Beijing, China).
min), the color index of supernatant was measured by UV spectrophotometer
at a wavelength of 420 nm.
2.2. Apple wine elaboration and experimental design

After cleaning, Fuji apples (approximately 60 kg) were squeezed by a

laboratory beating apparatus (HausElec, Qingdao, China) and the apple juice
was filtrated through gauze. Then, the apple juice (total soluble solid (TSS)
content, 14 °Brix; pH value, 3.85; GSH content, 24.61 mg/L; GSSG content,
3.73 mg/L) which had reached the labora-tory standard was collected and
transferred to 15 glass fermentation cylinders with the loading volume of 2 L.
Meanwhile, 60 mg/L SO2 was added to each of the glass fermentation
cylinders. Before alcoholic fermentation, GSH was added to the apple juice at
variable con-centrations (10, 20 and 30 mg/L) according to the experimental
design (supplementary table 1) and the S. cerevisiae (WLP775) was added
immediately. Another group was no GSH addition but inoculated the S.
cerevisiae with pre-incubation of GSH (GSH-WLP775, 100 mg/L GSH was
added to the liquid culture medium; cultured time: 24 h). The in-oculation
quantity of S. cerevisiae was 2% volume of apple juice. Fer-mentation
temperature was maintained at 20 °C and fermentation time was 7 days. In the
alcoholic fermentation process of apple wine, the changes of TSS content
(supplementary Fig. 1) were monitored so as to reflect the trend of
fermentation.
2.5. Analysis of phenolic compounds
Analysis of phenolic compounds using HPLC (LC-20A, Shimadzu,
Japan) was carried out with reference to literature (Webber et al., 2014). The
phenolic compounds were extracted from the wine samples using ethyl
acetate. After adjusting the pH to 7.0 with 0.5 M NaOH, 15 mL samples were
extracted with 20 mL ethyl acetate and repeated three times. Then, the pH of
above samples was adjusted to 2.0 with 1.0 M HCl and repeated extraction for
three times. The ethyl acetate extracts was mixed and rotationally evaporated.
Finally, the con-centrated liquid was dissolved in methanol and fixed volume
to 5 mL volumetric flasks. All samples were filtered with membrane of ester
mixture 0.45 μm, and 20 μL was injected to the chromatograph. HPLC
conditions: chromatographic column was ZORBAX SB-C18 column (4.6 ×
250 mm, 5 μm, Agilent, USA) and the column temperature was maintained at
30 °C. The wavelength of ultraviolet detector were 280 nm and 320 nm, and
the gradient elution with a flow rate at 0.8 mL/min was performed by mobile
phase A (2% acetic acid in deionized water) and solvent B (methanol; 0–10
min, 5–30%; 10–35 min, 30–50%; 35–40 min, 50–60%; 40–45 min, 60–
70%; 45–50 min, 70–5%; 50–55 min, 5%). The qualitation and quantification
of mono-phenols was based on the retention time and the standard curve,
respectively.

2
J. Xu, et al.
2.6. Analysis of aroma components
The aroma compounds were determined using headspace solid-phase
microextraction coupled with gas chromatography-mass spec-trometry (HS-
SPME-GC–MS, QP2010, Shimadzu, Japan) (Cai et al., 2014). Briefly, 5 mL
of apple wine was placed into a 20 mL headspace bottle and 2-octanol was
employed as the internal standard (6 μL of a 0.45 mg/mL solution in
methanol). Then, 1.5 g sodium chloride was added in order to inhibit
enzymatic degradation and facilitate the evolution of volatiles into the
headspace. Headspace bottles were equilibrated for 15 min at 40 °C in the
water bath and then a 50/30 μm DVB/CAR/PDMS fiber was inserted into the
headspace bottle (22 mm depth) for 10 min at 40 °C to collect the volatiles.
The volatiles of samples were separated using a DB-17MS chroma-
tographic column (60 m × 0.25 mm × 0.25 μm; Agilent, USA). Conditions for
GC–MS analysis were as follows: The temperature of injector, interface and
ion source was 250 °C, 230 °C and 230 °C re-spectively. The flow rate of
carrier gas (helium) was 1.0 mL/min and the oven temperature was held at 40
°C maintained for 3 min, then ramped at a rate of 4 °C/min to 120 °C, and
ramped up to 240 °C at a rate of 6 °C/min (the final temperature maintained
for 12 min). The data was collected at a scanning range of 35–500 amu
(atomic mass unit).
Identification was based on retention indices of reference standards and
mass spectra matching in the standard NIST 14 library. Quantification was in
accordance with the specific relation between peak areas and concentrations
of volatile compounds and the internal standard.
2.7. E-nose analysis
E-nose analysis of odor was carried out according to the previous report
with certain modifications (Xu, Yu, Liu, & Zhang, 2016). The PEN3 portable
E-nose (Win Muster Airsense Analytics Inc., Schwerin, Germany) equipped
with 10 metal oxide gas sensors (MOS sensors) based on different sensing
materials was used and the general de-scription of 10 sensors was shown in
Table 1. E-nose was calibrated with acetone, isopropanol and propanol
following the instruction of the machine before testing. Each sample of 1.0
mL was diluted to 10 mL and placed into a 30 mL glass vial with a
Teflon/silicone septum in the screw cap. The sample was equilibrated for 5
min to allow for head-space enrichment before analysis. And then, the
measurement phase lasted for 60 s, which was long enough for the sensors to
reach stable signal values. After each experiment, cleaning gas was pumped
into the sample gas path for 300 s, which was long enough to normalize
sensor signals. The sensor intensity is defined as G/G0, where G0 and G are
the resistance of the sensor in zero gas and sample gas, respectively.
2.8. Statistical analysis
All the samples were analyzed in triplicate. The principal
Table 1
Performance description of 10 sensors in PEN3 E-nose.
Array number Sensor name
1 W1C
2 W5S
3 W3C
4 W6S
5 W5C
6 W1S
7 W1W
8 W2S
9 W2W
10 W3S
3
Food Chemistry 300 (2019) 125130
Fig. 1. GSH and GSSH concentrations of apple wine with different treatments. CK –
control; GSH-10 – adding 10 mg/L GSH to apple juice; GSH-20 – adding 20 mg/L GSH
to apple juice; GSH-30 – adding 30 mg/L GSH to apple juice; GSH-100Y – inoculating
S. cerevisiae pre-incubated with 100 mg/L GSH. Different letters at the different
treatments indicate significant differences ac-cording to Duncan test (p < 0.05).
component analysis (PCA) and one way analysis of variance (ANOVA) using
the Duncan test at significance level p < 0.05 were performed to compare the
differences between 4 experimental groups and the control using SPSS 20.0
(Chicago, IL, USA).
3. Results and discussion
3.1. GSH content in apple wine
There were three main sources of GSH in apple wine: apple juice,
metabolism of S. cerevisiae and exogenous addition (Kritzinger et al., 2013).
As shown in Fig. 1, there was no significant difference in GSH content among
all apple wine samples. This result was similar to those found in the white
wines or sparkling wines (El Hosry, Auezova, Sakr, & Hajj-Moussa, 2009;
Webber et al., 2014).
For all apple wine samples, the content of GSSG was signi ficantly higher
than that of GSH, especially in the apple wine with exogenous GSH addition.
This might be attributed to the oxidation of GSH in the process of apple wine
brewing. The oxidation of GSH took place under condition of oxygen or
glutathione peroxidase (Gpo) existence to gen-erate GSSG (Margalef-Català
et al., 2016). According to Fracassetti (Fracassetti, Coetzee, Vanzo, Ballabio,
& Du Toit, 2013), GSH alone was not an effective oxygen consumption
accelerator so that small amounts of GSH addition could not reduce the level
of dissolved oxygen of wine and be eventually oxidized to GSSG. Besides,
GSH might have been lost by forming additional products with o-quinones
and carbonyl com-pounds (Sonni, Clark, et al., 2011; Sonni, Moore, et al.,
2011).
There was no significant difference in content of GSH between the
Performance description
Sensitive to aromatic compounds
Very sensitive, broad range, react on nitrogen oxides
Sensitive to aromatic compounds of ammonia
Main hydrogen, selectively
Alkanes, aromatic compounds, less polar compounds
Sensitive to methane, broad range, similar to No. 8
Reacts on many terpenes, organic sulfides and inorganic sulfide (H2S)
Detects alcohols, partially aromatic compounds, broad range
Sensitive to aromatic compounds, sulphur organic compounds
Sensitive to alkane (methane)
J. Xu, et al.
GSH-100Y and others (control, GSH-10, GSH-20 and GSH-30), but the
GSSG content was significantly higher than that of control. S. cerevisiae is the
promoter for the alcoholic fermentation and it can possibly alter the final GSH
level of apple wine by utilizing and secreting GSH during the metabolism
process (Penninckx, 2002). It is also suggested that as an intracellular
compound, GSH can be released by yeast autolysis at the end of fermentation
(Kritzinger et al., 2013). It might be true that the culture medium containing
100 mg/L GSH changed the metabolism of S. cerevisiae to some extent
(Kritzinger, 2012) as well as enhanced its ability to secrete GSH which was
oxidized to GSSG eventually.
3.2. Effect of GSH on total phenolic content and color index
Total phenolic content was shown in Fig. 2 A. Apple wine with GSH
addition had higher total phenolic content no matter what the additive content
was. Inoculating S. cerevisiae pre-incubated with 100 mg/L GSH did not
influence the total phenolic content compared with control. The results were
in agreement with that in Trebbiano and Bombino Bianco wines (Fragasso,
Antonacci, & Pati, 2010). There are two kinds of oxidation of phenolic
compounds in apple wine making processes: en-zymatic oxidation and
chemical oxidation (non-enzymatic oxidation). During apples crushing,
catalyzed by polyphenol oxidase (PPO), phe-nolic compounds could undergo
enzymatic oxidation reactions. During alcoholic fermentation, chemical
oxidation may occur for phenolic compounds (Kritzinger et al., 2013; Webber
et al., 2014). For the former type of oxidation, GSH addition hardly inhibited
the oxidation of polyphenols because the enzymatic oxidation had already
occurred before the addition of GSH. However, GSH could react to the
quinones generated from the enzymatic oxidation of polyphenols, which
could inhibit the browning of apple wine (Singleton, Zaya, Trousdale, &
Salgues, 1984). For the latter, GSH favored a reduced environment so that the
chemistry oxidation could be inhibited to a great extent (Webber et al., 2014).
This might be the main reason why GSH addition could increase the total
phenolic content.
However, there was an interesting phenomenon that the total phe-nolic
content of GSH-30 was less than GSH-20. The reason might be that some
phenols such as p-coumaric acid and ferulic acid, whose content decreased
significantly with the addition of 30 mg/L GSH (Table 2) could form the
adducts with excessive GSH. However, this is still a hypothesis that needs to
be confirmed by further research.
As shown in Fig. 2B, the addition of 10, 20 and 30 mg/L GSH sig-
nificantly decreased the color index of apple wine. The mechanism of GSH in
preventing browning primarily relates to its reactivity with o-quinones which
are the products of the oxidation of phenolic com-pounds (Rodríguez-
Bencomo et al., 2016). The unstable chemical properties of o-quinones lead to
polymerization of o-quinones between each other or the polymerization
between o-quinones and proteins, amino acid as well as phenolics, etc (Wu,
2017). These polymerization reactions with o-quinones can generate dark
polymers and result in browning of apple wine. According to the previous
study, GSH can react with o-quinones and generate the GRP which is not
brown and not a substrate for further oxidation by PPO (Singleton et al.,
1984). In this
4
Food Chemistry 300 (2019) 125130
Fig. 2. Total phenolic content (A) and color index (B) of
apple wine with different treatments. CK – control; GSH-
10 – adding 10 mg/LGSH to apple juice; GSH-20 –
adding 20 mg/L GSH to apple juice; GSH-30 – adding 30
mg/L GSH to apple juice; GSH-100Y – inoculating S.
cerevisiae pre-incubated with 100 mg/L GSH. Different
letters at the different treatments indicate significant
differences ac-cording to Duncan test (p < 0.05).
way, GSH can limits the formation of brown polymers. Inoculating S.
cerevisiae pre-incubated with GSH also inhibited the browning of apple wine
during alcoholic fermentation. The reason might be that GSH adsorbed on
surface of yeast was dissolved into the apple wine. What is more,
extracellular GSH can be absorbed by S. cerevisiae as an internal sulphur
source (Kritzinger, 2012). GSH also plays a role in defense against oxidative
stress and metal toxicity, which is conducive to the metabolism and
reproduction of S. cerevisiae (Perez, Sousa, Vankeersbilck, Machado, &
Soares, 2013). Therefore, the ability of S. cerevisiae to secrete GSH might be
enhanced and the significant increase (compared with the control) for GSSG
content in GSH-100Y wine could also corroborate this point indirectly.
3.3. Influence of GSH addition on mono-phenols
Ten phenolic compounds were identified and quantified in the apple wine
samples (Table 2). In this study, epicatechin, caffeic acid, chlorogenic acid
and catechin were relatively most abundant in Fuji apple wine. The result was
similar to previous studies which indicated that the content of chlorogenic
acid, caffeic acid and catechin were relatively high when compared with the
p-coumaric acid, ferulic acid and protocatechuic acid, etc. (Ye et al., 2014b).
Catechin and epicatechin belong to flavan-3-ols which are major
phenolics in apple juice and wine (Ramírez-Ambrosi et al., 2015; Xiang,
Apea-Bah, Ndolo, Katundu, & Beta, 2019). GSH addition and pre-in-cubated
S. cerevisiae significantly reduced the content of catechin. As for the content
of epicatechin, only the 20 mg/L and 30 mg/L GSH addition led to significant
decrease, but there was no significant dif-ference among the CK, GSH-10 and
GSH-100Y apple wines. Despite the fact that flavan-3-ols have a positive
effect on the taste of apple wine and they are capable of controlling
microbiological spoilage, lower content of flavan-3-ols also seems to be an
advantage in terms of the stability of apple wine. Because flavan-3-ols dimers
are involved in the formation of haze which can limit the shelf life of wine
(Ausse, Eudec, & Heynier, 2008). The content of caffeic acid showed the
same trend with catechin and their decrease might be attributed to the same
reason that catechin and caffeic acid can form adducts with GSH called glu-
tathionyl-catechin and glutathionyl-caffeic acid respectively (Sonni, Clark, et
al. 2011).
The content of p-coumaric acid and ferulic acid also decreased to some
extent with GSH addition. In addition to the reasons discussed in 3.2, there
might be special relations between their consumption and the increase of
GSSG level. Further research needs to be done in the future.
For chlorogenic acid and phloretin, GSH addition improved their content
to a large extent. Some studies revealed that chlorogenic acid was main
hydroxycinnamic acid found in apple products and it showed a significant
correlation (p < 0.001) with all antioxidant assays (r ≥ 0.58) (Zielinski,
Alberti, Viviana, Alvarez, & Nogueira, 2017). Phloretin, a natural
polyphenolic compound found in apples and pears, had been proven to
possess the functions of antioxidation and scaven-ging free radicals (Yang et
al., 2009). The thiol group of cysteine en-dows GSH with redox catalytic
properties which could protect the
J. Xu, et al. Food Chemistry 300 (2019) 125130

Table 2
Mono-phenols content of apple wine with different treatments.

Phenolics (mg/L) Experiments

CK GSH-10 GSH-20 GSH-30 GSH-100Y

Gallic acid 0.11a ± 0.05 0.12a ± 0.02 0.11a ± 0.04 0.12a ± 0.06 0.12a ± 0.10
Protocatechuic acid 0.80a ± 0.08 0.81a ± 0.05 0.81a ± 0.04 0.83a ± 0.04 0.88a ± 0.03
Catechin 8.13a ± 0.32 7.63b ± 0.31 6.23c ± 0.29 6.52c ± 0.25 7.47b ± 0.42
Epicatechin 24.44a ± 0.61 23.25a ± 0.30 21.47b ± 1.44 20.96b ± 0.66 23.40a ± 0.46
Phloridzin 1.56a ± 0.14 1.43a ± 0.07 1.33a ± 0.12 1.32a ± 0.191 1.40a ± 0.10
Chlorogenic acid 14.43c ± 0.70 15.71bc ± 0.76 17.24ab ± 1.22 18.25a ± 0.78 16.10b ± 0.57
Phloretin 4.85b ± 0.24 6.35a ± 0.63 6.55a ± 0.19 6.72a ± 0.28 4.42b ± 0.34
Caffeic acid 20.72a ± 0.42 17.80b ± 0.50 15.72bc ± 1.24 15.81c ± 0.37 19.86a ± 0.75
p-Coumaric acid 0.78a ± 0.02 0.68b ± 0.01 0.70b ± 0.03 0.54c ± 0.01 0.44d ± 0.02
Ferulic acid 1.71a ± 0.03 1.55ab ± 0.15 1.60a ± 0.17 1.37b ± 0.06 1.16c ± 0.01

CK – control; GSH-10 – adding 10 mg/L GSH to apple juice; GSH-20 – adding 20 mg/L GSH to apple juice; GSH-30 – adding 30 mg/L GSH to apple juice; GSH-100Y – inoculating
S. cerevisiae pre-incubated with 100 mg/L GSH. Values are means ± standard deviation (SD). Different letters in the same row means significant differences according to Duncan test (p
< 0.05).

chlorogenic acid and phloretin from oxidation to a great degree (Gaucher et
al., 2018). However, changes in the content of catechin, epicatechin and
caffeic acid could not be explained by the antioxidant activity of GSH simply.
The concentrations of gallic acid, protocatechuic acid, and phlor-idzin
showed no difference between the samples with or without GSH addition.
Inoculating S. cerevisiae pre-incubated with GSH also had no effect on
content of gallic acid and protocatechuic acid, while the phloridzin content
was significantly decreased.
3.4. Effects of GSH on aroma compounds
According to GC–MS results shown in Table 3, the main aroma
components of apple wine were alcohols, esters, acids, terpenes and other
volatile compounds. Significant increase was found in the con-tents of 2-
methyl-1-propanol, 3-methyl-1-butanol, 1-heptanol and phenylethyl alcohol
among the apple wine with different levels of GSH addition. 3-methyl-1-
butanol was the most abundant alcohols with the content ranging from 600 to
1200 μg/L. The formation of higher alco-hols seems to be influenced by the
redox status of the yeast cell. In the reductive state, aldehydes can be reduced
by NADH to the corre-sponding alcohols; as for the oxidative state, they can
+
be oxidized by NAD to volatile carboxylic acids (Webber et al., 2014). It
should be noted that GSH favors the reduction state of yeast cell. The
formation of higher alcohols can be achieved in another pathway called the
Ehrlich reaction in which amino acids are catabolized into specific higher al-
cohols. For example, valine converted to 2-methyl-1-propanol and leucine
converted to 3-methyl-1-butanol and phenylalanine translated into
phenylethyl alcohol (Styger, Prior, & Bauer, 2011).
Esters are one of the most important volatile constituents in the apple
wine. Although some of the esters are originally present in apple juice, most
of them are formed from the esterification process and secondary yeast
metabolism during alcoholic fermentation (Cai et al., 2014; Ye et al., 2014a).
In this study, esters, particularly ethyl capry-late, ethyl hexanoate, ethyl
caprate and isoamyl acetate were the pre-dominant free volatile compounds
accounting for 70–80% of total free volatile compounds. Ethyl benzoate, n-
butyl acetate and ethyl 9-de-cenoate positively contribute to wine quality by
imparting fruity ar-omas, their contents increased with GSH addition. Further,
most of other esters showed lower content with the GSH addition, which was
different from the previous research results that GSH could inhibit the
decrease of volatile esters during the storage of white wine (Papadopoulou &
Roussis, 2008). The difference may be ascribed to that GSH prevented the
acetaldehyde from being oxidized to the cor-responding carboxylic acids, so
that the esterification of alcohols and acids could be inhibited. However, in
the storage period of wine, most esters had already formed and the antioxidant
capacity of GSH
protected them efficiently.
Three kinds of volatile acids (acetic acid, 2-methyl butyric acid, decanoic
acid) were detected from the Fuji apple wine. Volatile acids can contribute to
the complexity of the wine bouquet even if present at low concentration,
while they have negative effects on wine aroma when above their thresholds
(Cai et al., 2014). Acetic acid has a pun-gent odor, and its threshold is about
200 mg/L. At low concentration, 2-methyl butyric acid has fruity aroma, but
at high level, the aroma be-comes an unpleasant pungent smell of goat cheese
(Styger et al., 2011). No matter how much GSH was added, apple wine
possessed a lower content of acetic acid and 2-methyl butyric acid than the
control wine and inoculating S. cerevisiae pre-incubated with 100 mg/L GSH
had the same effect. However, for the concentration of decanoic acid, GSH
addition made it higher than the control wine.
Terpenes mainly come from fruits and greatly contribute to ‘‘var-ietal
aroma’’ for fruit wines. With relative low thresholds, many ter-penes
contribute to the floral aroma of wine directly or through sy-nergistic effects
(Marais, 1983). According to the previous report, citronellol, geraniol and
linalool impart rose, citric/geranium and floral fragrance respectively (Cai et
al., 2014). The addition of GSH sig-nificantly increased the content of
citronellol, geraniol and linalool for the reason that most terpene alcohols are
replaced by corresponding terpene oxides during fermentation, whereas
linalool may be converted to α-terpineol, which has a much higher sensory
threshold (Roussis et al., 2010). The protection role of GSH on terpenes was
attributed to its thiol group, which confers unique redox and nucleophilic
properties (Kritzinger et al., 2013). GSH-100Y apple wine showed higher
levels of citronellol and geraniol, but there was no difference from the control
in the content of linalool.
Acetaldehyde is also an important aroma compound formed from the
primary metabolism of yeast during the fermentation or formed by the
oxidation of ethanol and it constitutes more than 90% of the total aldehyde
content of fruit wine (Styger et al., 2011; Webber et al., 2014). At low levels,
this compound imparts a pleasant fruity aroma to fruit wine, while this turns
into a pungent odor reminiscent of green grass or apples at higher
concentrations (Styger et al., 2011). The addition of GSH significantly
decreased the content of acetaldehyde, which could be attributed to the
following two reasons. On the one hand, GSH fa-vored a more reducing
environment so that the acetaldehyde could be reduced to ethanol; on the
other hand, the nucleophilic residues of GSH could be capable of forming
tetrahedral adducts with acetaldehyde (Lewis & Wolfenden, 1977; Webber et
al., 2014). The addition of GSH resulted in significant decrease of 2-
heptanone content, because GSH inhibited the oxidation of related alcohols to
2-heptanone (Kritzinger et al., 2013). However, 2-heptanone content of GSH-
100Y samples was higher than control, which might be related to the
metabolism of S. cerevisiae. 4-Vinylguaiacol which is characterized by spicy
and smoky,

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J. Xu, et al. Food Chemistry 300 (2019) 125130

Table 3
The aroma compounds content of apple wine with different treatments.

Compounds (μg/L) Experiments

CK GSH-10 GSH-20 GSH-30 GSH-100Y

Alcohols
2-Methyl-1-propanol 36.3d ± 0.9 40.8c ± 2.1 41.8bc ± 0.3 43.9b ± 0.9 61.0a ± 1.7
1-butanol 23.3b ± 0.8 21.7b ± 1.2 22.8b ± 0.1 21.8b ± 1.1 28.4a ± 1.0
3-Methyl-1-butanol 658c ± 11 678c ± 13 742b ± 15 731b ± 4 1198a ± 32
1-Heptanol 6.68d ± 0.42 8.23c ± 0.43 8.28c ± 0.39 9.16b ± 0.12 18.41a ± 0.40
2-Heptanol 2.38b ± 0.21 2.66b ± 0.16 2.50b ± 0.06 2.38b ± 0.19 4.05a ± 0.29
2-Ethyl-1-hexanol 3.65a ± 0.02 3.64a ± 0.02 3.61a ± 0.03 3.69a ± 0.02 3.57a ± 0.01
1-Octanol 16.33a ± 0.04 16.04a ± 0.24 16.31a ± 0.34 16.04a ± 0.34 15.94a ± 0.39
Phenylethyl alcohol 48.2c ± 1.1 54.6b ± 1.8 53.8b ± 0.3 54.1b ± 1.8 65.6a ± 1.0
1-Decanol 5.27c ± 0.12 5.99b ± 0.27 6.29ab ± 0.04 6.52a ± 0.12 ND

esters
Ethyl acetate 257a ± 2 235ab ± 5 232b ± 7 224b ± 9 230b ± 9
n-Propyl acetate 12.29a ± 0.34 12.50a ± 0.70 11.81a ± 0.12 12.33a ± 1.32 11.16a ± 0.79
n-Butyl acetate 36.7b ± 1.3 37.8b ± 0.4 39.0ab ± 1.2 40.7a ± 1.4 31.8d ± 1.0
Hexyl acetate 578a ± 4 562a ± 4 555a ± 19 550a ± 8 337b ± 2
Isobutyl acetate 18.68a ± 0.06 18.11a ± 0.11 17.32ab ± 0.52 15.94b ± 1.46 13.99c ± 1.07
Isoamyl acetate 767a ± 6 739ab ± 10 733b ± 8 727b ± 7 690c ± 11
Phenethyl acetate 16.37 ± 0.17 16.97a ± 0.96 15.95a ± 0.31 16.41a ± 1.05 13.75b ± 0.84
Ethyl propionate 15.41a ± 0.49 14.16bc ± 0.96 14.93ab ± 0.59 13.06 cd ± 0.48 12.32d ± 0.49
Ethyl butyrate 64.3a ± 0.8 55.9b ± 2.8 56.1b ± 0.5 55.6b ± 2.8 36.4c ± 0.6
Ethyl hexanoate 929a ± 17 819b ± 18 815b ± 23 766c ± 45 378d ± 8
Ethyl heptanoate 13.80a ± 0.06 10.64b ± 0.74 10.11b ± 0.75 9.71b ± 0.64 4.78c ± 0.55
Methyl caprylate 68.2a ± 2.0 61.6b ± 1.5 64.4a ± 1.4 57.2c ± 0.3 32.8d ± 1.7
Ethyl caprylate 2768a ± 85 2481b ± 58 2360c ± 26 2369c ± 42 1386d ± 48
Isoamyl caprylate 32.2a ± 1.1 27.4b ± 0.6 28.9b ± 0.6 24.1c ± 1.5 20.1d ± 1.0
Ethyl dodecanoate 65.2a ± 4.1 59.5a ± 3.2 60.3a ± 2.2 50.4b ± 2.9 40.5c ± 2.8
Methyl caprate 32.8a ± 2.0 27.7b ± 0.9 28.5b ± 0.3 27.5b ± 1.1 19.29c ± 0.08
Ethyl caprate 756a ± 21 717b ± 8 697b ± 5 647c ± 22 389d ± 6
Ethyl benzoate 2.85c ± 0.08 3.18b ± 0.09 3.17b ± 0.17 3.44a ± 0.10 2.77c ± 0.14
Ethyl phenylacetate 4.56a ± 0.19 4.71a ± 0.09 4.80a ± 0.16 4.35a ± 0.05 4.68a ± 0.45
Ethyl 9-decenoate 91.3e ± 0.6 95.4d ± 2.2 102.9c ± 2.6 118.1b ± 2.7 167.7a ± 2.1
Exyl 2-methylbutanoate 30.5a ± 0.8 25.2b ± 0.5 26.8b ± 1.1 16.12c ± 1.88 4.98d ± 0.12
2-Methybutyl acetate 168.1a ± 0.5 169.1a ± 1.2 160.5b ± 2.9 155.2b ± 4.9 117.2c ± 2.1
Butanoic acid, 2-methyl-, ethyl ester 71.6a ± 1.5 62.6b ± 1.3 61.5b ± 0.8 57.5c ± 0.8 35.9d ± 0.6
Undecylenic acid methyl ester 3.35b ± 0.19 3.33b ± 0.34 3.38b ± 0.35 3.81b ± 0.23 5.68a ± 0.07

Acids
Acetic acid 17.96a ± 0.80 14.16b ± 0.13 12.97bc ± 0.86 12.03c ± 0.56 12.28c ± 0.42
2-Methyl butyric acid 27.2a ± 1.5 25.8a ± 0.7 21.4b ± 0.4 20.7b ± 1.6 22.5b ± 0.7
Decanoic acid 12.51c ± 1.15 19.86b ± 0.28 21.6b ± 0.7 21.7b ± 0.8 53.6a ± 0.8

Terpenes
Citronellol 3.41c ± 0.09 3.79b ± 0.29 3.85b ± 0.01 3.93b ± 0.05 4.50a ± 0.29
Geraniol 1.96b ± 0.14 2.63a ± 0.23 2.56a ± 0.20 2.60a ± 0.01 2.54a ± 0.15
Linalool 4.77c ± 0.19 5.11bc ± 0.31 5.66a ± 0.44 5.34ab ± 0.23 4.55c ± 0.03

others
Acetaldehyde 3.41b ± 0.06 3.22c ± 0.04 2.91d ± 0.02 2.77e ± 0.10 6.54a ± 0.11
2-Heptanone 17.82b ± 0.66 13.62 cd ± 0.42 14.91c ± 0.15 11.65d ± 1.03 22.8a ± 1.2
4-Vinylguaiacol 2.94c ± 0.03 5.30b ± 0.16 5.02b ± 0.18 5.81a ± 0.26 4.78b ± 0.12

CK – control; GSH-10 – adding 10 mg/L GSH to apple juice; GSH-20 – adding 20 mg/L GSH to apple juice; GSH-30 – adding 30 mg/L GSH to apple juice; GSH-100Y – inoculating
S. cerevisiae pre-incubated with 100 mg/L GSH. Values are means ± standard deviation (SD). Different letters in the same row means significant differences according to Duncan test (p
< 0.05). ND means not detected.

clove-like odors has been identified as an important contributor to the varietal
aroma of apple wine (Ye et al., 2014a). Regardless of GSH concentrations,
GSH addition or inoculating S. cerevisiae pre-incubated with GSH
significantly increased the content of 4-vinylguaiacol in apple wine.
3.5. E-nose response to apple wine samples
In this study, the comprehensive flavor of apple wines with different
treatments was determined by E-nose equipped with 10 metal oxide
semiconductor (MOS) sensors. The typical sensors intensity curves of the
volatile compounds from apple wine with different treatments were presented
in Fig. 3A–E. After a low and stable resistance in initial period, the intensity
(conductivities) of sensors W1S, W1W, W2S, W2W and W5S increased
sharply and stayed stable to the end. For the rest of
sensors, especially W1C, W3C and W5C, the signal intensities were al-ways
at a low level and had no changes during the detection time.
As for the difference in the signal responses between 5 samples, the radar
graph (Fig. 3F) provides direct information. W1S is a methane-sensitive
sensor with a broad range and the W2S sensor is sensitive to a broad range of
alcohol partially aromatic compounds (Gómez, Hu, Wang, & Pereira, 2006).
GSH-100Y apple wine showed the highest signal response of these two
sensors and followed by the control. 10, 20 and 30 mg/L GSH significantly
reduced the intensity of W1S and W2S sensors and higher GSH
concentrations meant lower intensities. It in-dicated that the addition of GSH
decreased the production of methyl-containing volatile compounds during the
apple wine alcoholic fer-mentation, but inoculating the S. cerevisiae with pre-
incubation of GSH played the opposite role. The highest W2S signal response
of GSH-100Y was in accordance with the alcohols content in Table 3.
However, the

6
J. Xu, et al.
Fig. 3. E-nose sensors’ intensity for apple wine with different treatments (A: CK; B: GSH-10; C: GSH-20; D: GSH-30; E: GSH-100Y); The radar graph for 10 MOS sensors’ responses
to the wine samples of E-nose (F); PCA of E-nose data for apple wine with different treatments (G).
opposite result that higher alcohols contents caused lower W2S in-tensities
occurred with GSH addition. This finding could be explained by the lower
ethanol content of apple wine with GSH addition (shown in the
supplementary table 2). After all, ethanol dominated the volatile alcohols of
wine.
The W1W sensor reacts on sulfur compounds such as H 2S and is also
sensitive to terpenes as well as sulfur organic compounds (Gómez, Wang, Hu,
& Pereira, 2006). Apple wine with the addition of 30 mg/L GSH had the
highest value for the W1W intensity, on the contrary, the control group
maintained at the lowest level. On the one hand, GSH might be a potential
source of H2S because cysteine, a constituent amino acid, could be degraded
by cysteine desulfhydrase to form H 2S (Kritzinger et al., 2013); on the other
hand, GSH protected the terpenes from oxidation due to its free sulfhydryl
moiety (Roussis et al., 2010).
7
J. Xu, et al.
Food Chemistry 300 (2019) 125130
According to the content of terpenes in Table 3, there was no difference
between GSH-20 and GSH-30, but the W1W intensity of GSH-30 sample was
higher than that of GSH-20. It meant that more H 2S might be produced by the
addition of 30 mg/L GSH to apple wine. Based on this, 20 mg/L GSH
addition might be a good choice to protect the terpenes and produce less H2S.
Principal component analysis (PCA) is a multivariate statistical method
that uses the idea of dimensionality reduction to convert mul-tiple indicators
into several comprehensive indicators under the pre-mise of losing little
information (Xiang, Li, Ndolo, & Beta, 2019). In this study, ten variables
were reduced to two principal components (PC1 and PC2). As was shown in
the Fig. 3G, the variance of PC1 and PC2 accounted for 57.9% and 27.5% of
the total variance respectively and the sum of their variance was up to 85.4%.
The PCA results of the flavor
compounds of apple wines with different treatments was remarkably different
from the control group (All groups was clearly distinguishable from each
other by PCA analysis), suggesting that the addition of GSH or inoculating
the S. cerevisiae pre-incubated by GSH resulted in a no-ticeable change of
volatile flavor compounds. For PC1, apple wines with the addition of 20 or 30
mg/L GSH showed a great difference from control. With the exception of
GSH-20 and GSH-30, there was a sig-nificant distinction among all of the
apple wine samples on PC2, in-dicating that there was almost no difference
between the addition of 20 and 30 mg/L GSH on PC2, but they were
distinguished from the control group.
4. Conclusion
Based on the above results, the major functions of GSH could be
summarized as being a certain mono-phenols (such as chlorogenic acid and
phloretin) protector, browning inhibitor and flavor modifier. The addition of
GSH interfered more in total phenolic compounds and color index of apple
wine than inoculating S. cerevisiae pre-incubated with 100 mg/L GSH.
Further, according to the sensors’ response of E-nose, 20 mg/L GSH might be
a better choice for apple wine brewing in terms of the higher terpenes and
lower H2S content. However, the mechanism for the variation of some aroma
compounds content with GSH addition remains unclear, further research still
needs to be done in the future to understand the mechanism of how GSH
improve wine flavor and an-tioxidant.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests
or personal relationships that could have appeared to influ-ence the work
reported in this paper.
Acknowledgments
This work was supported by the program of Comprehensive utili-zation of
the by-products of horticultural crops processing (Grant No. 201503142-10)
and Shaanxi province science and technology co-ordi-nating innovation
project (Grant No. 2016KTCQ02-13).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foodchem.2019.125130.
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