Food Chemistry 363 (2021) 130351

Contents lists available at ScienceDirect

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

Study on the nutritional characteristics and antioxidant activity of

dealcoholized sequentially fermented apple juice with Saccharomyces
cerevisiae and Lactobacillus plantarum fermentation
Hongcai Li , Jintao Huang , Yaqin Wang , Xingnan Wang , Yichen Ren , Tianli Yue , Zhouli Wang ,
Zhenpeng Gao *
College of Food Science and Engineering, Northwest A&F University, 712100 Yangling, Shaanxi, People’s Republic of China

A R T I C L E I N F O A B S T R A C T

Keywords: For the preparation of non-alcoholic fermented apple juice products with high antioxidant activity, we evaluated
Apple juice the physicochemical indicators, functional components, aroma and antioxidant activity of products at different
Sequential fermentation stages of Saccharomyces cerevisiae fermentation, dealcoholization, and Lactobacillus plantarum sequential
Dealcoholization
fermentation. After S. cerevisiae fermentation, the total sugar was reduced by 7.46 g/100 mL and the ethanol
Nutritional properties
Antioxidant properties
reached 6.56% (v/v), which decreased to less than 0.50% (v/v) after dealcoholization. After L. plantarum
fermentation, the viable count reached 4.26 × 108 CFU/mL and flavonoids increased; vitamin C and B6 levels
peaked at 1.97 and 1.17 mg/100 mL respectively. Fermented by L. plantarum after dealcoholized, new volatiles
were produced (propanol, citronellol, ethyl lactate and hexyl butyrate), ketones and acids were enriched; anti­
oxidant activity was improved and had the highest free radical scavenging rate. The sequential fermentation of
apple juice with S. cerevisiae, dealcoholization, and L. plantarum enriched its nutritional properties and antiox­
idant activity.

1. Introduction have focused on distillation, membrane filtration, membrane reverse

Fruit juices are fermented by S. cerevisiae to prepare alcoholic fruit
wines. Fruit wines retain most of the nutrients in the original juice and
have higher levels of alcohols, vitamins, amino acids, minerals, antho­
cyanins, and other nutrients (Swami, Thakor, & Divate, 2014). Apples
are rich in sugar, inorganic salts, dietary fiber, vitamins, aromatic sub­
stances, and trace mineral elements. Apple juice can lower blood lipids,
prevent cancer, and enhance beauty due to the rich polyphenol content
in apples (Wolfe, Wu, & Liu, 2003). Apple juice fermentation by S.
cerevisiae alters its color, acidity, astringency, and sweetness, and in­
creases its volatile components, polyphenols, and antioxidant activity
(Wei et al., 2020). However, fermentation by S. cerevisiae produces
ethanol, which is the main carcinogen in alcoholic beverages. There is
no safe standard for drinking ethanol and any alcohol intake increases
the risk of cancer (Wood, Kaptoge, & Butterworth, 2018). As people pay
more attention to healthy diets, the demand for low- and non-alcohol
fruit wines has increased and non-alcoholic fermented apple juice has
been a research focus. Studies of non-alcoholic fermented apple juice
osmosis, and nanofiltration dealcoholization (Mangindaan, Khoiruddin,
& Wenten, 2018). Although the dealcoholization process retains the
vitamins, minerals, antioxidants, and other functional substances in the
wine, it is also accompanied by volatilization and loss of the aroma
components, which affects quality and taste. Therefore, the further
processing of dealcoholized products is the direction of future research
by scientific researchers.
Probiotics are live bacteria that produce beneficial effects after being
ingested, apple juice is a good substrate for fermentation by the pro­
biotic L. plantarum (Grover, Rashmi, Srivastava, & Batish, 2012).
Swarna, Srimathi, Madhavan, and Gomathi (2015) showed that Lacto­
bacillus promotes the dynamic balance of active microorganisms in the
host body, improves human immunity, and ameliorates the intestinal
flora. Dimitrovski, Velickova, Langerholc, and Winkelhausen (2015)
found that juice obtained by fermenting clear apple juice with L. plan­
tarum has better taste and acceptability, and the highest number of
viable cells. Etxeberria et al. (2013) proved that L. plantarum can de-
esterify, desugar, demethylate, and decarboxylate the bound phenols

* Corresponding author.
E-mail address: gzp5988@163.com (Z. Gao).

https://doi.org/10.1016/j.foodchem.2021.130351
Received 15 March 2021; Received in revised form 9 June 2021; Accepted 9 June 2021
Available online 11 June 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.
H. Li et al.
in fruit and vegetable juices and convert them into free phenols, which
are more easily absorbed by the body. Therefore, L. plantarum fermen­
tation can improve the antioxidant activity of apple polyphenols, which
are more easily absorbed. Apples are rich in various nutrients, and
different types of fermentation confer different flavor characteristics. W.
L. Liu, Fan, Sun, and Li (2020) studied the effect of mixed S. cerevisiae
and L. plantarum fermentation on the sensory quality of black raspberry
wine. Li et al. (2021) used S. cerevisiae and L. plantarum to co-ferment
cider and enhance its antioxidant activity. Minnaar, du Plessis, Paul­
sen, Ntushelo, Jolly, and du Toit (2017) showed that sequential
fermentation by S. cerevisiae, non-Saccharomyces yeasts, and lactic acid
bacteria improved the quality of wine. However, no study has reported
on the fermentation of apple juice by S. cerevisiae, followed by the
removal of ethanol to obtain non-alcoholic fermented apple juice, which
is then fermented by L. plantarum.
To assess S. cerevisiae and L. plantarum sequentially fermented non-
alcoholic apple juice products, we will determine the physicochemical
index, vitamin, polyphenol monomer, organic acid, and aroma compo­
nents, sensory evaluation, and antioxidant activity at each fermentation
stage. The results will provide a reference for the industrial development
of new non-alcoholic fermented apple juices with rich nutrition and high
antioxidant activity.
2. Materials and methods
2.1. Microorganisms and apples
The yeast (Saccharomyces cerevisiae PF14) and Lactobacillus (Lacto­
bacillus plantarum CICC21805) were purchased from China Agricultural
Culture Center (Beijing, China) and preserved by the Health Food
Manufacturing and Safety Laboratory of Northwest A&F University.
S. cerevisiae PF14 has weak ability to produce alcohol, giving it the po­
tential to produce low-alcohol wines (Ye, Yue, Yuan, & Wang, 2013).
L. plantarum CICC21805 has good acid and bile tolerance and good
hydrophobicity (Meng, Wu, Yue, & Gao, 2019), and it is a functional
probiotic with strong fermentation ability and rich metabolites (Wang
et al., 2021).
Aksu Fuji Apple was from a local fruit market (Shaanxi, China).
2.2. Reagent and standards
(i) De Man, Rogosa and Sharpe (MRS) medium was purchased from
Beijing Obosing Biotechnology Co., Ltd. (Beijing, China). Yeast extract,
peptone, and glucose (YPD) were purchased from Aobox (Beijing,
China). (ii) Organic acid standards (oxalic acid, tartaric acid, quinic
acid, pyruvic acid, malic acid, shikimic acid, lactic acid, formic acid,
acetic acid, citric acid and succinic acid), polyphenol standards (caffeic
acid, vanillic acid, catechins, Gallic acid, phloretin, phloridin, epi­
catechin, rutin, p-coumaric acid, ellagic acid, protocatechuic acid, iso­
ferulic acid and cinnamic acid), and vitamin standards (vitamin B1,
vitamin B2, vitamin B5, vitamin B6, vitamin C) were purchased from
Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Water
purified using a Milli-Q system from Millipore (Bedford, MA, USA),
acetonitrile (TEDIA, Fairfield, OH), and phosphoric acid (Kemiou,
Tianjin, China) were prepared for high-performance liquid chromatog­
raphy (HPLC) analysis. For gas chromatography–mass spectrometry
(GC–MS), 2-octanol (Aobox Beijing, China) was used as an internal
standard. The n-alkane solution comprising 24n-hydrocarbons
(C7–C30), which was used to calculate the retention index (RI), was
purchased from Supelco (Bellefonte, PA, USA).
2.3. Preparation of apple juice and inoculum
The fruit was washed with tap water, the pulp was chopped, and the
seeds were removed. Then the apple pulp was crushed using a food-
grade juicer and filtered through an 80 mesh gauze, after which
2
Food Chemistry 363 (2021) 130351
0.08% ascorbic acid was immediately added to prevent enzymatic
browning (Peng, Meng, Yue, Wang, & Gao, 2021). The apple juice in the
conical flask was heated in a water bath at 90 ◦ C for 15 s to inactivate the
polyphenol oxidase in the apple juice. After standing at room tempera­
ture (25 ◦ C), the juice by suction filtration with vacuum pump (SHZ-CS;
Gongyi Yuhua Instrument Co., Ltd., China), and then stored at − 20 ◦ C.
The S. cerevisiae was cultured in YPD medium in a shaker at 28 ◦ C and
120 rpm for 24 h, and L. plantarum was cultured in MRS broth at 37 ◦ C
for 16 h. The strains were placed in sterile glycerol (30% v/v) and stored
at − 80 ◦ C. The S. cerevisiae and L. plantarum were cultured for two
generations under anaerobic conditions ensure that microorganisms
grow in an oxygen-free environment. The cells were collected by
centrifugation at 7225×g for 15 min at 4 ◦ C (HC-30182; Anhui Zhongke
Zhongjia Scientific Co., Ltd., China). The cell pellet in the logarithmic
growth phase was washed three times with sterile water, and the final
concentration was 108 CFU/mL (i.e.OD600 = 1.12) (Carvalho, Bazana,
Ferrão, & Fuentefria, 2021). Then it was added to the apple juice to
prepare a seed suspension.
2.4. Apple juice fermentation process
The thawed apple juice was pasteurized at 80 ◦ C for 15 min and
cooled to room temperature (25 ◦ C). Then the seed liquid (35 mL) was
added to the apple juice (500 mL), this was performed in triplicate, and
the viable count in the juice was determined after fermentation.
Through optimization of experiments, the best fermentation conditions
are inoculation amount (7%), temperature (25 ◦ C) and time (6 days).
Fermentation is carried out under these conditions. The S. cerevisiae was
added to 500 mL apple juice at an inoculum of 7%. The initial sugar
concentration of apple juice was 18◦ Brix, and it was allowed to stand for
5–6 days in an incubator at 25 ◦ C. After S. cerevisiae fermentation, the
cider was subjected to rotary steaming and dealcoholization for 40 min
under a vacuum at a temperature of 30 ◦ C and a pressure of − 0.09 MPa
to remove the alcohol in the cider after S. cerevisiae fermentation
(Mangindaan, Khoiruddin, & Wenten, 2018), to obtain alcohol-free
apple juice. After dealcoholization, the apple juice was statically fer­
mented by 7% L. plantarum seed liquid, and for 16 h in an incubator at
39 ◦ C.
2.5. Analyses of physical and chemical indicators
The frozen apple juice was thawed and pasteurized at 80 ◦ C for 15
min, and then cooled to room temperature for microbial fermentation to
determine its physicochemical indicators. A pH meter (FE28; METTLER
TOLEDO, Shanghai, China) was used to measure the pH of the apple
juice, and a handheld refractometer was used to determine the total
soluble solid content (SSC) and expressed in ◦ Brix. The acid-base titra­
tion method was used to measure the total acid content and was
expressed by the malic acid conversion coefficient (Peng, Meng, Yue,
Wang, & Gao, 2021). The total sugar content was determined by titra­
tion and expressed as g/100 mL. The total phenol content was deter­
mined by the Folin–Ciocalteu method and the total flavonoid content
was measured using the aluminum chloride colorimetric method (Kwaw
et al., 2018).
2.6. Determination of phenol monomers and organic acid
The HPLC system (Shimadzu, Japan) was used for the quantitative
determination of polyphenols and organic acids. The C18 chromato­
graphic column (4.6 mm × 250 mm, 5 μm; Waters Co., Milford, MA,
USA) was used to separate the test compounds. The polyphenols were
determined using the mobile phase consisted of a 1% formic acid
aqueous solution (A) and acetonitrile (B). The gradient elution proced­
ure of phase B was: 0–5 min, 5%; 5–25 min, 12%; 25–40 min, 30%;
40–50 min, 45%; 50–60 min, 5%, flow rate, 1 mL/min; oven tempera­
ture, 30 ◦ C; and wavelength of the ultraviolet detector, 280 nm.
H. Li et al.
The organic acid was determined using 0.01 mol/L KH2PO4-H3PO4
(pH 2.7) and methanol as the mobile phase, and isocratic at a ratio of
97:3 mode for elution. The flow rate was 0.7 mL/min, and the UV–Vis
spectrum detection was set to 210 nm. The sample (1 mL) was filtered
through a 0.45 μm hydrophilic filter membrane, the injection volume
was 10 μL, and the oven temperature was 30 ◦ C. The peak areas of the
samples were compared with that of the internal standards and
quantified.
2.7. Determination of water-soluble vitamins
The sample (8 mL) was centrifuged at 7225×g for 6 min, after which
1 mL supernatant was removed and the filtrate was filtered through a
0.22 μm water-based filter (Bonna-Agela Technologies Venusil Tech­
nology Co., Ltd. Tianjin, China). The above process was done in the dark
that avoid vitamin decomposed by light. The conditions were as follows:
column, Alltima C18 (250 mm × 4.6 mm, 5 μm); mobile phase, 50
mmol•L− 1 ammonium dihydrogen phosphate solution (phosphoric acid
adjusted to pH 3.0)-acetonitrile (95:5); flow rate, 0.5 mL•min− 1;
detection wavelength, 275 nm (detection of vitamin B1, vitamin B6,
vitamin C, nicotinamide B3) and 210 nm (detection of pantothenic acid
B5); column temperature, room temperature; injection volume, 20 μL.
2.8. Determination of volatile substances
Volatile compounds were analyzed by GC–MS. First, 1 g NaCl was
added to a 15 mL glass bottle, followed by the addition of 5 mL sample
and 6 μL 2-octanol (Sigma-Aldrich, St. Louis, MO, USA) as the internal
standard (0.2 mg/mL). The first step was headspace solid phase micro­
extraction, and then the sample was equilibrated for 15 min at room
temperature (25 ◦ C) and then at 30 min at 40 ◦ C. The analyses were
conducted on a GC–MS system (Thermo Finnigan, San Jose, CA, USA)
equipped with the TR-5MS column (30 m × 0.25 mm, 0.25 μm; J&W
Scientific, Folsom, CA, USA) and quadrupole DSQ II MS. The oven
temperature program was maintained at 50 ◦ C for 3 min, and then
increased to 200 ◦ C at a rate of 5 ◦ C/min and maintained for 12 min.
Helium was used as the carrier gas at a flow rate of 1 mL/min. Quan­
titative analyses of volatile compounds were based on the internal
standard (2-octanol).
2.9. Determination of antioxidant activity
DPPH-scavenging ability was measured following Abirami, Nagar­
ani, Siddhuraju, and Wellness (2014). DPPH solution (3.9 mL 0.025 g/L)
was added to 0.1 mL sample, and reacted for 30 min in the dark at room
temperature. The absorbance was measured at a wavelength of 515 nm,
and 0.1 mL absolute ethanol and 3.9 mL DPPH solution were mixed as a
blank control. Each sample was tested three times in parallel. The
radical scavenging activity (RSA) represents the percentage of DPPH-
free radical inhibition, and was calculated as follows:
A0 -A1
DPPH RSA (%) = × 100 (1)
A0
here A0 is absorbance of the control group and A1 is absorbance of the
sample group.
The pyrogallol autooxidation method was slightly modified to
determine the ability of the test solution to remove superoxide anions.
Then 3 mL 0.05 mol/L Tris-HCL buffer solution (pH, 8.2) was placed in a
25 ◦ C water bath and pre-heated for 20 min, after which the same vol­
ume of test solution at different concentrations and 0.6 mL 30 mmol/L
pyrogallol were added. After mixing the solution, it was pre-heated in a
25 ◦ C water bath for 5 min, 0.5 mL 1 mol/L HCL was added to stop the
reaction, and the absorbance A1 at 420 nm was measured. The blank
control replaced the sample with the same volume of double distilled
water, and the absorbance was recorded as A0. Taking into account that
3
Food Chemistry 363 (2021) 130351
the test solution itself may have absorption at 420 nm, the pyrogallol
solution was replaced with the same volume of double distilled water to
obtain A2. RSA represents the inhibition percentage of ⋅O−2 free radical,
and was calculated as follows:
A0 -A1 + A2
Â⋅O−2 RSA (%) = × 100 (2)
A0
where A0 is absorbance of the control group, A1 is absorbance of the
sample group, and A2 is absorbance in the sample after replacement with
double distilled water. The iron ion reduction capacity of the sample was
determined, briefly, 0.5 mL sample was diluted 1:1 with deionized
water, mixed with 3 mL 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) working
solution, and reacted at 37 ◦ C for 4 min, after which the absorbance was
measured at a wavelength of 593 nm. TPTZ working solution consisted
of acetate buffer (pH, 3.6) and 10 mmol/L TPTZ solution (prepared with
40 mmol/L HCl), and was mixed at a 10:1:1 ratio to obtain iron chloride
solution (20 mmol/L). Trolox was used as the standard product and the
standard curve was constructed. The ferric ion reduction capacity of
fruit juice was expressed as mmol (Tolox)/L (juice). Each sample was
measured three times in parallel.
2.10. Sensory analyses
To evaluate the sensory quality of apple juice during fermentation,
15 teachers and student panel members with relevant experience and
background knowledge evaluated the color, posture, aroma, taste, and
sweetness of the apple juice. Sensory analyses were based on the method
by Wang, et al. (2021) with slight modifications.
2.11. Statistical analyses
Each experiment was repeated three times, and the results are
expressed as the mean ± standard deviation. SPSS 20.0 (Inc., Chicago,
IL) was used to analyze variance, and Duncan’s multiple comparison test
was used to analyze the differences between the experimental groups. p
< 0.05 was considered statistically significant.
3. Results and discussion
3.1. Changes in physical and chemical indexes during the fermentation of
apple juice
As shown in Table 1, the pH value showed a downward trend during
the fermentation of apple juice and the total acid showed an overall
upward trend, because the apple juice was fermented by S. cerevisiae,
which consumes reducing sugar. After dealcoholization following
S. cerevisiae and then L. plantarum fermentation, sugar in apple was
fermented by L. plantarum to produce acid. Therefore, the total acid
content in the fermentation broth showed an upward trend, and the pH
value continued to decrease. When the fermentation was complete, the
total acid content reached 3.32 mg/mL and the pH was 4.03.
The change in SSC and total sugar showed an overall downward
trend. During the fermentation process, SSC decreased from 20.5◦ Brix to
4.4◦ Brix and total sugar decreased from 19.39 g/100 mL to 5.68 g/100
mL. The rate of decline of SSC was lower during L. plantarum fermen­
tation than S. cerevisiae fermentation, because S. cerevisiae consumes a
lot of sugar during fermentation (Wei, et al., 2020). The content of
reducing sugars declines because probiotics need sugar metabolism for
energy to maintain their own growth and reproduction during fermen­
tation, and reducing sugars are continuously consumed as fermentation
substrates (Markowski, Baron, Le Quere, & Plocharski, 2015). After
S. cerevisiae fermentation, the alcohol content reached 6.56%, and less
than 0.5% (v/v) after dealcoholization. With the progress of fermenta­
tion time, the viable count increased rapidly and reaching 5.44 × 108
CFU/mL in the S. cerevisiae fermentation process and 4.26 × 108 CFU/
H. Li et al. Food Chemistry 363 (2021) 130351

Table 1 Table 2
Changes in physicochemical indexes and viable bacteria count during Changes in water-soluble vitamins during fermentation.
fermentation. Vitamins Content/(mg/100 mL)
Index Apple Yeast Dealcoholization Lactobacillus
Apple Yeast Dealcoholization Lactic acid
juice fermentation fermentation
juice fermentation fermentation
pH 4.21 ± 4.12 ± 0.02b 4.14 ± 0.04b 4.03 ± 0.01 a
Thiamin 0.62 ± 0.46 ± 0.002 a 0.63 ± 0.003 d 0.47 ± 0.002b
0.02c
0.003c
Soluble solids 20.50 6.70 ± 0.17c 6.10 ± 0.12b 4.40 ± 0.09 a
Riboflavin 17.02 ± 153.16 ± 83.07 ± 4.10b 68.69 ± 2.46 ab
(◦ Brix) ± 0.15
d 1.46 a 24.68c
Pantothenic 2.74 ± 3.05 ± 0.57 a 3.51 ± 0.46 a 4.88 ± 0.70 a
Viable 0.41 ± 5.44 ± 0.05 d 0.13 ± 0.01 a 4.26 ± 0.06c
acid 0.65 a
bacterias 0.03b
VitaminB6 0.04 ± 0.86 ± 0.04b 1.04 ± 0.45b 1.17 ± 0.01b
(108 CFU/
0.003 a
mL)
Vitamin C 0.92 ± 1.52 ± 0.04c 1.46 ± 0.02c 1.97 ± 0.03 d
Total acid 1.63 ± 2.59 ± 0.02b 2.53 ± 0.12b 3.32 ± 0.19c
0.014b
(mg/mL) 0.13 a
Total sugar (g/ 19.39 11.93 ± 11.26 ± 0.99b 5.68 ± 0.09 a Note: Different letters in the same row indicate statistically significant differ­
100 mL) ± 1.24 1.09b ences in the results (p < 0.05).
d

Reducing 59.25 6.38 ± 0.86b 4.06 ± 0.11b 2.06 ± 0.12 a

sugar (mg/ ± 1.81c antioxidant activity, and the content of vitamin C at the end of
mL) fermentation is equivalent to twice that of the original juice. After
Total 1.55 ± 3.66 ± 0.15c 1.19 ± 0.02 a 1.66 ± 0.015b fermentation, the antioxidant activity of the juice increases (Aliste & Del
polyphenols 0.07b
Mastro, 2004).
(mg/mL)
Total 0.12 ± 1.09 ± 0.06c 0.46 ± 0.03b 0.98 ± 0.01c
flavonoids 0.01 a
(mg/mL) 3.3. Changes in polyphenol monomer content during the fermentation of
Alcohol (v/v)/ ND 6.56 ± 0.53b 0.41 ± 0.02 a 0.37 ± 0.04 a apple juice
%

Note: Different letters in the same row indicate statistically significant differ­
ences in the results (p < 0.05).
ND: Not detected.
mL in the L. plantarum fermentation process, which met the re­
quirements for the viable count (Chapin & Lauderdale, 2003).
At the beginning of fermentation, the total flavonoids in apple juice
was 0.12 mg/mL, and increased to 0.984 mg/mL after L. plantarum
fermentation. The total polyphenol content in apple juice was 1.553 mg/
mL in the initial stage of fermentation. Wlodarska, Pawlak-Lemanska,
Gorecki, and Sikorska (2017) reported that the initial polyphenol con­
tent in apples is 0.74 mg/mL. It reaches 3.656 mg/mL after S. cerevisiae
fermentation, and the total phenolic acid content in the late fermenta­
tion period basically stabilizes. Phenolic acids mainly exist in the form of
covalent bonds in plants, the decrease of phenolic acid in apple juice
during fermentation may be that microbes decompose the covalently
bonded macromolecular phenolic acids into small molecules (McCue &
Shetty, 2005).
3.2. Changes in water-soluble vitamins during the fermentation of apple
juice
Water-soluble vitamins participate in the catalytic function of the
human body. B vitamins are the components of many kinds of co­
enzymes, which are responsible for the transfer of hydrogen, electrons,
or groups. They are involved in the metabolism of sugars, fats, proteins,
and nucleotides catalyzed by enzymes. Vitamin C participates in many
hydroxylation reactions and can promote immune system health and
calcium absorption. The changes in water-soluble vitamins in apple juice
during fermentation are shown in Table 2. The contents of riboflavin,
pantothenic acid, vitamin B6, and vitamin C in apple juice were
respectively 17.02, 2.74, 0.04, and 0.92 mg/100 mL at the beginning of
fermentation, and increased to 68.69, 4.88, 1.17, and 1.97 mg/100 mL
at the end of fermentation. After S. cerevisiae fermentation, the content
of riboflavin in apple juice was significantly increased (p < 0.05),
consistent with (Kariluoto, Vahteristo, Salovaara, Katina, Liukkonen, &
Piironen, 2004) showing that fermentation can lead to an increase in
water-soluble vitamins. Both pantothenic acid and vitamin B6 increase
to varying degrees during the fermentation process. Vitamin C has
Fruits are rich in polyphenols, which have many positive effects on
human health such as reducing the risk for cancer, anti-inflammatory
properties, and inhibiting carcinogenesis (Wolfe, Wu, & Liu, 2003). A
series of changes in polyphenol content occurred during apple juice
fermentation. As shown in Table 3, among the 13 monomeric phenolic
acids, the mass concentration of gallic acid at the initial fermentation
was 1.163 mg/L and at the end of S. cerevisiae fermentation reached
1.362 mg/L. After L. plantarum fermentation, the concentration was
1.646 mg/L, which significantly increased compared to the initial
fermentation concentration (p < 0.05), this because the hydrolysis of
polymerized tannins can generate gallic acid (Moreno-Arribas & Polo,
2009). Isoferulic acid was the precursor substance for the synthesis of
various aroma components by microorganisms (Abdelkafi, Sayadi, Gam,
Casalot, & Labat, 2006) and the content reaches 0.276 mg/L after
fermentation. In our study, the content of phloretin, rutin, and ellagic
acid showed a general downward, and the content changes before and
after fermentation were significantly different (p < 0.05). Among the
polyphenols in the original juice, the content of caffeic acid was highest,
reaching 74.434 mg/L. At the end of S. cerevisiae fermentation, the
content of caffeic acid reached a maximum value of 110.561 mg/L, due
to the release of free caffeic acid and tartaric acid through metabolic
hydrolysis of S. cerevisiae-fermented apple juice (Fracassetti, Lawrence,
Tredoux, Tirelli, Nieuwoudt, & Du Toit, 2011), and the content subse­
quently decreased in L. plantarum fermentation due to the increase in pH
of the juice. When the optimal pH of polyphenol oxidase reached
(4.0–6.0), the antioxidant activity of polyphenol oxidase was enhanced
and that of caffeic acid was degraded (Podsedek, Wilska-Jeszka, Anders,
& Markowski, 2000).
Protocatechins, catechins, caffeic acid, p-coumaric acid, isoferulic
acid, and cinnamic acid belong to the hydroxycinnamic acid group.
Compared with the fermentation of L. plantarum, the content of proto­
catechins in the original juice is lower. With the passage of fermentation
time, the content of catechins gradually increased from 0.287 mg/L
before fermentation to 0.608 mg/L. The content of cinnamic acid varied
from 0.089 mg/L to 0.149 mg/L. During the fermentation process, the
content reached the highest value after L. plantarum fermentation. Both
coumaric acid and isoferulic acid are derivatives of cinnamic acid.
Coumaric acid and isoferulic acid had a similar trend in content change
during the fermentation process, both increasing first and then
decreasing. During the analysis process, the relatively low content of

4
H. Li et al. Food Chemistry 363 (2021) 130351

Table 3 Table 4
Changes in polyphenols during the fermentation of apple juice. Changes in organic acids during fermentation.
Category Content /(mg/L) Category Content /(mg/L)

Apple Yeast Dealcoholization Lactobacillus Apple Yeast Dealcoholization Lactobacillus

juice fermentation fermentation juice fermentation fermentation

Caffeic acid 74.434 110.561 ± 78.173 ± 82.022 ± Oxalic 121.84 ± 27.76 ± 0.12b 36.93 ± 0.46b 41.19 ± 8.26b
± 0.745c 2.334ab 1.343b acid 3.89c
0.863a Tartaric 60.96 ± 190.89 ± 171.19 ± 1.11b 328.91 ± 13.81c
Vanillic acid 5.885 11.907 ± 12.996 ± 0.259c 5.858 ± 0.165 acid 3.39 a 0.75b
± 0.296 0.070b a
Quinic 144.72 ± 238.72 ± 169.38 ± 0.65 a 608.37 ± 15.70c
a
acid 11.48 a 5.02b
Cianidanol 2.287 1.054 ± 2.680 ± 0.100b 2.935 ± Formic 558.32 ± 138.85 ± 383.39 ± 0.82c 582.91 ± 10.57
± 0.436 a 0.035b acid 8.03 d 1.99b d

0.041b Pyruvic ND 433.40 ± 790.71 ± 1.07 d 118.66 ± 17.79b

Gallic acid 1.163 1.362 ± 1.788 ± 0.050c 1.646 ± acid 7.12c
± 0.087 0.003 ab 0.010bc Malic acid 2423.21 ± 2143.36 ± 3230.06 ± 0.84 e 249.76 ± 13.27b
a
25.42 d 17.22c
Phloretin 1.572 2.096 ± 0.982 ± 0.010 a 0.783 ± 0.019 Shikimic 4.56 ± 4.39 ± 0.07b 6.77 ± 0.005c 6.27 ± 1.27 bc
± 0.084c a
acid 0.21b
0.000b Lactic 165.29 ± 207.94 ± 1.81 446.19 ± 0.69b 5097.26 ±
Phlorizin 1.363 1.256 ± 2.291 ± 0.100c 1.670 ± acid 5.54 a a
32.23c
± 0.001 0.007 a 0.053b Acetic 67.64 ± ND 101.14 ± 6.92ab 1311.22 ±
a
acid 10.83 a 23.11ab
L-Epicatechin 1.008 2.200 ± 1.395 ± 0.361 a 0.893 ± 0.009 Citric acid 115.42 ± 56.19 ± 6.73b 264.78 ± 2.25b 330.82 ± 21.55b
± 0.012 0.016b a
0.02 ab
a
Succinic 13.89 ± 1802.17 ± 836.47 ± 11.16c 1764.39 ±
Rutin 0.500 0.127 ± 0.564 ± 0.008c 0.147 ± 0.012 acid 1.29 a 3.42b 41.29b
± 0.005 a a

0.018b Note: Different letters in the same row indicate statistically significant differ­
P-coumaric 0.382 0.341 ± 0.684 ± 0.018c 0.235 ± 0.021 ences in the results (p < 0.05).
acid ± 0.002 a a ND: Not detected.
0.000b
Ellagic acid 0.392 0.354 ± 1.583 ± 0.101b 0.267 ± 0.008
influences the sensory properties and nutritional quality of the apple
± 0.006 0.003 a a
a juice. Malic acid is the most important and highest organic acid in apple
Protocatechuic 0.287 0.312 ± 0.488 ± 0.050b 0.608 ± juice, and is an intermediate in the tricarboxylic acid cycle of organisms,
acid ± 0.004 0.009 a 0.019c it can participate in the fermentation engineering of microorganisms
a
and can be used as a carbon source for microorganism growth (Liu et al.,
Isoferulic acid 0.178 0.086 ± 0.190 ± 0.008 ab 0.276 ±
± 0.055 0.005 a 0.049b
2017). Malic acid in raw juice is the main organic acid. The initial mass
ab concentration of malic acid in apple juice was 2423.21 mg/L; the con­
Cinnamic acid 0.089 0.007 ± 0.117 ± 0.010c 0.149 ± tent peaked after S. cerevisiae fermentation, and decreased to 249.762
± 0.001 a 0.003d mg/L after L. plantarum fermentation. This was due to the decomposi­
0.001b
tion of malic acid by L. plantarum during malic acid-lactic acid
Note: Different letters in the same row indicate statistically significant differ­ fermentation, resulting in malic acid content decreases and lactic acid
ences in the results (p < 0.05). content increases. At the end of fermentation, the lactic acid content
reached the highest value of 5097.264 mg/L, which was significantly
polyphenols in apple juice due to polyphenol oxidase activity is caused higher than that before fermentation (p < 0.05). Succinic acid is an
by enzymatic hydrolysis or loss of cell wall matrix adsorbed to apple important product of alcohol fermentation, and its concentration de­
pomace during the pressing process (Nogueira, Guyot, Marnet, Lequere, pends on the yeast strain. During the wine brewing process, the con­
Drilleau, & Wosiacki, 2008), and the highest content occurs after centration of succinic acid is between 0.6 and 1.2 g/L (Satora, Sroka,
L. plantarum fermentation. Duda-Chodak, Tarko, & Tuszynski, 2008). This acid helps to increase the
The increase in content of p-coumaric acid may be due to the hy­ acidity of wine. Lactobacillus can synthesize succinic acid from the citric
drolysis of coumarate compounds, and the decrease in coumaric acid acid produced by the tricarboxylic acid cycle. At the same time, it also
content may be due to the generation of p-hydroxybenzoic acid through produces organic acids such as lactic acid, which directly affects the
degradation reactions. The decrease in isoferulic acid content may be acidity of the juice. The initial content of succinic acid in apple juice
because polysaccharides and proteins combine in the cell wall to form 13.893 mg/L. In this study, the concentration of succinic acid in the
the cytoskeleton, or because microbes use ferulic acid to synthesize S. cerevisiae fermentation process rapidly increased, the original juice
aroma substances. Rutin is the main flavonoid polyphenol in apple juice, content is 13.89 mg/L, and the mass concentration is 1802.172 mg/L at
and the content in apple juice is 0.50 mg/L. It decreased from 0.50 mg/L the end of fermentation (p < 0.05). During the fermentation process, the
to 0.147 mg/L during the fermentation process, this possibly because the content of tartaric acid increased from 60.958 mg/L to 328.9 mg/L. The
content of rutin in the peel is relatively high, and apple juice is mainly increase in tartaric acid content is caused by a sharp drop in the pH of
the clear juice after removing the pomace, which can easily cause the the juice, oxidative degradation, and precipitation of tartrate (Xiang,
loss of rutin. Phlorizin is a dihydrochal ketone, did not change signifi­ Zhu, Han, Li, Ge, & Lin, 2012).
cantly during the fermentation process. Quinic acid is an alicyclic organic acid that is unique to higher plants
in apple juice. The mass concentration of quinic acid at the beginning of
fermentation was 144.72 mg/L, and tended to level out during
3.4. Changes in organic acid content during the fermentation of apple S. cerevisiae fermentation. The content of quinic acid rapidly increased
juice during L. plantarum fermentation, and the mass concentration reached
608.374 mg/L at the end of fermentation, which was a significant in­
Table 4 shows the changes in organic acid content during the crease of 463.65 mg/L compared to the original juice (p < 0.05). Citric
fermentation of apple juice. The organic acid in apple juice significantly acid is an intermediate metabolite of the tricarboxylic acid cycle, and the

5
H. Li et al. Food Chemistry 363 (2021) 130351

initial content in apple juice was 115.421 mg/L. The content gradually
decreased during S. cerevisiae fermentation, possibly due to L. plantarum
during malic acid–lactic acid fermentation. Decomposition of citric acid
into pyruvic acid and acetic acid, and also into lactic acid, its metabolic
intermediate products, can also produce flavor compounds such as
acetoin and diacetyl, which have a greater influence on the flavor of
apple juice (Bandic, Zulj, Fruk, Babojelic, Jemric, & Jeromel, 2019).
Shikimic acid is present in a large number of higher plants and micro­
organisms, and its content in apple juice is low. The changing trend of
this process is relatively stable, which is similar to the changing trend of
shikimic acid in apple juice fermented by malolactic bacteria in coop­
eration with S. cerevisiae (Herrero, Garcia, & Diaz, 1999).
Different strains ferment to produce different organic acids, and
microorganisms can metabolize different substrates at different rates in
different growth environments, and even compete and assist. The
organic acids generated during the fermentation process not only reduce
the pH value and increase the stability of vitamin B but also participate
in the interaction between aroma substances and organic acids
(Dongmo, Procopio, Sacher, & Becker, 2016).
3.5. Changes in aroma substances during the fermentation of apple juice
A total of 58 aroma components were identified during the fermen­
tation of apple juice shows in Fig. 1 and Supplementary Table 1,
including 14 esters, 11 alcohols, 11 ketones, 7 acids, 6 aldehydes, 4
olefins, and 3 ethers. Among these substances, esters are the most
diverse and have the richest aroma in each fermentation group, followed
by alcohols. During the fermentation of apple juice, the main aroma
components and the characteristic aroma components with large aroma
values and significant contributions are ethyl acetate, hexyl acetate,
ethyl caprylate, isoamyl acetate, ethyl n-hexanoate, and ethyl caprate.
Esters are the main aroma substances in the fermentation process of
apple juice. Ethyl acetate, ethyl butyrate, and ethyl caprate are the main
esters in the fermentation process and produce the fruit aroma. In
addition to acetate, ethyl ester is also a major volatile substance, which
helps to improve the fruit flavor of fermented juice. The maximum
content of ethyl caprylate reached 2.25 mg/L. In this study, after
S. cerevisiae fermentation, the content of ethyl caprylate in the cider
peaked, indicating that yeast is an important producer of ethyl capry­
late. Ethyl caproate may provide fruity, green apple, brandy, banana and
similar aromas to the cider during the fermentation process (Peinado,
Moreno, Bueno, Moreno, & Mauricio, 2004).
Alcohols are one of the most abundant volatile compounds in ciders.
They are also a common final product in glucose degradation and amino
acid catabolism, and play a very important role in the overall aroma of
fermented juice (Di Cagno, Filannino, & Gobbetti, 2017). When the
concentration of higher alcohol exceeds 350 mg/L, it is usually consid­
ered an aroma defect in fruit wine; the concentration of higher alcohols
in this study was within the standard range. After S. cerevisiae fermen­
tation, the ethanol content increased to 6.56%, the content of deal­
coholization reached the requirement of low-alcohol wine, and the lactic
acid fermentation dropped to 0.37%. These results indicate that
L. plantarum fermentation has the role of degrading alcohol. The pres­
ence of 2-methylbutanol in cider may produce malt aroma. After
S. cerevisiae and L. plantarum fermentation, apple juice produces phe­
nethyl alcohol, which has the taste of rose and honey and can increase
the aroma of cider (Peinado, Moreno, Bueno, Moreno, & Mauricio,
2004).
Aldehydes are produced by alcohol oxidation or acid decarboxyl­
ation during the fermentation process. Although the amount is small
during the fermentation process, the aldehyde content increases
compared to before fermentation (p < 0.05), mainly because of the
production of acetaldehyde during the fermentation process. Acetalde­
hyde is an intermediate in the fermentation and metabolism of bacteria,
and is mainly produced by threonine metabolism (Chen, Zhao, Hao, Yu,
Tian, & Zhao, 2017). It is considered the main characteristic flavor

Pyridine 1.790
2,4-Di-tert-butylphenol
Ethyl caprylate
Decanoic acid
Acetic acid glacial
Hexanoic acid
DL-2-Methylbutyric acid
Isovaleric acid
Isobutyric acid
Eicosane
Tetradecane 1.148
Dodecane
Ethyl vinyl ether
3-Nonanone
4-Ethylacetophenone
Acetophenone
3-Hydroxy-2-butanone
2-Octanone
2-Heptanone
Isovaleraldehyde 0.5060
4-Ethylbenzaldehyde
Benzaldehyde
Decanal
Hexanal
Acetaldehyde
Ethyl laurate
Methyl n-caprate
Phenethyl acetate
Ethyl caproate
Isoamyl acetate -0.1360
Ethyl caprate
Ethyl caprylate
Hexyl acetate
banana oil
2-Methylbutyl acetate
Butyl acetate
Ethyl butyrate
Butyl formate
Ethyl acetate -0.7780
n-heptanol
1-Octanol
Phenethyl alcohol
(R)-(-)-1,3-Butanediol
(R,R)-2,3-Butanediol
2-Methyl-1-butanol
3-Methyl-1-butanol
Hexyl alcohol
1-Butanol
Ethanol -1.420

Apple juice Yeast fermentation Dealcoholization Lactobacillus

fermentation

Fig. 1. Changes in volatile substances in apple juice during fermentation.

6
H. Li et al. Food Chemistry 363 (2021) 130351

compound in fermented foods, which can help produce the fragrance of 1.0 FRAP mmol Trolox/L
60 90
fruits. Acetaldehyde is also the main aldehyde in cider (accounting for •O2- scavenging (% ) d
80
75.65–90.92% of the total aldehydes). Compared to S. cerevisiae DPPH scavenging (%) c d 50
0.8 70
fermentation, the content of acetaldehyde significantly decreased after

DPPH scavenging (%)

b

•O2- scavenging (% )
Lactobacillus fermentation. Acetaldehyde has a fruity taste, some char­

FRAP mmol Trolox/L

40 60
d
acteristic aroma compounds are also produced during the fermentation 0.6 c
50
process. Other trace volatiles, such as hexanal, have a sweet taste and a c 30
b 40
orange aroma (Welke, Zanus, Lazzarotto, & Zini, 2014). 0.4 b
a
Regarding ketones, 2-octanone is constantly changing during the 20 30
sequential fermentation of S. cerevisiae and L. plantarum, and the content a 20
0.2
reaches 0.0501 mg/L after fermentation. 3-hydroxy-2-butanone is 10
10
considered relatively tasteless in wine (Romano & Suzzi, 1996). The
acetone content after L. plantarum fermentation reached 0.152 mg/L. At 0.0 0 0
Yeast Dealcoholization Lactobacillus
the beginning of fermentation, no acid was detected in the apple juice. Apple juice
fermentation fermentation
Acidic substances increased after L. plantarum fermentation. The content
of acetic acid was highest, reaching 0.136 mg/L. Capric acid and cap­ Fig. 2. Changes in the antioxidant activity of apple juice during fermentation.
rylic acid were only found in L. plantarum fermentation, which means
that these two acids may only be produced by L. plantarum fermentation. that in the original juice before fermentation (0.4 mmol Trolox/L),
The aroma characteristics of fruit wine during fermentation include similar to a previous study on the effects of L. plantarum fermentation on
hundreds of aroma compounds, many of which have a very low con­ phenolic characteristics and various polyphenol compounds (Kwaw,
centration threshold (μg/L) (Carrau, Gaggero, & Aguilar, 2015), but et al., 2018). Compared to unfermented apple juice, the antioxidant
these substances have an effect on fruit wine. Flavor plays a significant activity of the apple juice after sequential fermentation was significantly
role and affects the quality of fruit wine. different (p < 0.05).

3.6. Comparative analyses of sensory evaluation in different fermentation 4. Conclusion

stages
During the fermentation of apple juice, the color, aroma, taste,
sweetness and posture all affect its sensory quality. Supplementary Fig. 1
shows the sensory score of apple juice during the fermentation process.
At the end of L. plantarum fermentation, apple juice has the richest color,
aroma, and taste. The sensory evaluation at the end of the fermentation
of L. plantarum is better than that of the original juice and S. cerevisiae in
the fermentation stage. The sour taste produced by L. plantarum
fermentation has a typical fermented taste (sourness) and emits a
pleasant smell (Zhao et al., 2016). Fermentation makes the taste of apple
juice more pleasant, edible after fermentation, and may be beneficial to
health.
3.7. Comparative analyses of antioxidant activity of different
fermentation stages
Apple juice is rich in vitamins, polyphenols, and flavonoids, which
have strong antioxidant activity. The activity of DPPH-free radical su­
peroxide anion and iron-reducing power in each fermentation stage of
probiotics have been studied. Apple juice in different fermentation
stages has different antioxidant activities, but overall, the antioxidant
activity continues to increase. The free radical scavenging rate of apple
juice after L. plantarum fermentation was higher than that of S. cerevisiae
fermentation and the original juice.
Fig. 2 shows the DPPH free radical scavenging rate of apple juice
after S. cerevisiae dealcoholization and L. plantarum fermentation was
higher than that before fermentation, and it gradually increased during
the fermentation process. The DPPH scavenging rate of apple juice was
77.45%, which was 36.08% higher than that of unfermented apple juice,
because L. plantarum fermentation can enhance the utilization of poly­
phenol compounds with proton donor properties (dos Santos et al.,
2019). The superoxide anion free radical scavenging rate of apple juice
after fermentation by L. plantarum is similar to that of DPPH free radical
scavenging ability, showing a gradual upward trend. When the
L. plantarum fermentation reached over 45.07%, which was 26.08%
higher than before fermentation. At the end of fermentation, the iron-
reducing ability of apple juice was higher than that of the pre-
fermentation stages. During the fermentation process, the iron-
reducing ability of apple juice continued to increase. After
L. plantarum fermentation, it reached 0.56 mmol Trolox/L, higher than
This thesis studied the changes in the physicochemical characteris­
tics, functional properties, and sensory and antioxidant activity of apple
juice during fermentation by S. cerevisiae, dealcoholization, and
sequential fermentation by L. plantarum. The results showed that during
the fermentation process, 70.70% of the total sugar was consumed, and
the ethanol content reached 6.56% (v/v). The ethanol content was less
than 0.5% (v/v) after dealcoholization. Compared with the original
juice, flavonoids increased by 87.76%, the viable count reached 4.26 ×
108 CFU/mL, and vitamin C increased by 1.02 mg/100 mL. New vola­
tiles (propanol, citronellol, ethyl lactate, and hexyl butyrate) were
produced after fermentation by L. plantarum, and ketones and acids were
enriched. Sequential fermentation improved the antioxidant activity of
apple juice, including the DPPH free radical (77.45%) and superoxide
anion (45.07%) scavenging abilities and iron ion reduction ability (0.56
mmol Trolox/L). This research provides a basis for the industrial
application of a new type of non-alcoholic fermented apple juice. The
shortcoming of this study is that S. cerevisiae removes ethanol, while also
removing some aroma substances after fermenting apple juice. In the
future, molecular distillation will be considered to separate ethanol and
aroma substances, and the aroma substances will be returned to the non-
alcoholic juice to increase its aroma.
CRediT authorship contribution statement
Hongcai Li: Formal analysis, Data curation, Writing - original draft,
Writing - review & editing, Visualization. Jintao Huang: Methodology,
Validation, Formal analysis. Yaqin Wang: Investigation. Xingnan
Wang: Data curation. Yichen Ren: Conceptualization. Tianli Yue:
Resources. Zhouli Wang: Methodology. Zhenpeng Gao: Writing - re­
view & editing, Supervision, Project administration, Funding
acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

7
H. Li et al. Food Chemistry 363 (2021) 130351

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