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*Corresponding author: E-mail: xjg71@163.com; 
 
 
 

Asian Journal of Immunology 
 
4(1): 85-95, 2021; Article no.AJI.71615 
 

 
 

 

 

Fermented Apple Juice by Two Commercial 
Lactobacillus species: Changes of Physicochemical 

Composition and Antioxidant Activity 
 

Wen-Sheng Yan1, Yu-Ru Guo1, Huan-Yang Li1 and Jian-Guo Xu1* 
 

1
School of Food Science, Shanxi Normal University, Linfen, China. 

 
Authors’ contributions  

 
This work was carried out in collaboration among all authors. All authors read and approved the final 

manuscript. 
 

Article Information 
 

Editor(s): 
(1) Prof. Cynthia Aracely Alvizo Báez, Autonomous University of Nuevo Leon,  Mexico. 

Reviewers: 
(1) Abdul Manab, , Brawijaya University, Indonesia.  

(2) Valcineide Oliveira de Andrade Tanobe, Federal University of Paraná, Brazil. 
Complete Peer review History: https://www.sdiarticle4.com/review-history/71615 

 
 
 
 

Received 01 June 2021  
Accepted 04 August 2021 
Published 10 August 2021 

 
 

ABSTRACT 
 

The present study was aimed to compare the changes of physiochemical composition, antioxidant 
activities of fermented apple juice with non-fermented apple juice by two commercial lactic acid 
bacteria (LAB), Lactobacillus casei CICC 20975 and Lactobacillus bulgaricus CICC 21101. The 
antioxidant activity was evaluated by three systems including DPPH, ABTS free radical scavenging 
methods and Fe

3+ 
reducing power. The results showed that fermentation significantly increased the 

content of total phenols in apple juice (P<0.05). After fermentation, all malic acid was converted into 
lactic acid during fermentation with the lactic acid content up to 381.78 mg/kg. Free proline 21.55 
mg/kg and lysine 21.99 mg/kg were also significantly increased. Similarly, fermented apple juice 
showed significantly higher antioxidant activities when compared to non-fermented apple juice. The 
scavenging activity of DPPH, ABTS

 
free radical

 
and the reducing power of Fe

3+
 in fermented apple 

juice increased by 22.4%, 35.0%, 9.7%, respectively. In conclusion, fermented apple juice by two 
commercial lactic acid bacteria (L. casei CICC 20975 and L. bulgaricus CICC 21101) exhibited a 
more satisfied property and possessed great application potentials. 

 

 
Keywords: Fermented apple juice; lactic acid bacteria; physicochemical composition; antioxidant 

activity. 

Original Research Article 



 
 
 
 

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86 

 

1. INTRODUCTION 
 

Lactic acid bacteria (LAB) was a group of 
probiotics that can utilize carbohydrate of matrix 
to produce a large amount of lactic acid, 
including Lactococcus, Lactobacillus, 
Leuconostoc, Pediococcus, Streptococcus and 
Oenococcus species [1]. With an extremely wide 
carbohydrate utilizing profiles ranging from 
common monosaccharides to complex plant 
polysaccharides, LABs were especially suitable 
to ferment various vegetable and fruits juice. 
Fermented vegetable and fruit juice combined 
the beneficial role of probiotics with bioactive 
phytochemical compounds in plants such as 
flavonoids and polyphenols, becoming a kind of 
unique functional food. Nowadays, fermentation 
by different LAB was an effective technology that 
had been used not only to improve the flavor and 
stability of food but also to improve the economic 
and nutritional value of the original product [2,3].  
 

Apple was one of the special local fruit products 
in China. According to the latest statistics, China 
had become the world’s largest producer of 
apples, apple planting area and output 
accounted for more than 50% of the world. 
However, most of the apple products sold were 
fresh food with low processing output, and mainly 
concentrated apple juice, apple vinegar, which 
had a single processing type, single taste and 
low additional value. Among various choices, 
fermented apple juice was a good choice to add 
economical value for apples. 
 

In many literature, Lactic acid bacteria such as L. 
acidophilus, L. rhamnosus, L. casei, L. plantarum 
in fermented apple juice had been reported [4,5]. 
Previous studies in our laboratory optimized 
various LAB to ferment apple juice and 
compared their consumer preferences and the 
number of viable bacteria (data not published). 
The result showed that apple juice fermented 
with L. casei CICC 20975 and L. bulgaricus 
CICC 21101 had typical apple flavor, soft taste, 
delicious with slight sweetness and sour. On the 
basis of the above test, in the present study, we 
compared the physicochemical compounds, 
antioxidative abilities as well as aroma profile of 
the fermented apple juice with non-fermented 
apple juice to indicate the nutritional and 
functional changes before and after fermentation.  
  
2. MATERIALS AND METHODS 
 

2.1 Microorganisms and Culture 
 

Both L. casei CICC 20975 and L. delbrueckii 
subsp. bulgaricus (termed L. bulgaricus 

hereafter) CICC 21101 were purchased from 
China’s Industrial Microbial Preservation 
Management Center (CICC). All bacterial 
cultures were stored frozen at -20°C in MRS 
medium (Aoboxing Biotech Co. Ltd, Beijing, 
China) containing 20% glycerol. The strains were 
reactivated by means of double passage on MRS 
when needed. 
 

2.2 Raw Materials and Reagents 
 

In this work, fuji apples were purchased from 
orchard worker in Jixian, Shanxi, China. The fuji 
apples were cleaned with filtered water and 
chopped into small pieces. Then they were 
soaked in color protector liquid (mixed solution of 
ascorbic acid concentration of 3.5 mg/L and citric 
acid concentration of 7.5 mg/L) for 30 minutes. 
The apple juice was obtained through 
mechanical process by pressing the pulp in juice 
extractor, and then apple juice was heated by 
microwave heating for 2 min. The heated fuji 
apple juice was stored frozen (-20°C) prior to 
use. No additive was added to the juice. 
 

Methanol (HPLC grade) and acetonitrile (HPLC 
grade) were purchased from Merck (Germany); 
MRS broth medium, AGAR and nutrient broth 
medium (NB) were purchased from Beijing 
Aoboxing Biotech Co. Ltd. (Beijing, China); 
Fructose, maltose, sucrose and free amino acid 
were purchased from Sigma Chemical Co. 
Sigma (USA); amino acid standards were 
purchased from Shanghai Anpel laboratory 
Technology Co., Ltd. All other chemicals and 
reagents used in the experiments were of 
analytical grade. 
 

2.3 Preparation of Fermented Apple Juice 
 

L. casei CICC 20975 and L. bulgaricus CICC 
21101 were cultivated on MRS broth at 37°C for 
24 h. Cells at the late exponential phase was 
obtained, harvested by centrifugation (10,000×g, 
10 min, 4°C), washed twice with 50 mM sterile 
phosphate buffer solution (PBS, pH 7.0), and 
then re-suspended in sterile distilled water to the 
final optical density. After that, L. casei CICC 
20975 (3.0×10

6
 CFU/mL) and L. bulgaricus CICC 

21101 (3×10
6
 CFU/mL) were inoculated into 200 

mL freshly prepared apple juice. Mixtures were 
statically cultured at 38°C for 60 h. And the final 
live bacterial counting was performed in MRS 
and the results showed that the live LAB 
numbers in fermented apple juice were up to 1.5-
3.0×10

9
 CFU/mL. In the present study, an 

uninoculated apple juice was used as an 
experimental control. 



 
 
 
 

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2.4 Determination of Free Sugars 
 
The concentration of free sugars was determined 
by HPLC (1200 Series, Agilent, USA) equipped 
with a refractive index detector (RID) according 
to a modified version of the method described by 
Mousavi with some modifications [6]. The mobile 
phase was acetonitrile and water at a volume 
ratio of 7:3. Chromatographic analysis was 
achieved using a column (Agilent amino column 
4.6 mm×250mm, 5 μm) maintained at 35°C. The 
injection volumes of 10 μL were 
chromatographically separated at a flow rate of 
1.0 mL/min for both samples and standards. 
Sugar content was calculated using external 
standards. The following chromatographic grade 
free sugars were used as standards.  
 

2.5 Analysis of Organic Acids  
 
The chromatographic system was used to 
quantify the organic acids consisted of a Diode 
array detector (DAD) (1200 Series, Agilent, USA) 
according to a modified version of the method 
described by Belguesmia et al. with some 
modifications [7]. The mobile phase was K2HPO4 
(10 mM, pH 2.55). One milliliter of sample was 
added 5 mL of mobile phase, ultrasonic extracted 
for 30 min and left for 1 h at 60 °C water bath. 
After being centrifuged for 10 min at 12000×g at 
4 °C, the supernatant was kept for 1 h at 4 °C 
before filter-sterilization (0.45 μm pore size). The 
samples were then kept at –80 °C until analysis. 
Chromatographic analysis was achieved using a 
column (Agilent AQ 4.6 mm×250 mm, 5 μm) 
maintained at 30 °C. The injection volumes of 10 
μL were chromatographically separated at a flow 
rate of 0.5 mL·min

-1
 for both samples and 

standards. The wavelength was set at 210 nm to 
detect the organic acids. The following 
chromatographic grade organic acids were used 
as standards. To ensure accuracy, the working 
standards were prepared daily. The HPLC 
results were qualitatively analyzed by peak 
retention time and quantified by peak area using 
the external standard method. Extractions and 
injections were conducted in triplicate for each 
fermentation replicate. 
 

2.6 Determination of Free Amino Acids  
 
FAAs analysis was carried out using acid 
hydrolysed (0.2 mM HCl) samples by reverse-
phase high-performance liquid chromatography 
(HPLC) after precolumn derivatization by 
phenylisothiocyanate (PITC), by a modified 
method adapted from Zhao [8].  

Extraction of FAAs from apple juice: One gram of 
sample was added with 8 mL of 0.2 mM 
hydrochloric acid, shook in a vortex for 5 min, 
then extracted by ultrasound for 10 min. After 
standing in the dark for 2 h, it was centrifuged at 
2057×g for 10 min, taking the supernatant for 
later use. 
 
Instrument method: The FAAs contained in the 
samples were separated using an amino acid 
SHISEIDO C18 column, 5 μm (250×4.6 mm) 
attached to Agilent 1260 Chromatography 
system equipped with UV-Vis detector monitoring 
at 254 nm. The injection volume was set at 10 μL 
and the column was kept at 40°C. The gradient 
mobile phase, consisting of eluent A (prepared 
by mixing the 0.1 mol/L sodium acetate with 
acetonitrile at a ratio of 97: 3) mix well and adjust 
pH to 6.5 (31.815 g sodium acetate plus 3880 
mL water plus 120 mL acetonitrile) and eluent B 
(80% acetonitrile, 20% Milli-Q water) was 
injected at a flow rate of 1 mL / min throughout 
the experiment. The gradient program was 
defined as follows: 100 % A at start, 85 % A and 
15 % B at 14 min, 66 % A and 34 % B at 29 min, 
0 % A and 100 % B at 30 min and for 7 min, 
100% A at 38 min and for 9 min, 100 % A at 45 
min, allowing the column to equilibrate for 15 min 
until the 60th min. 
 

2.7 Determination of the Total Phenolic 
Content 

 
Total phenolic content was determined based on 
the Folin-Ciocalteu colorimetric method as 
described by Xu et al. [9]. Gallic acid was used 
as a reference standard, and the values of total 
phenols were expressed as milligram of gallic 
acid equivalent (GAE) per milliliter of fruit juice. 
The juice was diluted with a mixture of methanol 
(80%)-water and centrifuged at 6650×g for 10 
min. The supernatant was used as a test sample 
for determining the total phenolic content. Briefly, 
an aliquot (0.5 mL) of appropriately diluted apple 
juice, 2.5 mL of deionized water and 0.5 mL of 
1.0 M Folin-Ciocalteu reagent were mixed within 
10 mL volumetric flasks and vortexed. After 
8min, 1.5mL of 7.5% sodium carbonate solution 
was added and mixed thoroughly. The 
absorbance of the reaction mixtures was 
measured using a spectrophotometer at 765 nm 
wavelength after incubation for 2 h at room 
temperature. Methanol was used as the blank, 
and gallic acid (GA) was used for calibration of 
the standard curve (0-500 mg·L

-1
). Phenolic 

content was expressed as gallic acid equivalents 
(milligrams of GAE per gram juice). 



 
 
 
 

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2.8 Determination of the Content of Total 
Flavonoid 

 
The total flavonoid was measured by the method 
of by Feng and Xu [10]. Rutin was used as a 
reference standard, and the total flavonoid 
content was calculated as the equivalent of rutin 
content per mL of sample.  
 
2.8.1 Determination of the content of ascorbic 

acid (Vc), total titratable acidity (TTA), pH, 
and total soluble solids (TSS) 

 
The content of Vc was determined by 2, 6-
dichlorophenol titration method according to the 
publication of AOAC [11]. TAA content was 
measured by using the method of AOAC [11]. 
Briefly, samples were 10-fold diluted, and then 
titrated with 0.1 N NaOH with phenolphthalein as 
indicator. The pH of the juice was measured 
using a pH meter at 25℃. TSS in the juice 
sample was determined by a benchtop digital 
refractometer (PAL-1, Atago, Japan).  
 

2.9 Determination of Antioxidant Activity 
 
2.9.1 DPPH free radical scavenging activity 
 
The DPPH free radical scavenging activity 
method according to the method as previously 
described by Guo et al. [12]. Briefly, each of 
sample solutions was serially diluted to various 
concentrations in methanol respectively, and 
then a 0.5 mL of samples was mixed with 2.5 mL 
of 60 μM DPPH dissolved in methanol. The 
mixture was shaken vigorously and left to stand 
for 30 min in the dark, and the absorbance was 
measured at 517 nm against a solvent blank. 
The scavenging rate on DPPH free radical was 
calculated according to the formula: Scavenging 
rate (%) = [1- (Asample – Ablank) /Acontrol] 
×100. Where, A was the absorbance of the 
sample, blank, or control, as indicated. The 
DPPH free radical scavenging activity of apple 
juice was expressed in mM of Vc. 
 
2.9.2 ABTS free radical scavenging activity 
 
The ABTS

 
free radical scavenging activity 

method as described by Xu et al. [13]. ABTS free 
radical cation was generated by a reaction of 7 
mmol/L ABTS and 2.45 mmol/L potassium 
persulfate. The reaction mixture was allowed to 
stand in the dark at room temperature for 16−24 
h before use and was used within 2 days. The 
ABTS solution was diluted with methanol to an 
absorbance of 0.700 ± 0.050 at 734 nm. One 

hundred microliters of the diluted samples were 
mixed with 2.0 mL of diluted ABTS solution. The 
mixture was allowed to stand for 6 min at room 
temperature, and the absorbance was 
immediately recorded at 734 nm. The 
scavenging rate were calculated using the 
equation described for DPPH assay. The ABTS 
free radical scavenging activity of apple juice was 
expressed in mM of Vc. 
 
2.9.3 Determination of Fe

3+
 reducing power 

 
The Fe

3+
 reducing power method as described 

by Kwaw et al. [14]. An aliquot of the apple juice 
(1 mL, diluted at 1:100) was mixed with 0.05 mL 
of HCl (0.01 M), 0.4 mL of potassium ferricyanide 
(0.02 M), 0.4 mL of 0.02 M FeCl3 and 0.7 mL of 
distilled H2O. The mixture was consequently 
incubated at 37 

o
C in the dark for 30 min and the 

absorbance read at 720 nm. The Fe
3+

 reducing 
power of apple juice was expressed in mM of Vc. 
 

2.10 Electronic Nose Measurements 
 
The aroma profiles of the juice samples were 
analyzed using an electronic nose system 
(PEN3, Airsence, Germany) [15]. Briefly, each 
sample (15 mL) was put into a 100 mL glass jar. 
Then the glass jar was sealed with three layers 
of plastic wrap and the headspace inside it was 
equilibrated for 30 min at 45°C under agitation at 
a speed of 500 rpm. The headspace volatiles 
were put into the electronic nose for 9 sec at a 
rate of 7.7 mL·min

-1
. To acquire stable signals, 

the acquisition duration for the sensors was 90s. 
The analysis system was purged with processed 
dry and pure air before each analysis. Each 
sample was tested 6 times to ensure the 
accuracy of the data, and the last measured 
three data were used in the subsequent analysis. 
The serial number, main applications, and 
references of the ten sensors were listed in Table 
1 [16]. 
 

3. RESULTS AND DISCUSSION 

 

3.1 Changes of Sugars Content in Apple 
Juice 

 
The content of the free sugars and total soluble 
solids (TSS) in fermented and non-fermented 
apple juice were shown in Table. 2. As we can 
see from Table. 2, the levels of TSS, fructose 
and glucose decreased significantly (P<0.05). 
The soluble solid (TSS) of apple juice decreased 
by 3.4% during fermentation. The soluble solids 
utilization was high in fermented apple juice 



 
 
 
 

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89 

 

compared to non-fermented apple juice due to 
the higher microbial load [17]. There was 
literature reported that the decrease in sugar 
concentrations during fermentation was largely 
due to not only bioconversion into lactic acid, but 
also the utilization for growth and metabolism of 
lactic acid bacteria [18]. Besides, 5.3% of 
fructose and 1.3% glucose were utilized during 
fermentation, respectively. Similar results were 
reported for Sohiong juice [19] and Pomegranate 
Juice [7]. It had been reported that fructose and 
glucose were efficient carbon and energy source 
for most of Lactobacillus strains [20]. Among the 
free sugars, the concentrations of sucrose and 
maltose showed no significant difference when 
compared fermented juice with non-fermented 
juice (P>0.05). This may be the result that 
sucrose and maltose were not utilized by lactic 
acid bacteria during fermentation generally. In 
addition, there were no detectable lactose in 
fermented and non-fermented juices. 
 

3.2 Changes of Acid Substances of Apple 
Juice  

 
The total acid (TA), pH and the content of nine 
free sugars in fermented and non-fermented 
apple juice were shown in Tab. 3. As shown in 
Table. 3, the total acid content of fermented 
apple juice increased significantly (P<0.05), and 
the pH content decreased significantly (P<0.05). 
The total titratable acid concentration decreased 
by 28.1% from 0.69 g·kg

-1
 to 0.96 g·kg

-1
. At the 

meantime, the pH decreased by 29.4% from 5.21 
to 3.68. The low pH and high acidity were in 
agreement with the findings reported by Ibanoglu 
et al. [21].  
 

3.3 Changes of Free Amino Acid Content 
of Apple Juice  

 
It had been reported that soluble substances, 
some fat and some vitamins in fruit juice can be 
metabolized by probiotics into amino acids, fatty 
acids and so on [22]. The varieties and contents 
of free amino acids in fermented and non-
fermented apple juice were shown in Table. 4. 
According to Table. 4, a total of 17 amino acids 
were detected in fermented and non-fermented 
apple juice, among which 7 were essential amino 
acids for human body. Changes in the content of 
nonessential amino acids (NEAA) were similar to 
total free amino acids (TFAA), there were no 
significant differences in fermented and non-
fermented apple juice (P>0.05). Among the 17 
detected amino acids, aspartic acid was the most 
abundant, and there was no significant change 

after fermentation, followed by glutamic acid and 
serine, which decreased significantly from 49.13 
mg·L

-1
 to 14.58 mg·L

-1
 and decreased from 

293.68 mg·L
-1

 to 255.86 mg·L
-1

, respectively. 
Similar results were reported by Xu et al. [23]. A 
total of seven total essential amino acids were 
detected and represented 4.6% and 3.6% of the 
total free amino acids in fermented and non-
fermented apple juice, respectively. Meanwhile, 
cysteine, phenylalanine and methionine were not 
detected. Moreover, the content of proline was 
up to 21.55 mg·kg

-1
 and 3.7 times higher than 

the juice without fermentation. The similar trend 
was also found in the content of lysine with a 
concentration of 21.99 mg·kg

-1
 in fermented 

juice. 
 

3.4 Changes of Phytochemical Contents 
in the Apple Juice 

 
The contents of phytochemical compounds, 
including total polyphenols, flavonoids and Vc, 
fermented and non-fermented apple juice were 
shown in Fig. 1. As indicated in Fig. 1, except for 
the content of total polyphenols, the contents of 
the other two active compounds in the juice with 
and without fermentation showed no significant 
differences. Interestingly, total polyphenols 
significantly increased by 14.4% and the value 
was up to 0.97 milliequivalent of gallic acid. 
Previous studies showed that vegetable or fruits 
juice fermented by Lactobacillus showed higher 
contents of polyphenols and flavonoids, 
suggesting a bio-conversion role of LAB in this 
process [24]. As for the change of Vc content, 
Kaprasob et al. also reported that cashew apple 
juice fermented with L. plantarum retained a 
matchable level. This may be due to the 
protective effect of LAB fermentation and they 
may prevent the degradation of Vc. All of these 
results indicated that LAB fermentation can 
biotransform some natural botanical compounds 
into bioactive compounds or prevent the loss of 
bioactive phytochemicals, and finally enhance 
the quality of products.  
 

3.5 Changes of Antioxidant Activity of 
Apple Juice 

 
The changes of antioxidant activity of in 
fermented and non-fermented apple juice were 
shown in Fig. 2. It can be seen from Fig. 2 that 
the antioxidant activity of fermented apple juice 
was significantly increased (P<0.05). After 
fermentation, the DPPH free radical scavenging 
rate reached up to 56.9% equivalent to 7.32 
mmol·L

-1
 Vc and it was 1.3 times of that non-  



 
 
 
 

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90 

 

Table 1.  Sensors used and their main applications in PEN 3 
 

Number in Array Sensor name General description Reference (ppm) 

S1 W1C Aromatic compounds  Toluene,10 
S2 W5S Very sensitive, broad range sensitivity, react on nitrogen oxides, very sensitive with negative signa NO2, 1 
S3 W3C Ammonia, used as sensor for aromatic compounds Benzene, 10 
S4 W6S Mainly hydrogen, selectively (breath gases) H2, 0.1 
S5 W5C Alkenes, aromatic compounds, less polar compounds Propane, 1 
S6 W1S Sensitive to methane broad range CH3, 100 
S7 W1W Reacts on sulfur compounds, sensitive to many terpenes and sulfur organic compounds, which are important for smell, 

limonene, pyridine 
H2S, 1 

S8 W2S Detects alcohols, partially aromatic compounds, broad range CO, 100 
S9 W2W Aromatics compounds, sulfur organic compounds H2S, 1 
S10 W3S Reacts on high concentrations, sometime very selective (methane) CH3, 100 

 
Table 2. Changes of sugar content in fermented and non-fermented apple juice 

 

Sample Soluble solids (
o
Brix) Free sugar 

Fructose (g·100g
-1

) Glucose (mg·kg
-1

) Sucrose (g·100g
-1

) Maltose (ug·g
-1

) Lactose (g·100g
-1

) 

AJ 12.8±0.5a 6.24±0.20a 4.46±0.23a 0.77±0.09a 0.12±0.04a NF 
FAJ 9.4±0.3b 5.91±0.19b 3.80±0.16b 0.79±0.21a 0.15±0.03a NF 

Different letters within a column indicate statistically significant differences between the means (P<0.05); NF, not found; AJ, apple juice; FAJ, fermented apple juice. 
 

Table 3. Changes of acid substances of fermented and non-fermented apple juice 
 

Sample TTA (g·kg
-1

) pH Organic acid (ug·g
-1
） 

oxalic acid tartaric acid malic acid lactic acid acetic acid citric acid oxalic acid maleic acid fumaric acid 

AJ 0.69±0.12b 5.21±0.64a NF 0.59±0.18a 204.30±9.21 NF  NF NF NF NF NF 
FAJ 0.96±0.06a 3.68±0.43b NF 0.45±0.09b NF  381.78±7.89 NF NF NF NF NF 
Numbers represent mean values of three independent replicates ± SD; TTA, Abbreviations of total titratable acid; NF, not found; AJ, apple juice; FAJ, fermented apple juice; Different letters within a column indicate 

statistically significant differences between the means (p < 0.05). 



 
 
 
 

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91 

 

Table 4. Changes of free amino acid content (mg·kg
-1

) of fermented and non-fermented apple 
juice 

 

Amino acids AJ FAJ Amino acids AJ FAJ 

aspartic acid 415.61±10.91a 418.13±9.88a proline 5.84±0.38b 21.55±0.52a 
glutamate 49.13±5.14a 14.58±2.12b tyrosine NF NF 
cystine NF NF valine 7.02±0.23 NF 
serine 293.68±9.35a 255.86±8.89b methionine NF NF 
 glycine 4.48±0.23 NF isoleucine 6.11±0.41a 2.42±0.34b 
histidine NF NF leucine 1.53±0.08a 1.43±0.19a 
arginine NF NF phenylalanine 

(Phe)△ 

NF NF 

threonine 2.52±0.21a 1.66±0.17a lysine 20.3±3.18b 21.99±2.91a 
alanine 12.04±0.75a 10.61±2.21a    
TEAA 37.48±3.34a 28.92±2.26b    
NEAA 780.78±18.23a 782.31±26.39a    
TFAA 818.26±10.45a 811.23±12.18a    

Numbers represent mean values of three independent replicates ± SD; NF, not found; AJ, apple juice; FAJ, 
fermented apple juice; Abbreviations of amino acids (TEAA, total essential amino acids; NEAA, nonessential 
amino acids; TFAA, total free amino acids); Different letters within a column indicate statistically significant 

differences between the means (P<0.05). 
 

 
 

Fig. 1. Changes of antioxidant substances of fermented and non-fermented apple juice 
Different letters within a column indicate statistically significant differences between the means (P<0.05); AJ, 

apple juice; FAJ, fermented apple juice. 
 

 
 

Fig. 2. Changes of antioxidant activity of fermented and non-fermented apple juice 
Different letters within a column indicate statistically significant differences between the means (p < 0.05); AJ, 

apple juice; FAJ, fermented apple juice 



 
 
 
 

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92 

 

fermented apple juice. As for ABTS free radical 
scavenging rate, the activity reached up to 58.9% 
equivalent to 13.45 mmol·L

-1
 Vc and had a 50% 

increase. The Fe
3+

 reducing power of fermented 
apple juice was 82.3% equivalent to 9.35 
mmol·L

-1
 Vc and it was 1.1 times of that non-

fermented apple juice. Similar results reported 
that Noni juice fermented with Bifidobacterium 
longum had greater antioxidant activity than non-
fermented noni juice.  
 

3.6 Analysis of Aroma Composition of 
Apple Juice  

 

The aroma profiles of apple juice during 
fermentation were examined using an electronic 
nose. Fig. 3 displayed a typical response of ten 
sensors during measurement of apple juice (0h) 
and fermented apple juice (60h) in which each 
curve represented a different sensor response 
with time. The ordinate represented the changing 
ratio between G and G0. It was apparent that, the 
responsive values of the sensors, after an initial 
period of low responsive values, increased 

sharply and then stabilized after 60 s. In this 
research, the responsive values of each sensor 
at 60 s point were used in analysis.  

 
As shown in Fig. 3, compared with Fig. (a), the 
(b) of response values of W5S, W1S, W1W, 
W2S, W6S and W2W were enhanced to varying 
degrees. With the increase of fermentation time, 
the response values of W1W and W2W 
decreased at first (0h~12h) and then increased 
(12h~60h), while the response values of W5S, 
W1S and W2S gradually increased to stable with 
the increase of fermentation time, but the 
response values of other sensors did not change 
obviously. The differences in flavor of apple juice 
during different fermentation stages may be due 
to the changes in the substances represented by 
the sensors. Therefore, the characteristic flavor 
of fermented apple juice may be derived from 
esters, nitrogen, methane, sulfur compounds, 
alcohols, hydrogen, alcohols, and the dynamic 
changes of these substances affected the overall 
flavor. 

 

 
(a). 0h; (b). 60h 

 
Fig. 3. Response curves of electronic nose sensors to fermented and non-fermented apple 

juice 

0

5

10

15

20

0 20 40 60 80 100

Time(s)

R
es

po
ns

e
 v

al
ue

s 
G

(G
0
)

 

 

 

 

 

 

 

 

 

 

W1C

W5S

W3C

W6S

W5C

W1S

WIW

W2S

W2W

W3S



 
 
 
 

Yan et al.; AJI, 4(1): 85-95, 2021; Article no.AJI.71615 
 

 

 
93 

 

 
 

Fig. 4. Changes in response curves of electronic nose sensors to apple juice at different 
fermentation stages 

 

4. CONCLUSION 
 
The results showed that the LAB might had 
utilized carbohydrates and produced large 
amounts of organic acid, thus lowering the pH of 
the samples during fermentation. Lactic acid 
bacteria fermentation can release the content of 
organic acids, free amino acids and convert 
phenolic compounds to enhance the antioxidant 
activity. In addition, the characteristic flavor of 
fermented apple juice may be derived from 
nitrogen, methane, sulfur compounds, alcohols, 
hydrogen, alcohols. 
 
These findings highlighted the beneficial effect of 
probiotic fermentation on the quality of apple 
juice. Probiotic fermentation changed the 
phytochemical composition of fruits juice, which 
thus enhanced their antioxidant activity. All of 
these contribute to a more satisfied quality of the 
final product from both stability and nutrition 
perspectives. Based on the findings in this study, 
apple juice fermented by L. casei CICC 20975 
and L. bulgaria CICC 21101 was satisfied 
leavening agents for a health beverage. 
However, additional research was needed on the 
control of the fermentation process and the 
identification of the key active compounds and 
flavor substances produced during fermentation. 
All these works assist in obtaining more desirable 
organoleptic qualities in fermented food products. 

DISCLAIMER 
 
The products used for this research are 
commonly and predominantly use products in our 
area of research and country. There is absolutely 
no conflict of interest between the authors and 
producers of the products because we do not 
intend to use these products as an avenue for 
any litigation but for the advancement of 
knowledge. Also, the research was not funded by 
the producing company rather it was funded by 
personal efforts of the authors. 
 

CONSENT  
 
Not applicable. 
 

ETHICAL APPROVAL 
 
Not applicable. 
 

COMPETING INTERESTS 
 

Authors have declared that no competing 
interests exist. 
 

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provided the original work is properly cited. 

 
 

 

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