ORIGINAL RESEARCH

published: 09 February 2022

doi: 10.3389/fnut.2022.833906

Fermentation and Storage

Characteristics of “Fuji” Apple Juice
Using Lactobacillus acidophilus,
Lactobacillus casei and Lactobacillus
plantarum: Microbial Growth,
Metabolism of Bioactives and in vitro
Bioactivities
Jie Yang 1,2 , Yue Sun 1,2 , Tengqi Gao 1,2 , Yue Wu 3 , Hao Sun 1,2 , Qingzheng Zhu 1,2 ,
Chunsheng Liu 1,2 , Chuang Zhou 4 , Yongbin Han 5 and Yang Tao 5*
1
Jiangsu Key Laboratory of Marine Bioresources and Environment/Jiangsu Key Laboratory of Marine Biotechnology, Jiangsu
Ocean University, Lianyungang, China, 2 Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean
University, Lianyungang, China, 3 Sonochemistry Group, School of Chemistry, The University of Melbourne, Parkville, VIC,
Australia, 4 Department of Animal Husbandry and Veterinary Medicine, Jiangsu Vocational College of Agriculture and Forestry,
Jurong, China, 5 College of Food Science and Technology, Whole Grain Food Engineering Research Center, Nanjing
Agricultural University, Nanjing, China
Edited by:
Yu Xiao,
Hunan Agricultural University, China Fruit juices have been widely used as the substrates for probiotic delivery in non-dairy
Reviewed by: products. In this study, three lactic acid bacteria (LAB) strains, including Lactobacillus
Hossein Kiani,
acidophilus, Lactobacillus casei and Lactobacillus plantarum, were selected to ferment
University of Tehran, Iran
Kit Wayne Chew, apple juice. During 72-h of fermentation, these LAB strains grew well in the apple juice
Xiamen University Malaysia, Malaysia with significant increases in viable cell counts (from 7.5 log CFU/mL to 8.3 log CFU/mL)
*Correspondence: and lactic acid content (from 0 to 4.2 g/L), and a reduction of pH value (from 5.5
Yang Tao
[email protected]
to around 3.8). In addition, the antioxidant and antibacterial capacities of fermented
apple juice in vitro were significantly improved through the phenolic and organic acid
Specialty section: metabolisms. After storage at 4◦ C for 30 days, the total amino acid content of fermented
This article was submitted to
apple juice was significantly increased, although the viable cell counts and total phenolic
Food Chemistry,
a section of the journal content were decreased (p < 0.05). Furthermore, the stored fermented apple juices still
Frontiers in Nutrition possessed antibacterial and in vitro antioxidant activities. Overall, all the selected LAB
Received: 12 December 2021 strains could be suitable for apple juice fermentation and can effectively improve their
Accepted: 17 January 2022
Published: 09 February 2022
biological activities.
Citation: Keywords: lactic acid bacteria, apple juice, fermentation, physicochemical properties, bioactivities
Yang J, Sun Y, Gao T, Wu Y, Sun H,
Zhu Q, Liu C, Zhou C, Han Y and
Tao Y (2022) Fermentation and INTRODUCTION
Storage Characteristics of “Fuji” Apple
Juice Using Lactobacillus acidophilus,
Probiotics are living microorganisms and potential functional foods, which have a positive impact
Lactobacillus casei and Lactobacillus
plantarum: Microbial Growth,
on the human body by improving the microbial balance in the human intestine (1). Lactic acid
Metabolism of Bioactives and in vitro bacteria (LAB) are important probiotics, and they are often used in yogurt and other dairy products.
Bioactivities. Front. Nutr. 9:833906. In recent years, the researches on fermented fruit and vegetable juices by lactic acid bacteria have
doi: 10.3389/fnut.2022.833906 gradually emerged. Meanwhile, the related products in the Chinese market are still limited, which

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Yang et al. Lactobacillus Fermented Apple Juice

could not meet the consumer’s demand. After the fermentation organic acid standards (oxalate acid, pyruvic acid, malic acid,
of fruit and vegetable juices by LAB strains, their nutritional and citric acid, succinic acid and lactic acid), phenolic acid standards
functional values are improved, which not only gives fruit and (gallic acid, protocatechuic acid, catechin, procyanidin B2 ,
vegetable juice a special taste and taste, but also extends their shelf chlorogenic acid, hydroxybenzoic acid, caffeic acid, ferulic acid,
life (2, 3). rutin and quercetin), amino acid standards (Asp, Thr, Ser, Glu,
Apple is one of the most consumed fruits in the world, Gly, Ala, Cys, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg, Pro) and
especially in “fuji” and related varieties which are widely accepted sugar standards (fructose, sorbitol, glucose and sucrose) were all
by consumers because of its excellent sensory properties (4, 5). bought from Yuanye Biotechnology Co., Ltd (Shanghai, China).
They are characterized by high sugar content and low acidity
(6). With the improvement of living standards, consumers are Lactic Acid Bacteria Strains and Inoculum
more concerned about the freshness, originality, physicochemical Preparation
properties, and nutritional values of food products including Three commercial LAB strains L. acidophilus BNCC 185342, L.
fruit juice (7). Apple juice has some positive impacts on human casei ATCC 393, L. plantarum BNCC 337796 were provided
health due to its rich contents of polyphenols such as isoflavones, by Beijing Beina Chuanglian Biotechnology Research Institute
flavonoids and phenolic acids (8). Although fruit juices are rich (Beijing, China). All stains were activated in MRS broth (Bo
in minerals, sugars and vitamins, they contain low amounts of Microbiology Technology Co., Ltd. Shanghai) at 37◦ C for 24 h
free peptides and amino acids, which is adverse to the growth and to achieve a final concentration at 9.0 log CFU/mL.
metabolism of human intestinal microorganisms (9). To address
this problem, LAB strains are widely used for the fermentation of Preparation of Apple Juice and
fruit and vegetable substrates due to their excellent tolerability Fermentation
in acidic environments. In addition, the nutrients of apples Fresh apples (“Red Fuji” apple from Xuanwu Fruit Store,
are retained due to the fermentation by probiotics, resulting in Nanjing, Jiangsu Province) were washed, peeled, nucleated, and
unique flavor and efficacy (8). In the literature, Chen et al. (10) cut into small pieces. Next, 0.15% (w/w) ascorbic acid (also
studied the effect of four LAB strains on the flavors of fermented known as vitamin C) was added during crushing. Then, apple
apple juice. The results showed that the total acid concentration slurries were filtered through four layers of gauze and centrifuged
and microbial activity under the fermentation of lactic acid at 6,000 × g for 20 min. The supernatant was collected, and the
bacteria increased markedly, and new volatile compounds with soluble solid content and pH were adjusted to 13% using glucose
important flavors were produced. Kwaw et al. (11) reported and 6.5 using 1N NaOH, separately.
that the effects of lactic acid bacteria on the color, phenolics Apple juice was sterilized at 110◦ C for 10 min before
(such as anthocyanins) and antioxidant activity of mulberry fermentation. After cooling to room temperature, 8 mL of
juice. The results found that lactic acid fermentation can enrich inoculum containing various LAB strains were added into
the bioactive components in mulberry juice. Similarly, Vivek et 400 mL of apple juice, respectively. The initial viable bacterial
al. (12) found an increase trend of antioxidant activity, total count in apple juice was about 7.5 log CFU/mL. Next, apple
phenolic and anthocyanin contents in “Sohiong” juice fermented juice with different LAB strains were placed into an incubator
by L. plantarum, confirming the suitability and feasibility of to ferment for 72 h at 37◦ C. After that, fermented juices were
developing a non-dairy based probiotic drinks from Sohiong immediately stored at 4o C for 30 days. Unfermented apple juice
juice. Kaprasob et al. (13) also reported an increase in phenolic was selected as a control group. Samples were collected for
compounds, vitamin C and other antioxidants in cashew apple analysis during fermentation at 0, 12, 24, 48, 72 h and storage at
juices fermented by L. casei, L. plantarum and L. acidophilus. 10, 20, 30 d. Before the physicochemical analysis, the collected
In theory, by using the selected three lactic acid bacteria, apple samples were centrifuged at 4◦ C and 12,000 × g for 15 min.
juice can be processed into value-added products. However,
there is still limited researches about the effect of LAB on the Determination of Viable Cell and pH
fermentation and preservation of apple juice. The number of viable bacteria was determined by the
In this study, three potential probiotics, L. plantarum, L. plate counting method. Specifically, 1 mL fermented juices
acidophilus and L. casei, were selected to ferment apple juice. were diluted serially with sterile saline to 104 -1010 dilutions.
The changes of organic acids, sugars, free amino acids, and Inoculation of 0.1 mL of the diluted samples were plated on
phenolics during fermentation and storage (4◦ C for 30 days) were the MRS agar. The plates were incubated at 37◦ C for 36–48 h.
evaluated. Finally, this study can provide the basic knowledge of Plates containing 30–300 colonies were counted and recorded
the biotransformation of relevant components in apples by LAB as log CFU/mL (14). The pH of fermented apple juice was
fermentation, and guide the development of fermented apple monitored by a digital pH meter (PHS-3C, Shanghai INESA
juice products with high nutritional value and safety. Scientific Instrument Co., Ltd, Shanghai, China).

MATERIALS AND METHODS Determination of the Browning Index

The browning index of unfermented and fermented apple juice
Materials and Chemicals samples were measured referring to Tiwari et al. (15). 3 mL
Fresh apple was provided by Xuanwu Fruit Store, Nanjing, of centrifuged apple juice was thoroughly mixed with 3 mL of
Jiangsu Province. Ethanol, acetonitrile, Folin-Ciocalteu reagent, anhydrous ethanol, and then the mixture was centrifuged at 4◦ C

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Yang et al. Lactobacillus Fermented Apple Juice

and 12,000 × g for 15 min. The supernatant was collected, and B; 25–45 min, 40–65% B; 45–55 min, 65–95% B; 55–58 min, 95–
its absorbance was measured at 420 nm in a spectrophotometer 100% B; 58–65 min, 100% B. Flavonoids and phenolic acids in
(LabTech UV9100, USA). samples were detected at 350 and 280 nm respectively. The results
were expressed in mg/L.
Determination of Organic Acid Contents
Organic acids were determined by a LC-2010A system (Shimadzu Determination of Antioxidant Activities
Company, Japan) according to the previously reported method in vitro
(16). The mobile phase is KH2 PO4 (0.08 mol/L, pH 2.9). An The free radical scavenging activity of apple juice was assessed
Agilent TC-C18 chromatographic column (250 × 4.6 mm i.d., by the ABTS·+ method, as described in previous paper (21). The
5 mm) was applied for the separation of organic acids and it calibration curve was made using Trolox as the standard, and
was maintained at 30◦ C. The injection volumes of the samples the results were expressed as mmol Trolox/L. Ferric reducing
or standards were 20 µL, and the flow rate was 0.7 mL/min. antioxidant power (FRAP) was determined according to our
Organic acids were detected using an ultraviolet detector (DAD) previously reported method (21). FeSO4 was employed as the
at 210 nm. The content of each organic acid was expressed standard to obtain the calibration curve, and the results were
as mg/L. expressed as mmol Fe 2+ /L.

Determination of Sugar Contents Statistical Analysis

The sugars were determined by a HPLC-ELSD system (Agilent The experiments were repeated three times. The test data were
Technologies, USA., Alltech-ELSD 3300 evaporative light analyzed by Excel, Origin 8.0, Minitab, and SPSS 20.0 software.
scattering detector: Otai Technology, USA) coupled with a Grace Significance was defined at p < 0.05, and Duncan’s test was used.
Prevail Carbohydrate ES column (250 × 4.6 mm i.d., 5 mm)
according to the previously reported method (17). The mobile RESULTS AND DISCUSSION
phase consisted of 25% (v/v) H2 O and 75% (v/v) acetonitrile. The
solvent flow rate was 1.0 mL/min and the injection volume was Bacterial Growth and Change in pH During
10 µL. The column temperature was maintained at 30◦ C. The LAB Fermentation and Storage
drift tube temperature of the ELSD detector was 80◦ C, the flow Changes in viable cell counts and pH value of apple juice
rate of nitrogen gas was 1.5 L/min. The content of each sugar was fermented with L. acidophilus, L. casei and L. plantarum during
expressed as mg/L. 72-h fermentation and the subsequent 30-day storage are shown
in Figure 1, which can reflect the growth status of these
Determination of Free Amino Acids strains. As can be seen from Figure 1A, L. acidophilus, L. casei
and L. plantarum all grew well in the apple juice, and there
Contents
was no significant difference in their viable cell count during
The apple juice collected at different time intervals was filtered
the fermentation process (p ≥ 0.05). At the latter period of
through 0.45 µm membranes. The amino acids were analyzed
fermentation (from 48 to 72 h), the viable cell counts of L.
by a L-8900 automatic amino acid analyzer (Hitachi Technology
acidophilus, L. casei and L. plantarum were still above 8.3
Company, Japan). The content of each free amino acid was
log CFU/mL. This result demonstrates that the growth of L.
expressed as mg/L.
acidophilus, L. casei and L. plantarum metabolized vigorously.
Meanwhile, this result also confirmed that apple juice can be
Determination of the Contents of Total a suitable substrate for the fermentation of the selected strains.
Phenolics and Individual Phenolic Additionally, the viable cell counts of three LAB strains were
Compounds maintained above 8.0 log CFU/mL in apple juice during the first
The method for the determination of total phenols was the Folin- 20 days of storage at 4◦ C. However, during the storage of 20–
Ciocalteu method (18). Specifically, 0.2 mL of diluted apple juice 30 days, the viable cell counts of L. casei decreased rapidly to
sample was mixed with 1.5 mL of 7.5% (w/v) Na2 CO3 and 1.5 mL 5.4 ± 0.04 log CFU/mL, following L. acidophilus (5.6 ± 0.13 log
of Folin-Ciocalteu (10-fold dilution). After standing at 25◦ C in CFU/mL) and L. plantarum (5.7 ± 0.04 log CFU/mL). These
darkness for 40 min, and the absorbance was measured at 765 nm. results are in line with the findings reported by Yoon et al.
The total phenolic content was standardized against gallic acid (22), which states that L. casei and L. plantarum were able to
and expressed as gallic acid equivalents/L. grow in cabbage juice without supplementing nutrients. With
The phenolic substances were analyzed using a Shimadzu- the increase of storage time, the substances that can be used by
HPLC system coupled with an Inertsil ODS-3 column (250 × LAB in the apple juice were dramatically reduced with a marked
4.6 mm i.d., 5 mm) (LC-2010A, Shimadzu Corporation, Japan) accumulation of the metabolites (13). Eventually, the growth of
according to the previously reported method (19, 20). The the LAB reached the death phase, when the viable cell counts of
column was maintained at 25◦ C, and the injection volume was LAB rapidly decreased.
20 µL. The binary mobile phase consisted of (A) glacial acetic LAB strains can metabolize sugar and produce organic acids,
acid aqueous (1%, v/v) and (B) glacial acetic acid methanol (1%, thus changing the pH value of apple juice during fermentation
v/v). The samples were eluted at a flow rate of 0.6 mL/min with and storage (23). The change in pH values of apple juice
the following gradients: 0–10 min, 10–26% B; 10–25 min, 10–26% fermented with L. acidophilus, L. casei and L. plantarum is shown

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Yang et al. Lactobacillus Fermented Apple Juice

significant difference among the juices fermented by the three

different strains (p ≥ 0.05). The variations of the pH values
were consistent with the report of Chen et al. (10), who studied
the effects of four LAB strains on the flavor characteristics of
fermented apple juice.

Change in Browning Index During LAB

Fermentation and Storage
Color is an important quality indicator for apple juice, and
the fermentation of LAB in fruit and vegetable substrates can
change their color (11). The change in color of apple juice
fermented with L. acidophilus, L. casei and L. plantarum during
the 72-h fermentation and the subsequent 30-day storage are
shown in Figure 1C. The Browning Index value of apple juice
increased slightly during the first 12 h of fermentation, and then
decreased rapidly during the fermentation between 12 and 24 h,
which was consistent with the variation of pH. The Browning
Index value showed a downward trend during the period of
24-h fermentation and 30-day storage at 4◦ C. This may be
due to the metabolism of phenolics in apple juice caused by
LAB fermentation and the decrease of pH in apple juice, which
inhibited the color change caused by Maillard reaction (24).

Changes in Organic Acids and Sugars

Contents During LAB Fermentation and
Storage
The changes in organic aicds and sugars in apple juice fermented
with L. acidophilus, L. casei and L. plantarum during the 72-h
fermentation and the subsequent 30-day storage are summarized
in Supplementary Table 1. Organic acids are important products
derived from LAB metabolism, which can improve the flavor
and palatability of fruit juices (25). Meanwhile, organic acids
can increase the acidity of the juice and inhibit the growth of
spoilage and pathogen microorganisms. Therefore, organic acids
have a profound influence on the storage characteristics and
flavor of fermented fruit and vegetable juices (25). As shown in
Supplementary Table 1, six organic acids including oxalic acid,
pyruvic acid, malic acid, lactic acid, citric acid, and succinic acid
can be identified in the fermented apple juice with L. acidophilus,
L. casei and L. plantarum.
Lactic acid content was increased significantly after
fermentation owing to the biosynthesis pathway of LAB
metabolism, and the highest concentration was 4,191.6 ± 31.6
FIGURE 1 | Evolutions of viable cell counts (A), pH value (B) and browning mg/L by L. casei. Malic acid is the main source of sour flavor in
index (C) in apple juices fermented by the three LAB strains during
unfermented apple juice. Before fermentation, it had the highest
fermentation and refrigerated storage (4◦ C, 30 days).
content of 1,166.5 ± 14.7 mg/L in apple juice (26, 27). Malic
acid can be bio-transferred to lactic acid under the catalysis of
the malolactic enzyme produced by LAB, thus weakening the
in Figure 1B. The pH values of apple juice fermented by the three acidity and providing a mildly sour taste of apple juice (28). The
LAB strains had a minor change in the first 12 h of fermentation, existence of this metabolic pathway can be confirmed by the
and then experienced a rapid drop between 12 and 72 h (from similarity between the increasing amount of lactic acid content
5.5∼4.0). To be specific, after 48-h fermentation, the maximum and the decreasing amount of malic acid content.
reduction of pH value was obtained in apple juice fermented Citric acid content (730.7 ± 13.1 mg/L in unfermented juice)
with L. casei (3.8 ± 0.02), following that fermented with L. was decreased after lactic acid fermentation as a result of its
acidophilus (3.9 ± 0.01) and L. plantarum (4.0 ± 0.02). In the decomposition into various products, such as diacetyl, lactic acid,
storage period, the pH value remained stable, and there was no and acetic acid (29). Pyruvate is an intermediate product of the

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Yang et al. Lactobacillus Fermented Apple Juice

basic metabolic pathways of organisms such as tricarboxylic acid components, PC1 and PC2, were extracted that accounted for
cycle, glycolysis, and other metabolic pathways. The pyruvic acid 67.7% and 19.7% of the total variance in seventeen variable
content experienced an increasing trend and reached a maximum systems, respectively. The unfermented sample was distributed
concentration of 130.2 ± 1.4 mg/L after fermentation by L. on the negative side of PC1. During 72-h fermentation, a
plantarum for 24 h, and then followed a decreasing trend. It has significant shift of the distribution could be observed among
been confirmed that pyruvic acid can be produced from glucose fermented apple juice (Figure 2A). This phenomenon indicates
through glycolysis, which may be related to the rising phase of that the profile of amino acids in apple juice changed obviously
pyruvic acid. In addition, pyruvic acid can also be used to form during 72-h fermentation. For example, the PC1 value of
the amino acid alanine and converted into lactic acid or ethanol unfermented apple juice was−7.31. After fermentation for 72 h,
via fermentation, which may be associated with its decline at the the PC1 values for apple juice fermented with L. acidophilus,
end of fermentation (30). The trends in the content of oxalic acid L. plantarum, and L. casei increased to 2.42, 2.49, and 2.47.
and succinic acid in fermented apple juice were similar to that According to the loading plot (Figure 2B), Leu, Ala, Val, His,
of pyruvic acid. In addition, the content of total organic acids Ile, Met, Thr and Phe distributed on the negative side of PC1,
increased during fermentation and then remained stable during and Tyr, Lys, Pro, Arg, Glu, Asp, Cys and Gly distributed on
the storage period, which may be due to the inhibition of the the positive side. These amino acids placed to the right such as
metabolism of lactic acid bacteria at low temperatures. Pro, Cys and Gly in the loading plot were close and positively
Before fermentation, fructose is the major sugar (52.0 ± correlated. However, amino acids on the negative side were
2.3 mg/L) in apple juice. During the fermentation process, negatively correlated. In addition, some amino acids including
the content of glucose in all fermented samples experienced a Leu, Val, Ala, His, Ile, Met, Thr, Phe, Pro, Cys, Gly and Asp
continuous decrease from 17.7 ± 0.8 mg/L to about 11.7 ± distributed far from the origin of the first PC. Therefore, LAB
0.1 mg/L, although there was no significant difference in the fermentation of apple juice may have a higher influence on
content of glucose among the apple juice fermented with different the content of these amino acids. Based on the locations of
microbial strains (p ≥ 0.05). This trend can be explained by samples and the attributes, it can be found that the contents of
the reason that the fermentation of the selected LAB strains is Leu, Ala, Val, His, Ile, Met, Thr and Phe in apple juice were
facultative heterologous fermentation, and glucose and fructose generally decreased with fermentation. In contrast, the lactic acid
are converted into lactic acid through the Embden-Meyerhof fermentation led to the increment of the amounts of Tyr, Lys,
pathway. The content of sucrose in apple juice fermented Pro, Arg, Glu, Asp, Cys and Gly. Besides, it can be observed that
with L. acidophilus, L. casei and L. plantarum was reduced the fermented apple juice by the three microbial strains at the
continuously, which can be attributed to the hydrolysis of sucrose same stage had similar PC1 values, indicating that PC2 is mainly
by galactosidase in LAB strains. This trend is consistent with responsible for the discrimination of the samples fermented
the results of Wang et al. (31) who used LAB strains to ferment by different strains. For example, after 72-h fermentation, the
soy milk. The contents of glucose, fructose and sorbitol were contents of Asp, Cys and Gly in apple juice fermented with L.
also increased significantly (P < 0.05) during storage. This may acidophilus, L. plantarum, and L. casei were 979.4 ± 1.0 mg/L,
be due to the inhibition of the respiration of LAB strains by 972.9 ± 1.0 mg/L, 969.3 ± 16.8 mg/L; 6.2 ± 0 mg/L, 6.2 ± 0.2
monosaccharides in an environment of low temperature and high mg/L, 5.8 ± 0.1 mg/L and 80.3 ± 1.2 mg/L, 86.9 ± 1.7 mg/L, 85.3
acidity. Meanwhile, the degradation of sucrose by galactosidase ± 1.2 mg/L, respectively. And the PC2 values of Asp, Cys and
may also take place in LAB strains (32–34). Gly were−0.17, 0.17, and 0.10 (Figure 2B). This result implies
that different strains have different capacities to metabolize some
amino acids such as Asp, Cys and Gly and others.
Change of Free Amino Acids During LAB The contents of Asp and Gly were increased significantly
Fermentation and Storage during fermentation (P < 0.05), which may improve the flavor
Amino acids are important flavoring compounds and their of fermented apple juice (37). LAB can metabolize threonine to
compositions have a profound influence on the evaluation of pyruvate and ammonia through threonine dehydratase, resulting
sensory properties of apple juice (35). For LAB fermentation, in a significant decrease in threonine content (P < 0.05) during
amino acids may first undergo decarboxylation, deamination, the first 24-h fermentation. It has been reported that lactic acid
transamination, and desulfurization reactions, followed by amine bacteria can alleviate environmental acid stress by increasing
conversion. Eventually, the aldehydes generated from amino the expression of enzymes consuming cytoplasm (38). Ammonia
acid metabolism are oxidized or reduced to the corresponding produced by amino acid metabolism can accept protons and
carboxylic acids and alcohols (36). Seventeen free amino acids promote acid-base balance in microbial cells (29), which is
could be detected in the apple juice samples fermented by the confirmed by the increase in ammonia contents from 3.5 ± 0.1
three LAB strains. Among them, Thr possessed the highest mg/L to 121.0 ± 0.2 mg/L, 121.0 ± 2.4 mg/L, 120.2 ± 0.01 mg/L
amount in unfermented samples (695.3 ± 13.2 mg/L). To clarify during fermented by L. acidophilus, L. plantarum, and L. casei,
the effect of LAB strains on the metabolism of amino acids, two- respectively. Moreover, the contents of Phe, Val, Ile and Met also
dimensional analysis of the principal component analysis (PCA) showed a downward trend throughout fermentation, which may
was used to analyze the profile of free amino acids during 72- be related to the decarboxylase (4) and transaminase (39, 40)
h fermentation. Meanwhile, the original data about the content produced by LAB. Decarboxylase decomposes free amino acids
of each amino acid are prepared in Table 1. Two principal to produce primary amine and release carbon dioxide, while

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Yang et al. Lactobacillus Fermented Apple Juice

transaminases can catalyze the inter-conversion of amino acids

into alpha-ketonic acid (38) and the transamination reaction
between branched-chain amino acids (i.e., leucine) and alpha-
keto-beta-methyl valerate/alpha-keto isopropionate (41).
During storage, almost all the mentioned amino acids were
increased significantly during the first 10-day storage (P < 0.05).
To be specific, the contents of Asp, Phe and Gly in apple juice
fermented by L. plantarum increased from about 972.9 ± 1.0
mg/L, 7.2 ± 0.1 mg/L and 86.9 ± 1.7 mg/L to approximately
1496.4 ± 20.2 mg/L, 11.7 ± 0.1 mg/L and 106.8 ± 1.6 mg/L,
respectively. Meanwhile, the contents of Met, Ile, Leu had no
obvious changes in the early period of storage. In the contrast,
the content of Thr was decreased from 370.6 mg/L to below 1
mg/L. In the subsequent storage period, there was no significant
change in the content of all the amino acids (P ≥ 0.05).
Besides, the content of total amino acids in apple juice
fermented by three LAB strains was studied. The results are
shown in Figure 2C. The total amino acid content in fermented
apple juice was decreased slightly in the first 24 h of fermentation,
and then increased gradually. During the first 10 days of storage
at 4◦ C, the content of total amino acids in apple juice fermented
by L. acidophilus, L. plantarum, and L. casei were increased from
1.88 ± 0.002 g/L, 1.88 ± 0.01, and 1.88 ± 0.04 g/L to 2.26 ±
0.02 g/L, 2.22 ± 0.02 g/L, and 2.27 ± 0.03 g/L, respectively. Then
maintained at this level until the end of storage.

Evolution of Phenolic Profile During LAB

Fermentation and Storage
Gallic acid, protocatechuic acid, catechin, proanthocyanidin B2 ,
chlorogenic acid, p-hydroxybenzoic acid, coffee acid, syringic
acid, phloxic acid, p-coumaric acid, ferulic acid, cinnamaldehyde,
p-vinyl guaiacol, rutin and quercitrin can be tentatively identified
in the fermented apple juice. Gallic acid, protocatechuic acid,
catechin, proanthocyanidin B2 , caffeic acid, rutin ferulate and
quercetin were the main phenolic substances in fermented apple
juice, and their contents were summarized in Table 2. After 48-
h fermentation by L. acidophilus, L. plantarum, and L. casei, the
content of ferulic acid was declined by 11.1, 18.5, and 11.1%,
and the caffeic acid content was reduced by 41.7, 50, and 50%,
respectively. These results indicated that the selected LAB strains
were able to biotransform ferulic acid and caffeic acid. This
phenomenon can be explained by two possible pathways. One
is that phenolic acid decarboxylases synthesized by LAB can
catalyze the non-oxidative decarboxylation of phenolic acids to
generate their corresponding p-vinyl derivatives (42). Another
reason is that ferulic acid and caffeic acid could be transformed
to dihydrocaffeic acid and dihydroferulic acids through the side
chain hydrogenation (43–46). The ferulic acid content in apple
FIGURE 2 | Principal component analysis of free amino acid profile in apple
juice during fermentation by the three LAB strains [(A), score plot; (B), loading
juice fermented by L. plantarum was decreased faster than those
plot] and changes in total free amino acids (C). “Control,” Unfermented apple fermented by L. acidophilus and L. casei (P < 0.05), guessing
juice; “La,” Apple juice fermented by L. acidophilus; “Lp”, Apple juice that L. plantarum was more capable to synthesize the enzymes
fermented by L. plantarum; “Lc”, Apple juice fermented by L. casei. In (A), the involved in the metabolism of ferulic acid.
number followed with the name of strain indicated the time of
The content of ferulic acid was significantly decreased from
fermentation.
around 4.7 mg/L to about 0.8 mg/L (P < 0.05) after storage for

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Yang et al. Lactobacillus Fermented Apple Juice

TABLE 1 | Changes in the contents of free amino acids (mg/L) of apple juice during lactic acid fermentation and the subsequent refrigerated storage period (4◦ C, 30 days).

Amino Strains Time

acid
Fermentation Fermentation Fermentation Fermentation Fermentation Storage for 10 d Storage for 20 d Storage for
for 0 h for 12 h for 24 h for 48 h for 72 h 30 d
(Storage for
0 d)

Asp L. acidophilus 678.8 ± 31.8Ad 662.3 ± 1.9Bd 834.4 ± 15.7Ac 974.1 ± 14.2Ab 979.4 ± 1.0Ab 1,529.7 ± 12.5Aa 1522.8 ± 1.6Aa 1512 ± 4.5Aa
Ad ABd Ac Bb Ab Ba Ba
L. casei 678.8 ± 31.8 672.4 ± 7.3 835.3 ± 17 933.4 ± 5.7 969.3 ± 16.8 1,443 ± 20.9 1460.3 ± 6.2 1,458.2 ±
2.5Ca
L. plantarum 678.8 ± 31.8Ad 676.6 ± 1.5Ad 852.9 ± 33.6Ac 926.7 ± 7.2Bb 972.9 ± 1.0Ab 1,496.4 ± 20.2ABa 1492.7 ± 17.4ABa 1,489.3 ±
9.8Ba
Thr L. acidophilus 695.3 ± 13.2Aa 522.7 ± 2.7Ab 404.3 ± 6.2Ac 385.5 ± 1.8Ad 373.9 ± 2.6Ad ND ND ND
L. casei 695.3 ± 13.2Aa 521.5 ± 4.6Ab 400.2 ± 0.3Ac 379.4 ± 2.3Ad 371.4 ± 2.9Ad ND ND ND
L. plantarum 695.3 ± 13.2Aa 533.6 ± 8.2Ab 412.9 ± 8.6Ac 378.2 ± 5.3Ad 370.6 ± 6.8Ad ND ND ND
Ser L. acidophilus ND ND ND ND ND ND ND ND
L. casei ND ND ND ND ND ND ND ND
L. plantarum ND ND ND ND ND ND ND ND
Glu L. acidophilus 200.9 ± 3.2Ad 213.4 ± 2.5Ac 184.6 ± 0.8Ae 205.3 ± 1.5Bd 218.4 ± 1.6Bb 292.2 ± 3.1Aa 294.7 ± 1.3Ba 294.1 ± 0.4Ba
L. casei 200.9 ± 3.2Ad 211.2 ± 1.1Ac 185 ± 2.3Ae 212 ± 0.6Ac 227.7 ± 1.7Ab 293.7 ± 6.1Aa 300.5 ± 2.2Aa 299.9 ± 2.4Aa
Ad Ac Ae Bcd Ab Aa Aa
L. plantarum 200.9 ± 3.2 210.1 ± 1.8 189.6 ± 4.8 206.1 ± 2.8 224.3 ± 0.5 298.9 ± 1.1 300.1 ± 0.5 301.1 ± 1.1Aa
Af Be Bd Bc Bb Ba Ba
Gly L. acidophilus 4 ± 0.1 47 ± 0 72.5 ± 0.9 77.8 ± 1.6 80.3 ± 1.2 100.2 ± 0.6 100.1 ± 1.3 101.3 ± 1.2Ba
Ae Bd ABc Ab Ab ABa Aa
L. casei 4 ± 0.1 46.9 ± 0.2 76.8 ± 1.8 83 ± 1.4 85.3 ± 1.2 104.9 ± 2 106.2 ± 1.9 106.5 ± 1.2Aa
Ae Ad Ac Ab Ab Aa Aa
L. plantarum 4 ± 0.1 48.2 ± 0.1 77.6 ± 1.7 84.8 ± 1.4 86.9 ± 1.7 106.8 ± 1.6 107.7 ± 1.7 108.9 ± 1.8Aa
Ab Aa Ac Ad Ad
Ala L. acidophilus 33.3 ± 0.1 37.8 ± 0.9 8.3 ± 0.8 1.9 ± 0.2 1.9 ± 0 ND ND ND
L. casei 33.3 ± 0.1Ab 36.8 ± 0.9Aa 7.7 ± 0.4Ac 1.9 ± 0Ad 1.9 ± 0.2Ad ND ND ND
L. plantarum 33.3 ± 0.1Ab 39 ± 1.1Aa 9.9 ± 1Ac 2 ± 0.2Ad 1.9 ± 0.1Ad ND ND ND
Cys L. acidophilus 3.1 ± 0.1Ad 5.1 ± 0Ac 6.5 ± 0.3Aab 6.5 ± 0Aa 6.2 ± 0Ab ND ND ND
L. casei 3.1 ± 0.1Ad 5 ± 0Ac 6 ± 0.1Aa 5.9 ± 0Bab 5.8 ± 0.1Bb ND ND ND
L. plantarum 3.1 ± 0.1Ac 5.2 ± 0Ab 6.4 ± 0.4Aa 6.4 ± 0.2Aa 6.2 ± 0.2Aa ND ND ND
Val L. acidophilus 31.5 ± 0.1Ab 35.3 ± 0.3Aa 8.7 ± 0.9Ac 2.1 ± 0.2Ad 2.7 ± 0.2Ad 8.4 ± 0.4Ac 6.9 ± 3.5Ac 8.2 ± 0.7Ac
L. casei 31.5 ± 0.1Ab 34.2 ± 0.4Aa 6 ± 0.3Bd 1.5 ± 0.1Be 2.2 ± 0.2Ae 7.5 ± 0.4Ac 8 ± 0.4Ac 7.7 ± 0.7Ac
L. plantarum 31.5 ± 0.1Ab 34.8 ± 0.5Aa 7.7 ± 0.8ABc 1.7 ± 0Be 2.3 ± 0.2Ae 4.1 ± 0.3Bde 6.3 ± 3.4Acd 8.3 ± 0.5Ac
Met L. acidophilus 10.4 ± 0.5Aa 8 ± 0Aab 2.9 ± 1Abc 1.1 ± 0.5Ac 1.2 ± 0.3ABc 4.3 ± 1.1Abc 4.7 ± 1.3Abc 11.4 ± 6.4Aa
Aab Bb Ac Ac Bc Aab Aa
L. casei 10.4 ± 0.5 7.7 ± 0 0.9 ± 0.5 0.6 ± 0 0.9 ± 0 9.7 ± 3.8 13 ± 1.1 10.3 ± 5Aab
Aab ABabc Ac Ac Ac Abc Aab
L. plantarum 10.4 ± 0.5 7.9 ± 0.2 1.3 ± 0 1.1 ± 0.5 1.5 ± 0 3.5 ± 0.1 9.6 ± 8.8 13.8 ± 2.2Aa
Aa Ab Ac Ad Ad Ad Acd
Ile L. acidophilus 59.6 ± 1.6 49.1 ± 0.5 7.4 ± 0.3 1.4 ± 0.1 1.8 ± 0.2 2.4 ± 0.6 3.9 ± 3.1 4 ± 2.6Acd
Aa Ab Bc Bd Ad Bd Ad
L. casei 59.6 ± 1.6 46.9 ± 0.6 3.9 ± 0.2 0.7 ± 0.1 1.2 ± 0.1 0.9 ± 0.2 1.6 ± 1 1.8 ± 0.6Ad
Aa Ab Ac ABd Ad ABd Acd
L. plantarum 59.6 ± 1.6 48.4 ± 1.1 5.9 ± 0.9 0.9 ± 0.3 1.5 ± 0.3 1.3 ± 0.3 3.4 ± 3.2 3.4 ± 3.2Acd
Ab Aa Acd Ad Ad Acd Ac
Leu L. acidophilus 16.7 ± 0.3 22.6 ± 0.4 4.1 ± 0.1 1.8 ± 0.3 2.6 ± 0.3 3.9 ± 0.4 7.1 ± 2.8 6.5 ± 3.1Ac
Ab Aa Bc Bf Ae Ad Ad
L. casei 16.7 ± 0.3 21.8 ± 0.5 3.1 ± 0.2 0±0 1.2 ± 0.6 2.2 ± 0.5 2.2 ± 0.5 2.4 ± 0.1Acd
L. plantarum 16.7 ± 0.3Ab 22.4 ± 0.2Aa 3.9 ± 0.3Acde 1.4 ± 0.5Ae 2.1 ± 0.6Ade 3.4 ± 0.7Acde 5.8 ± 4.1Acd 6.7 ± 2.9Ac
Tyr L. acidophilus ND 3.2 ± 0.1Aa 3.5 ± 0.2Aa 3.9 ± 0.1Aa 4 ± 0Aa ND 3.6 ± 0.6Aa 2 ± 2.8Aab
L. casei ND 3 ± 0Ad 3.7 ± 0.1Ab 3.4 ± 0.1Bc 4.1 ± 0.2Aa ND ND ND
L. plantarum ND 7.9 ± 7Aa 3.3 ± 0Aab 3.9 ± 0.1Aab 3.9 ± 0Aab ND 2 ± 2.9Aab 3.7 ± 0.4Aab
Phe L. acidophilus 12.9 ± 0.1Aa 8.9 ± 0.3Ac 7.1 ± 0Ad 7.1 ± 0.1Ad 7.2 ± 0.1Ad 12 ± 0.3Ab 12.1 ± 0.1Ab 11.8 ± 0Bb
L. casei 12.9 ± 0.1Aa 8.9 ± 0.3Ac 7.2 ± 0.1Ad 6.8 ± 0.1Be 7.1 ± 0.2Ade 11.7 ± 0.1Ab 11.8 ± 0.1ABb 11.9 ± 0Ab
L. plantarum 12.9 ± 0.1Aa 9 ± 0.4Ac 7.2 ± 0Ad 7 ± 0Ad 7.2 ± 0.1Ad 11.7 ± 0.1Ab 11.7 ± 0.1Bb 11.9 ± 0.1ABb
Lys L. acidophilus 25.4 ± 0.1Ad 51 ± 0.7Ab 51.8 ± 0.6Ab 46.9 ± 0.1ABc 46 ± 0.1Ac 60.9 ± 0.8Aa 61.1 ± 0.5Aa 60.6 ± 0.2Aa
Ad Ab Ab Bc Ac Aa Aa
L. casei 25.4 ± 0.1 49.9 ± 0.7 49.6 ± 0.1 44.8 ± 0.4 44 ± 0.5 57.8 ± 0.6 58.2 ± 1 58 ± 0.1Aa
Ac Ab Ab Ab Ab Aa Aa
L. plantarum 25.4 ± 0.1 50.1 ± 0.5 49.4 ± 3.9 47.4 ± 1.3 46.5 ± 1.4 60.9 ± 1.7 61.1 ± 1.5 60.7 ± 1.9Aa
Aa Ab Ad Ae Ae Ac Ac
His L. acidophilus 26 ± 0.1 25.5 ± 0.2 21.1 ± 0.1 19.4 ± 0 19.5 ± 0 24 ± 0.3 24.1 ± 0.1 24 ± 0.1Ac
Aa Ab Ad Be Be Bc Bc
L. casei 26 ± 0.1 25 ± 0.2 20.5 ± 0.2 18.8 ± 0.1 19 ± 0.1 23 ± 0.2 23.3 ± 0.4 23.3 ± 0.2Bc
Aa Ab Ad Ae ABe ABc ABc
L. plantarum 26 ± 0.1 25.4 ± 0.2 20.8 ± 0.5 19.3 ± 0.1 19.4 ± 0.2 23.6 ± 0.2 23.6 ± 0.1 23.6 ± 0.1ABc
Ae Aa Ac Ad Ad Ab Ab
Arg L. acidophilus 10.8 ± 0.1 45.3 ± 0.7 36.3 ± 0.4 33.5 ± 0.3 34.2 ± 0.5 40.7 ± 0.8 41.6 ± 0.5 41.2 ± 0.2Ab

(Continued)

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Yang et al. Lactobacillus Fermented Apple Juice

TABLE 1 | Continued

Amino Strains Time

acid
Fermentation Fermentation Fermentation Fermentation Fermentation Storage for 10 d Storage for 20 d Storage for
for 0 h for 12 h for 24 h for 48 h for 72 h 30 d
(Storage for
0 d)

L. casei 10.8 ± 0.1Ae 44.4 ± 0.7Aa 34.9 ± 0.2Ac 32.3 ± 0.5Ad 32.9 ± 0.4Ad 38.9 ± 0.6Ab 39.5 ± 1Ab 39.5 ± 0.4Bb
Ae Aa Ac Ad Ad Ab Ab
L. plantarum 10.8 ± 0.1 44.4 ± 0.6 35.4 ± 1 32.9 ± 0.4 33.3 ± 0.7 39.9 ± 0.5 40.1 ± 0.5 39.8 ± 0.8ABb
Ac Ac Ab Ab Aa Aa
Pro L. acidophilus ND 6.3 ± 0.6 6.7 ± 0.1 9.9 ± 0.6 11.1 ± 0.6 53.7 ± 0.1 54.8 ± 2.8 55.1 ± 0.4Aa
Acd Ade Abc Ab Aa Aa
L. casei ND 5.8 ± 0.3 2.6 ± 3.7 9 ± 0.2 10.7 ± 0.5 55.5 ± 2.2 56 ± 0.4 56 ± 0.3Aa
Ac Ac Ab Ab Aa Aa
L. plantarum ND 5.8 ± 0.7 6.9 ± 0.3 8.9 ± 0.9 10.4 ± 0.9 53.5 ± 1.7 53.9 ± 0.7 53.4 ± 0.4Ba

Results are expressed as mean ± standard deviation from the three replicates. ND refers to not detected. Values with different letters indicated a significant difference (p < 0.05).

10 days, which may also be related to the metabolism of ferulic implying that PC1 can be considered as a contributor to
acid into 4-vinyl guaiacol and other substances (43). Also, caffeic distinguish the unfermented and fermented samples. In the
acid content increased rapidly, which could be attributed to the loading plot of phenolic acids (Figure 3B), three phenolics,
conversion of dihydrocaffeic acid to caffeic acid (47). Meanwhile, including cinnamaldehyde, phloretin, and p-vinyl guaiacol, were
the content of catechin in apple juice fermented by L. acidophilus, located on the negative side of PC1. In contrast, the other 13
L. plantarum, and L. casei were increased from 10.1 ± 0.1 mg/L, phenolic acids were located on the positive side of PC1. Among
9.9 ± 0.3 mg/L, 10.5 ± 0.4 mg/L to 12.6 ± 0.9 mg/L, 14.0 ± 0.5 them, p-vinyl guaiacol, phloretin, cinnamaldehyde, quercetin
mg/L, and 14.7 ± 0.3 mg/L while the content of protocatechuic and ferulic acid were placed nearby the origin of first PC.
acid were decreased from 24.4 ± 0.09 mg/L, 22.8 ± 2.6 mg/L, Therefore, LAB fermentation may have minor effects on these
25.1 ± 0.3 mg/L to 16.2 ± 0.8 mg/L, 18.9 ± 2.5 mg/L, and 18.2 phenolic acids. However, protocatechuic acid, gallic acid, p-
± 0.6 mg/L in the first 10-day storage. It has been reported that hydroxybenzoic acid, p-coumaric acid, chlorogenic acid and
LAB can metabolize protocatechuic acid to catechin through the syringic acid were all placed far from the origin of first PC and
decarboxylation reaction (48). The study of Annalisa et al. also positively correlated. This result implied that LAB fermentation
demonstrated the metabolic correlation between protocatechuic has some positive effects on the phenolic acids in apple juice.
acid and catechin (49). It was also found that the changes of rutin The changes in total phenolic content in apple juices
and quercetin were almost the same during fermentation and fermented by the three LAB strains are shown in Figure 3C.
storage at 4◦ C. Moreover, the contents of rutin and quercetin Overall, the total phenolic content was declined after 72-h
were almost stable throughout fermentation and there was no fermentation (P < 0.05). During the first 12 h of fermentation,
significant difference among the three LAB fermented samples the total phenolic contents of apple juice fermented with different
(P ≥ 0.05). However, the contents of rutin and quercetin in all strains all declined from 1.37 g/L to around 1.27 g/L. However,
the samples decreased significantly after storage for 10 days (P the total phenolic content in apple juice fermented by L. casei
< 0.05), which may be attributed to the uncoupling of beta- was increased to 1.35 ± 0.01 g/L after 72 h of fermentation (P
glucosidase in lactic acid bacteria (50). Meanwile, rutin and < 0.05), while that in apple juice fermented by L. plantarum
quercetin could be biotransformed to low molecular weight was decreased to 1.27 ± 0.02 g/L. The loss of phenolics may
metabolites, such as 2-(3,4-dihydroxyphenyl) acetic acid, 2- be due to the interaction between phenolics and proteins in
(3-hydroxyphenyl) acetic acid, 3,4-dihydroxybenzoic acid and apple juice during fermentation, which could produce insoluble
others (51). The gallic acid content was increased during 72-h complexes (4). Another potential reason is that phenolics are
fermentation, but decreased during storage at 4◦ C for 30 days. the inhibitors of the growth of L. plantarum, and LAB can
In contrast, the content of proanthocyanidin B2 was decreased synthetize a series of enzymes (benzyl alcohol dehydrogenase,
rapidly within the first 12 h of fermentation and then stayed decarboxylase, tannase) to degrade phenolics (42, 52). The total
stable in the following 36 h of fermentation. Similar to gallic acid, phenolic contents in apple juice fermented by the three LAB
the proanthocyanidin B2 content was decreased significantly strains were close after fermentation. In the first 10 day of
during storage. storage, the total phenolic content decreased continuously to
The changes of individual phenolic compounds during 72- about 0.95 g/L. After that, no significant change in the total
h fermentation were analyzed by PCA (Figures 3A,B). PC1 phenolic content was observed in the subsequent 20-day storage.
and PC2 explained 94% of the total variance of all the “fuji” Similar results were found by Wang et al. (53), the total phenolic
apple juices. According to Figure 3A, the unfermented sample content of fermented mango juice experienced a rapid decrease
was located on the positive side of PC1. And apple juices during the first 10-d storage and then stable. This phenomenon
fermented with L. acidophilus, L. plantarum, and L. casei at may be caused by the presence of dissolved oxygen which
different stage were distributed on the different PC1 region, resulted in the oxidation of phenolic compounds during the

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Frontiers in Nutrition | www.frontiersin.org

Yang et al.
TABLE 2 | Changes in the contents of individual phenolic compounds (mg/L) of apple juices during lactic acid fermentation and the subsequent refrigerated storage period (4◦ C, 30 days).

organic strains Time

Fermentation Fermentation Fermentation Fermentation Fermentation for Storage for 10 Storage for 20 Storage for
for 0 h for 12 h for 24 h for 48 h 72 h (Storage for d d 30 d
0 d)

Gallic acid L. acidophilus 15.4 ± 0.06ef 12.8 ± 0.54de 16.8 ± 0.11f 19.8 ± 0.10gh 21.3 ± 0.23h 9.9 ± 2.23c 4.5 ± 0.45a 3.2 ± 0.13a
ef ef f gh h cd a
L. casei 15.4 ± 0.06 14.3 ± 1.94 16.5 ± 0.11 19.6 ± 0.05 21.6 ± 0.16 10.5 ± 2.57 4.3 ± 0.63 2.8 ± 0.42a
ef d f g gh b a
L. plantarum 15.4 ± 0.06 12.2 ± 0.37 16.5 ± 0.32 18.9 ± 0.14 20.8 ± 0.14 7.5 ± 1.57 3.2 ± 0.52 2.9 ± 0.15a
ab c c c c ab b
Protocatechuic L. acidophilus 17.1 ± 0.50 16.0 ± 1.45 24.9 ± 0.40 23.9 ± 0.87 24.4 ± 0.09 16.2 ± 0.84 18.8 ± 1.19 17.4 ± 1.02ab
acid L. casei 17.1 ± 0.50 ab
20.1 ± 1.70 bc
23.0 ± 2.31 c
24.4 ± 0.73 c
25.1 ± 0.31 c
18.2 ± 0.60 ab
19.9 ± 1.23 bc
17.9 ± 0.68ab
L. plantarum 17.1 ± 0.50ab 17.4 ± 0.95ab 20.0 ± 0.97bc 22.5 ± 2.69c 22.8 ± 2.57c 18.9 ± 2.50b 19.4 ± 0.37b 18.1 ± 1.24ab
Catechin L. acidophilus 8.9 ± 0.08a 8.6 ± 1.73a 10.3 ± 0.16c 9.6 ± 0.35bc 10.1 ± 0.14bc 12.6 ± 0.89d 13.5 ± 0.65de 12.6 ± 0.68d
L. casei 8.9 ± 0.08a 9.7 ± 0.27bc 9.9 ± 0.14bc 9.7 ± 0.36bc 10.5 ± 0.37c 14.7 ± 0.29e 13.5 ± 0.18de 12.9 ± 0.35de
L. plantarum 8.9 ± 0.08a 7.7 ± 0.18a 9.6 ± 1.17bc 9.0 ± 0.77b 9.9 ± 0.27bc 14.0 ± 0.48e 13.7 ± 0.17de 12.8 ± 0.63d
Procyanidin B2 L. acidophilus 78.7 ± 6.78f 43.1 ± 8.73c 58.9 ± 1.08e 56.5 ± 2.40de 57.4 ± 0.38de 28.4 ± 1.77 27.7 ± 1.58ab 24.9 ± 1.59ab
L. casei 78.7 ± 6.78f 52.3 ± 0.73d 57.4 ± 1.43de 57.6 ± 1.65de 59.4 ± 0.49e 31.9 ± 1.47b 28.1 ± 0.73ab 24.7 ± 1.34ab
f c de de de b ab
L. plantarum 78.7 ± 6.78 42.7 ± 0.99 56.9 ± 3.48 55.8 ± 2.29 57.8 ± 0.60 30.0 ± 0.88 28.1 ± 0.54 23.9 ± 2.67a
g c e de de ab ab
Chlorogenic acid L. acidophilus 116.0 ± 0.26 97.8 ± 2.66 111.0 ± 0.43 107.8 ± 1.25 106.8 ± 0.32 99.0 ± 2.90 98.7 ± 1.76 96.3 ± 0.89ab
g d de de e b ab
L. casei 116.0 ± 0.26 99.7 ± 0.53 110.5 ± 0.57 108.7 ± 1.10 107.9 ± 0.31 105.2 ± 2.65 97.9 ± 0.36 95.8 ± 0.79ab
g c de de de b ab
L. plantarum 116.0 ± 0.26 94.2 ± 0.76 110.0 ± 2.08 107.4 ± 1.48 106.9 ± 0.52 100.9 ± 1.59 98.2 ± 0.49 96.1 ± 1.09a
9

d a cd de e a bc
P- L. acidophilus 19.3 ± 0.18 15.0 ± 1.64 18.2 ± 0.15 20.1 ± 2.36 21.4 ± 0.14 15.2 ± 0.82 15.8 ± 0.42 15.2 ± 0.50a
hydroxybenzoic L. casei 19.3 ± 0.18d 15.9 ± 0.17bc 17.8 ± 0.22c 21.6 ± 0.50e 21.5 ± 0.19e 16.9 ± 0.43c 15.7 ± 0.17bc 15.3 ± 0.27b
acid
L. plantarum 19.3 ± 0.18d 13.7 ± 0.14a 17.4 ± 0.96c 20.6 ± 1.20de 21.2 ± 0.10e 16.3 ± 0.43bc 15.9 ± 0.12bc 15.4 ± 0.42bc
Caffeic acid L. acidophilus 1.2 ± 0.27b 1.0 ± 0.81b 1.2 ± 0.09b 0.7 ± 0.42ab 1.6 ± 0.07bc 2.0 ± 0.50c 3.0 ± 0.22d 3.0 ± 0.42d
L. casei 1.2 ± 0.27b 1.6 ± 0.15bc 1.0 ± 0.11b 0.6 ± 0.29ab 2.0 ± 0.09c 3.0 ± 0.13d 3.1 ± 0.08d 3.2 ± 0.26d
L. plantarum 1.2 ± 0.27b 0.0 ± 0.05a 0.4 ± 0.58ab 0.6 ± 0.42ab 1.9 ± 0.04c 3.0 ± 0.19d 3.3 ± 0.12d 3.0 ± 0.34d
Ferulic acid L. acidophilus 5.4 ± 0.08e 5.1 ± 0.93d 5.0 ± 0.05d 4.8 ± 0.05cd 4.6 ± 0.19cd 0.4 ± 0.19a 0.8 ± 0.10a 0.7 ± 0.18a
L. casei 5.4 ± 0.08e 5.4 ± 0.32d 4.9 ± 0.02cd 4.8 ± 0.06cd 4.8 ± 0.04cd 0.9 ± 0.02a 0.9 ± 0.03a 0.8 ± 0.11a
e b c c cd a a
L. plantarum 5.4 ± 0.08 3.7 ± 0.00 4.4 ± 0.56 4.4 ± 0.53 4.7 ± 0.04 0.8 ± 0.12 0.9 ± 0.03 0.8 ± 0.03a
bc b c c c a a
Rutin L. acidophilus 2.3 ± 0.00 2.2 ± 0.05 2.5 ± 0.27 2.4 ± 0.19 2.6 ± 0.24 0.3 ± 0.02 0.3 ± 0.05 0.3 ± 0.02a
bc bc bc bc bc a a
L. casei 2.3 ± 0.00 2.3 ± 0.08 2.2 ± 0.01 2.3 ± 0.02 2.3 ± 0.01 0.3 ± 0.02 0.3 ± 0.02 0.3 ± 0.03a
February 2022 | Volume 9 | Article 833906

bc bc bc bc bc a a
L. plantarum 2.3 ± 0.00 2.2 ± 0.02 2.2 ± 0.01 2.3 ± 0.02 2.3 ± 0.00 0.4 ± 0.19 0.3 ± 0.00 0.3 ± 0.20a
bc bc c bc c a a
Quercetin L. acidophilus 2.1 ± 0.00 2.1 ± 0.12 2.2 ± 0.21 2.2 ± 0.18 2.2 ± 0.19 0.2 ± 0.01 0.2 ± 0.03 0.2 ± 0.02a

Lactobacillus Fermented Apple Juice

bc b bc bc bc a a
L. casei 2.1 ± 0.00 2.0 ± 0.01 2.1 ± 0.01 2.1 ± 0.01 2.1 ± 0.01 0.2 ± 0.01 0.2 ± 0.01 0.2 ± 0.02a
bc bc bc bc bc a a
L. plantarum 2.1 ± 0.00 2.0 ± 0.02 2.1 ± 0.01 2.1 ± 0.02 2.1 ± 0.01 0.2 ± 0.04 0.2 ± 0.03 0.2 ± 0.05a

Results are expressed as mean ± standard deviation from the three replicates. Values with different letters indicate a significant difference (p < 0.05).
Yang et al. Lactobacillus Fermented Apple Juice

FIGURE 4 | The antioxidant activity of unfermented and LAB-fermented apple

juice in vitro. (A) ABTS·+ radical scavenging activity, (B) ferric reducing
antioxidant power.

Change in the Antioxidant and

Antimicrobial Activities in vitro During LAB
Fermentation and Storage
The antioxidant properties of apple juice are mainly related
to vitamin C and phenolics (55, 56). ABTS·+ method (free
radical scavenging capacity) and FRAP method (iron reduction
capacity) were used to evaluate the effect of LAB fermentation
on the antioxidant activity of apple juice in vitro. The results
of antioxidant activity of fermented apple juice are shown in
Figure 4. Generally, LAB fermentation enhanced the ABTS·+
FIGURE 3 | Principal component analysis of phenolic profile in apple juice radical scavenging capacity of the apple juice fermented by L.
during fermentation by the three LAB strains [(A), the score plots; (B), the acidophilus, L. plantarum, and L. casei were increased from
loading plots] and changes in total phenolic content (C). “Control,”
36.41 ± 1.1 mmol Trolox/L to 49.22 ± 3.0 mmol Trolox/L,
Unfermented apple juice; “La,” Apple juice fermented by L. acidophilus; “Lp”,
Apple juice fermented by L. plantarum; “Lc”, Apple juice fermented by L. casei. 54.03 ± 1.7 mmol Trolox/L, 51.94 ± 2.4 mmol Trolox/L, which
The number followed with the name of strain indicated the time of was consistence with the trend in pomegranate juice (57) and
fermentation. jussara pulp (58) fermented by LAB. On the other hand, both
ABTS·+ radical scavenging capacity and ferric reducing power
of fermented apple juice declined significantly during the first
first 10-d storage (54). However, phenolic compounds were 10-day storage, which could be related to the aforementioned
more stable with the consumption of oxygen during the later decrease of total phenolic content in fermented apple juice. In
storage stage. the next 20-day storage, their trends have different performance.

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Yang et al. Lactobacillus Fermented Apple Juice

The ferric reducing power was stable, whereas the ABTS·+

radical scavenging capacity was even increased. Overall, the
ferric reducing ability of fermented apple juice has similar trend
with the change of total phenolic acid content whereas the
ABTS·+ radical scavenging capacity of fermented apple juice
were increased during fermentation. Such inconsistent changes
in ferric reducing ability and ABTS·+ radical scavenging capacity
of fermented juice were also found by Wang et al. (9) who
used LAB to ferment apple juice. There are two possible reasons
for this phenomenon. First one is that the dissolved oxygen
and microbial strains can affect the ABTS·+ radical scavenging
capacity of fermented apple juice (9). Another one is that the
bioconversion of phenolic acids during fermentation (59). Ozgen
et al. (60) compared the ABTS·+ radical scavenging activities
of four phenolic acids (gallic acid, chlorogenic acid, caffeic acid
and ascorbic acid), and reported that gallic acid had the highest
ABTS·+ radical scavenging activities, followed by caffeic acid.
Zhang et al. (61) illustrated that gallic acid and caffeic acid had
more contribution to the ABTS·+ radical scavenging activity as
compared to ferulic acid. And in our study, we also found an
increase in these phenolic acid contents (gallic acid, caffeic acid
and so on).
In addition, microbial contamination is an important reason
of food spoilage. E. coli (G− ) and S. aureus (G+ ) are the
most common microorganisms in contaminated food (62).
In this study, the antimicrobial activities of unfermented and
fermented apple juice against the growth of E. coli and S. aureus
were evaluated, and the results are shown in Figure 5. Before
fermentation, the samples had weak antibacterial activities. After
72-h fermentation, the diameters of the inhibition circles against
E. coli and S. aureus were 23.78 ± 0.1 mm and 17.53 ± 1.0 mm
for the apple juice fermented by L. plantarum, 23.74 ± 1.1 mm
and 16.81 ± 0.6 mm for the juice fermented by L. casei, and
23.76 ± 0.9 mm and 17.13 ± 0.8 mm for the juice fermented FIGURE 5 | The antimicrobial activity of unfermented and LAB-fermented
by L. acidophilus. It is supposed that the antibacterial properties apple juice against the growths of (A) E. coli and (B) S. aureus in vitro. Values
of fermented apple juice may be related to bacteriocin, diacetyl, with different letters within each samples indicated a significant difference in
organic acid and hydrogen peroxide produced by lactic acid content (p < 0.05).

bacteria metabolism (63). Although LAB fermentation improved

the antimicrobial activities, there was no significant difference
in the areas of inhibition zone of apple juice fermented by the antimicrobial activities after fermentation and storage may be
three LAB strains after 72-h fermentation. Interestingly, after influenced by the change of organic acids and viable cell counts
storage for 30 days, the diameter of the inhibition circle against of LAB.
E. coli for all the juices even increased. The apple juice fermented
with L. acidophilus had the largest inhibition circles (26.41 ± CONCLUSION
0.3 mm) followed by those fermented with L. plantarum (25.03
± 0.6 mm) and L. casei (24.31 ± 0.9 mm). In addition, the In this study, three commercial LAB strains including L.
diameter of the inhibition circle against S. aureus for the samples acidophilus, L. casei, and L. plantarum were utilized to ferment
fermented with L. acidophilus did not change significantly after apple juice. All the microbial strains presented satisfactory
30-day storage, whereas the antimicrobial ability of samples growth capacities in the studied apple juice environment.
fermented with L. casei and L. plantarum against S. aureus got In general, LAB fermentation improved the antioxidant
weakened with storage. Generally, the antimicrobial activities and antimicrobial capacities by metabolizing phenolics
of unfermented and fermented apple juice can be contributed and producing lactic acid. In addition, the changes in the
by the acid production, these acids can lower pH and create physicochemical properties of fermented apple juice during
an unfavorable environment for pathogens and deteriorating storage at 4◦ C for 30 days was also evaluated. The results
microorganisms (64). Additionally, LAB can produce some showed that the total amino acid content was significantly
antimicrobial compounds such as H2 O2 and bacteriocin, which increased, but the total phenolic content was significantly
can inhibit microbial growth (21). Therefore, such changes in decreased. Overall, these results prove that lactic acid

Frontiers in Nutrition | www.frontiersin.org 11 February 2022 | Volume 9 | Article 833906

Yang et al. Lactobacillus Fermented Apple Juice

fermentation is a useful method to enhance the nutritional FUNDING

value of apple juice.
This work was financially supported by grants from the National
DATA AVAILABILITY STATEMENT Natural Science Foundation of China (No. 32100037), Natural
Science Research General Project of Jiangsu Higher Education
The original contributions presented in the study are included Institutions (No. 20KJB550008), Science and Technology
in the article/Supplementary Material, further inquiries can be Program of Kizilsu Kirghiz Autonomous Prefecture in 2021 (No.
directed to the corresponding author/s. 3-25), Jiangsu Planned Projects for Postdoctoral Research Funds
(No. 2019K015), Priority Academic Program Development of
Jiangsu Higher Education Institutions (PAPD), and the Research
AUTHOR CONTRIBUTIONS
and Practice Innovation Program for College Graduates of
JY performed the experiments, analyzed the data, and wrote Jiangsu Province (No. KYCX20_2888).
the manuscript. YS, TG, and YW analyzed the data and wrote
the manuscript. HS, QZ, and CL analyzed and discussed the SUPPLEMENTARY MATERIAL
data. CZ, YH, and YT provided samples and discussed the
data. YT designed the research content, analyzed the data, and The Supplementary Material for this article can be found
modified the manuscript. All authors read and approved the online at: https://www.frontiersin.org/articles/10.3389/fnut.2022.
final manuscript. 833906/full#supplementary-material

REFERENCES activities of lactic-acid-fermented mulberry juice. Food Chem. (2018)

250:148–54. doi: 10.1016/j.foodchem.2018.01.009
1. Yuasa M, Shimada A, Matsuzaki A, Eguchi A, Tominaga M. Chemical 12. Vivek K, Mishra S, Pradhan RC, Jayabalan R. Effect of probiotification with
composition and sensory properties of fermented citrus juice Lactobacillus plantarum MCC 2974 on quality of Sohiong juice. LWT-Food
using probiotic lactic acid bacteria. Food Biosci. (2020) 39:100810. Sci Technol. (2019) 108:55–60. doi: 10.1016/j.lwt.2019.03.046
doi: 10.1016/j.fbio.2020.100810 13. Kaprasob R, Kerdchoechuen O, Laohakunjit N, Sarkar D, Shetty K.
2. Dogan K, Akman PK, Tornuk F. Role of non-thermal treatments Fermentation-based biotransformation of bioactive phenolics and volatile
and fermentation with probiotic Lactobacillus plantarum on in vitro compounds from cashew apple juice by select lactic acid bacteria. Process
bioaccessibility of bioactives from vegetable juice. J Sci Food Agr. (2021) Biochem. (2017) 59:141–9. doi: 10.1016/j.procbio.2017.05.019
101:4779–88. doi: 10.1002/jsfa.11124 14. Balestra GM, Misaghi IJ. Increasing the efficiency of the plate counting
3. Wang Z, Feng Y, Yang N, Jiang T, Xu H, Lei H. Fermentation of method for estimating bacterial diversity. J Microbiol Methods. (1997) 30:111–
kiwifruit juice from two cultivars by probiotic bacteria: bioactive phenolics, 7. doi: 10.1016/S0167-7012(97)00056-0
antioxidant activities and flavor volatiles. Food Chem. (2022) 373:131455. 15. Tiwari BK, Muthukumarappan K, Odonnell CP. Colour degradation
doi: 10.1016/j.foodchem.2021.131455 and quality parameters of sonicated orange juice using response
4. Ankolekar C, Johnson K, Pinto M, Johnson D, Labbe RG, Greene D, surface methodology. LWT-Food Sci Technol. (2008) 41:1876–83.
et al. Fermentation of whole apple juice using lactobacillus acidophilus doi: 10.1016/j.lwt.2007.11.016
for potential dietary management of hyperglycemia, hypertension, and 16. Lima M, Silani I, Toaldo IM, Correa LC, Biasoto ACT, Pereira GE. Phenolic
modulation of beneficial bacterial responses. J Food Biochem. (2012) 36:718– compounds, organic acids and antioxidant activity of grape juices produced
38. doi: 10.1111/j.1745-4514.2011.00596.x from new Brazilian varieties planted in the northeast region of Brazil. Food
5. Varela P, Salvador A, Fiszman S. Shelf-life estimation of ’Fuji’ apples: Sensory Chem. (2014) 161:94–103. doi: 10.1016/j.foodchem.2014.03.109
characteristics and consumer acceptability. Postharvest Biol Tec. (2005) 38:18– 17. Wasik A, Mccourt J, Buchgraber M. Simultaneous determination of
24. doi: 10.1016/j.postharvbio.2005.05.009 nine intense sweeteners in foodstuffs by high performance liquid
6. Zhu G, Tian C. Determining sugar content and firmness of "Fuji’ chromatography and evaporative light scattering detection—Development
apples by using portable near-infrared spectrometer and diffuse and single-laboratory validation. J Chromatogr A. (2007) 1157:187–96.
transmittance spectroscopy. J Food Process Eng. (2018) 41:e12810.1–10. doi: 10.1016/j.chroma.2007.04.068
doi: 10.1111/jfpe.12810 18. A MRR, B RCB. Quantification of total phenols in bio-oil using the Folin–
7. Ross JA, Kasum CM. Dietary flavonoids: bioavailability, Ciocalteu method–sciencedirect. J Anal Appl Pyrol. (2013) 104:366–71.
metabolic effects, and safety. Annu Rev Nutr. (2002) 22:19–34. doi: 10.1016/j.jaap.2013.06.011
doi: 10.1146/annurev.nutr.22.111401.144957 19. Zhang W, Han F, He J, Duan C. HPLC-DAD-ESI-MS/MS analysis and
8. Wang X, Han M, Zhang M, Wang Y, Ren Y, Yue T, et al. In vitro evaluation antioxidant activities of nonanthocyanin phenolics in mulberry (Morus
of the hypoglycemic properties of lactic acid bacteria and its fermentation alba L). J Food Sci. (2008) 73:C512–518. doi: 10.1111/j.1750-3841.2008.
adaptability in apple juice. LWT- Food Sci Technol. (2021) 136:110363. 00854.x
doi: 10.1016/j.lwt.2020.110363 20. Abid M, Jabbar DS, Wu T, Hashim MM, Hu B, Lei S. Sonication enhances
9. Wang H, Tao Y, Li Y, Wu S, Li D, Liu X, et al. Application of ultrasonication polyphenolic compounds, sugars, carotenoids and mineral elements of apple
at different microbial growth stages during apple juice fermentation by juice. Ultrason Sonochem. (2013) 21:93–7. doi: 10.1016/j.ultsonch.2013.06.002
Lactobacillus plantarum: Investigation on the metabolic response. Ultrason 21. Wang Y, Tao Y, Zhang X, Shao S, Han Y, Chu DT. Metabolic profile of ginkgo
Sonochem. (2021) 73:105486. doi: 10.1016/j.ultsonch.2021.105486 kernel juice fermented with lactic aicd bacteria: a potential way to degrade
10. Chen C, Lu Y, Yu H, Chen Z, Tian H. Influence of 4 lactic acid bacteria ginkgolic acids and enrich terpene lactones and phenolics. Process Biochem.
on the flavor profile of fermented apple juice. Food Biosci. (2019) 27:30–6. (2019) 76:25–33. doi: 10.1016/j.procbio.2018.11.006
doi: 10.1016/j.fbio.2018.11.006 22. Yoon KY, Woodams EE, Hang YD. Production of probiotic cabbage
11. Kwaw E, Ma YK, Tchabo W, Apaliya MT, Wu M, Sackey AS. Effect of juice by lactic acid bacteria. Bioresour Technol. (2006) 97:1427–30.
lactobacillus strains on phenolic profile, color attributes and antioxidant doi: 10.1016/j.biortech.2005.06.018

Frontiers in Nutrition | www.frontiersin.org 12 February 2022 | Volume 9 | Article 833906

Yang et al. Lactobacillus Fermented Apple Juice

23. Ricci A, Cirlini M, Levante A. Volatile profile of elderberry juice: Effect 43. Knockaert D, Raes K, Wille C, Struijs K, Camp JV. Metabolism of ferulic acid
of lactic acid fermentation using L. plantarum, L rhamnosus and L during growth of Lactobacillus plantarum and Lactobacillus collinoides. J Sci
casei strains. Food Res Int. (2018) 105:412–22. doi: 10.1016/j.foodres.2017. Food Agr. (2012) 92:2291–6. doi: 10.1002/jsfa.5623
11.042 44. Filannino P, Bai Y, Cagno R, Gobbetti M, Gaenzle MG. Metabolism
24. Verardo V, Moreno-Trujillo TR, Caboni MF, Garcia-Villanova B, Guerra- of phenolic compounds by Lactobacillus spp. during fermentation
Hernandez EJ. Influence of infant cereal formulation on phenolic compounds of cherry juice and broccoli puree. Food Microbiol. (2014) 46:272–9.
and formation of Maillard reaction products. J Food Compos Anal. (2021) doi: 10.1016/j.fm.2014.08.018
104:104187. doi: 10.1016/j.jfca.2021.104187 45. Sanchez-Maldonado AF, Schieber A, Ganzie MG. Structure-function
25. Pimentel TC, Madrona GS, Garcia S, Prudencio SH. Probiotic viability, relationships of the antibacterial activity of phenolic acids and their
physicochemical characteristics and acceptability during refrigerated storage metabolism by lactic acid bacteria. J Appl Microbiol. (2011) 111:1176–84.
of clarified apple juice supplemented with Lactobacillus paracasei ssp. doi: 10.1111/j.1365-2672.2011.05141.x
paracasei and oligofructose in different package type. LWT-Food Sci Technol. 46. Huang J, Depaulis T, May J. Antioxidant effects of dihydrocaffeic acid
(2015) 63:415–22. doi: 10.1016/j.lwt.2015.03.009 in human EA. hy926 endothelial cells. J Nutr Biochem. (2005) 15:722–9.
26. Toit M, Engelbrecht L, Lerm E, Krieger-Weber S. Lactobacillus: the next doi: 10.1016/j.jnutbio.2004.07.002
generation of malolactic fermentation starter cultures—an overview. 47. Ricci A, Cirlini M, Calani L. In vitro metabolism of elderberry juice
Food Bioprocess Tech. (2011) 4:876–906. doi: 10.1007/s11947-010- polyphenols by lactic acid bacteria. Food Chem. (2019) 276:692–9.
0448-8 doi: 10.1016/j.foodchem.2018.10.046
27. Ye M, Yue T, Yuan Y. Polyphenols and organic acids evolution during apple 48. Tabasco R. Snchez-pat NF, Monagas M, Bartolome B, Moreno-Arribas MV,
cider fermentation. J Sci Food Agr. (2014) 94:2951–7. doi: 10.1002/jsfa.6639 Pelaez C. Effect of grape polyphenols on lactic acid bacteria and bifidobacteria
28. Zhang H, Zhou F, Ji B, Nout RMJ, Yang FZ. Determination of organic growth: Resistance and metabolism. Food Microbiol. (2011) 28:1345–52.
acids evolution during apple cider fermentation using an improved doi: 10.1016/j.fm.2011.06.005
HPLC analysis method. Eur Food Res Technol. (2008) 22:1183–90. 49. He L, Xu H, Liu X, He WH, Yuan F, Hou ZQ, et al. Identification of
doi: 10.1007/s00217-008-0835-9 phenolic compounds from pomegranate (Punica granatum L) seed residues
29. Dudley EG, Steele JL. Succinate production and citrate catabolism by and investigation into their antioxidant capacities by HPLC–ABTS+ assay.
cheddar cheese nonstarter lactobacilli. J Appl Microbiol. (2010) 98:14–23. Food Res Int. (2011) 44:1161–7. doi: 10.1016/j.foodres.2010.05.023
doi: 10.1111/j.1365-2672.2004.02440.x 50. Svensson L, Sekwati-monang B, Lopeslutz D, Schieber AG, Nzle MG.
30. Abdelrahman MA, Tashiro Y, Sonomoto K. Recent advances in lactic acid Phenolic acids and flavonoids in nonfermented and fermented red Sorghum
production by microbial fermentation processes. Biotechnol Adv. (2013) (Sorghum bicolor L Moench). J Agr Food Chem. (2010) 58:9214–20.
31:877–902. doi: 10.1016/j.biotechadv.2013.04.002 doi: 10.1021/jf101504v
31. Wang YC, Yu RC, Yang HY, Chou CC. Sugar and acid 51. Attri S, Sharma K, Raigond P, Goel G. Colonic fermentation of polyphenolics
contents in soymilk fermented with lactic acid bacteria alone or from sea buckthorn (Hippophae rhamnoides) berries: assessment of effects
simultaneously with bifidobacteria. Food Microbiol. (2003) 20:333–8. on microbial diversity by principal component analysis. Food Res Int. (2018)
doi: 10.1016/S0740-0020(02)00125-9 105:324–32. doi: 10.1016/j.foodres.2017.11.032
32. Ortiz-Cortés LY, Ventura-Canseco L, Abud-Archila M, Ruíz-Valdiviezo VM, 52. Hashemi SMB, Khaneghah AM, Barbaf FJ, Nemati Z, Shokofti SS, Alizadeh
Velázquez-Ríos IO, Alvarez-Gutiérrez PE. Evaluation of temperature, pH F. Fermented sweet lemon juice (Citrus limetta) using Lactobacillus
and nutrient conditions in bacterial growth and extracellular hydrolytic plantarum LS5: chemical composition, antioxidant and antibacterial activities.
activities of two alicyclobacillus spp. strains Arch Microbiol. (2021) 203:1–14. J Functional Foods. (2017) 38:409–14. doi: 10.1016/j.jff.2017.09.040
doi: 10.1007/s00203-021-02332-4 53. Wang J, Xie B, Sun Z. Quality parameters and bioactive compound
33. Hughes DB, Hoover DG. Viability and enzymatic activity of Bifidobacteria in bioaccessibility changes in probiotics fermented mango juice using ultraviolet-
Milk. J Dairy Sci. (1995) 78:268–76. doi: 10.3168/jds.S0022-0302(95)76634-6 assisted ultrasonic pre-treatment during cold storage. LWT-Food Sci Technol.
34. KMital B, Shallenberger RH, Steinkraus K. α-Galactosidase (2021) 137:110438. doi: 10.1016/j.lwt.2020.110438
activity of Lactobacilli. J Appl Microbiol. (1973) 26:783–8. 54. Filho A, Freitas HV, Rodrigues S, Virgínia Kelly Gonalves Abreu, Pereira
doi: 10.1128/am.26.5.783-788.1973 A. Production and stability of probiotic cocoa juice with sucralose as sugar
35. Bermdez R, Franco D, Carballo J. Influence of muscle type on the evolution substitute during refrigerated storage. LWT-Food Sci Technol. (2019) 99:371–
of free amino acids and sarcoplasmic and myofibrillar proteins through 8. doi: 10.1016/j.lwt.2018.10.007
the manufacturing process of Celta dry-cured ham. Food Res Int. (2014) 55. Queirozc C, Lopes M, Fialho E, Valente-Mesquita VL. Changes in bioactive
56:226–35. doi: 10.1016/j.foodres.2013.12.023 compounds and antioxidant capacity of fresh-cut cashew apple. Food Res Int.
36. Mcsweeney P, Sousa M. Biochemical pathways for the production of flavour (2011) 44:1459–62. doi: 10.1016/j.foodres.2011.03.021
compounds in cheeses during ripening: a review. Lait. (2000) 80:293–324. 56. Xiao Y, Wu X, Yao X, Chen Y, Ho CT, He C, et al. Metabolite profiling,
doi: 10.1051/lait:2000127 antioxidant and α-glucosidase inhibitory activities of buckwheat processed by
37. Mau JL, Tseng YH. Nonvolatile taste components of three strains of Agrocybe solid-state fermentation with Eurotium cristatum YL-1. Food Res Int. (2021)
cylindracea. J Agr Food Chem. (1998) 46:2071–4. doi: 10.1021/jf971016k 143:110262. doi: 10.1016/j.foodres.2021.110262
38. Zuljan FA, Mortera P, Alarcón SH, Blancato VS, Espariz M, Magni C. Lactic 57. Mousavi Z, Mousavi M, Razavi S, Hadinejad M, Emam-Djomeh Z, Mirzapour
acid bacteria decarboxylation reactions in cheese. Inter Dairy J. (2016) 62:53– M. Effect of Fermentation of pomegranate Juice by Lactobacillus plantarum
62. doi: 10.1016/j.idairyj.2016.07.007 and Lactobacillus acidophilus on the antioxidant activity and metabolism
39. Liesbeth R, Mireille Y, Richard van K, Pascal C, Annette V, Emilie C, et of sugars, organic acids and phenolic compounds. Food Biotechnol. (2013)
al. Lactococcal aminotransferases Arat and Bcat are key enzymes for the 27:1–13. doi: 10.1080/08905436.2012.724037
formation of aroma compounds from amino acids in cheese. Int Dairy J. 58. Braga A, Mesquita L, Martins P, Habu S, Rosso VD. Lactobacillus fermentation
(2003) 13:805–12. doi: 10.1016/S0958-6946(03)00102-X of jussara pulp leads to the enzymatic conversion of anthocyanins
40. Tanous C, Gori A, Rijnen L, Chambellon E, Yvon M. Pathways for - increasing antioxidant activity. J Food Compos Anal. (2018) 69:162–70.
ketoglutarate formation by Lactococcus lactis and their role in amino acid doi: 10.1016/j.jfca.2017.12.030
catabolism. Int Dairy J. (2004) 15:759–70. doi: 10.1016/j.idairyj.2004.09.011 59. Wu Y, Li S, Tao Y, Li D, Han Y, Show PL. et al. Fermentation of blueberry and
41. Thierry A, Mailard MB, Yvon M. Conversion of L-Leucine to isovaleric acid blackberry juices using Lactobacillus plantarum, Streptococcus thermophilus
by Propionibacterium freudenreichii TL 34 and itgp23. Appl Environ Microb. and Bifidobacterium bifidum: Growth of probiotics, metabolism of phenolics,
(2002) 68:608–15. doi: 10.1128/AEM.68.2.608-615.2002 antioxidant capacity in vitro and sensory evaluation. Food Chem. (2021)
42. Rodriguez H, Curiel JA, Landete JM. Food phenolics and lactic acid bacteria. 348:129083. doi: 10.1016/j.foodchem.2021.129083
Int J Food Microbiol. (2009) 132:79–90. doi: 10.1016/j.ijfoodmicro.2009. 60. Ozgen M, Reese RN, Tulio AZ, Scheerens JC, Miller AR. Modified 2,2-
03.025 azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method to measure

Frontiers in Nutrition | www.frontiersin.org 13 February 2022 | Volume 9 | Article 833906

Yang et al. Lactobacillus Fermented Apple Juice

antioxidant capacity of selected small fruits and comparison to ferric reducing Conflict of Interest: The authors declare that the research was conducted in the
antioxidant power (FRAP) and 2,2’-diphenyl-1-picrylhydrazyl (DPPH) absence of any commercial or financial relationships that could be construed as a
methods. J Agri Food Chem. (2006) 54:1151–7. doi: 10.1021/jf051960d potential conflict of interest.
61. Zhang B, Xia T, Duan W, Zhang Z, Wang M. Effects of organic acids, amino
acids and phenolic compounds on antioxidant characteristic of Zhenjiang Publisher’s Note: All claims expressed in this article are solely those of the authors
aromatic vinegar. Molecules. (2019) 24:3799. doi: 10.3390/molecules2420 and do not necessarily represent those of their affiliated organizations, or those of
3799 the publisher, the editors and the reviewers. Any product that may be evaluated in
62. Thuibaut S, Caillon J, Huart C, Grandjean G, Lombrail P, Potel
this article, or claim that may be made by its manufacturer, is not guaranteed or
G. Susceptibility to the main antibiotics of Escherichia coli and
endorsed by the publisher.
Staphylococcus aureus strains identified in community acquired infections
in France (MedQual, 2004–2007). Med Mal Al Infec. (2010) 40:74–80.
doi: 10.1016/j.medmal.2009.01.011 Copyright © 2022 Yang, Sun, Gao, Wu, Sun, Zhu, Liu, Zhou, Han and Tao. This is an
63. Reis JA, Paula AT, Casarotti S, Penna A. Lactic acid bacteria antimicrobial open-access article distributed under the terms of the Creative Commons Attribution
compounds: characteristics and applications. Food Eng Rev. (2012) 4:124–40. License (CC BY). The use, distribution or reproduction in other forums is permitted,
doi: 10.1007/s12393-012-9051-2 provided the original author(s) and the copyright owner(s) are credited and that the
64. Pereira A, Maciel TC, Rodrigues S. Probiotic beverage from cashew apple original publication in this journal is cited, in accordance with accepted academic
juice fermented with Lactobacillus casei. Food Res Int. (2011) 44:1276–83. practice. No use, distribution or reproduction is permitted which does not comply
doi: 10.1016/j.foodres.2010.11.035 with these terms.

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