Evaluation of a strawberry fermented

beverage with potential health benefits

Zhiqiao Zhao1 ,* , Xulong Wu2 ,* , Hong Chen3 , Yuntao Liu3 , Yirong Xiao4 ,
Hui Chen1 , Zizhong Tang1 , Qingfeng Li1 and Huipeng Yao1
1
College of Life Sciences, Sichuan Agricultural University, Ya’an, China
2
Chengdu Agricultural College, Chengdu, China
3
College of Food Sciences, Sichuan Agricultural University, Ya’an, China
4
Sichuan Agricultural University Hospital, Ya’an, China
*
These authors contributed equally to this work.

ABSTRACT
Background. Functional fermented beverages are popular worldwide due to their
potential to promote health. Starter culture is the main determinant of the final quality
and flavor of fermented beverages. The co-cultivation of lactic acid bacteria (LAB)
and yeast makes a significant contribution to the safe flavor of fermented beverages.
However, the research on the potential of antioxidant, antimicrobial, and anti-biofilm
formation of strawberry fermented beverage obtained by combining the LAB and yeast
as starter cultures has not been well explored.
Methods. In this study, LAB and yeast were combined as starter culture to obtain
strawberry fermented beverage. Fourier transform infrared (FTIR ) spectroscopy
was used for the qualitative analysis of the fresh strawberry juice and fermented
beverage. From the changes in antioxidant content, free radical scavenging ability,
total superoxide dismutase (T-SOD) activity and total antioxidant capacity (T-AOC)
to evaluate the antioxidant capacity of fermented beverage in vitro. The antibacterial
ability was tested by the Oxford cup method. The biofilms of Escherichia coli ATCC
25922, Staphylococcus aureus ATCC 6538 under fermented beverages treatment was
observed by Fluorescence microscope. In addition, sensory analysis was conducted in
this study.
Submitted 21 April 2021
Results. In this study, the absorption peaks of Fourier transform infrared between
Accepted 24 July 2021 1,542 cm−1 and 976 cm−1 , suggest the existence of organic acids, sugars and ethanol.
Published 23 August 2021 The total phenols and total flavonoids content decreased by 91.1% and 97.5%,
Corresponding author respectively. T-SOD activity increased by 33.33%.The scavenging ability of fermented
Zizhong Tang, [email protected] beverage on superoxide anion free radicals was enhanced, and the scavenging ability
Academic editor on DPPH free radicals, hydroxyl free radicals, and ABTS free radicals was weakened.
Charles Okpala However, the T-AOC increased from 4.15 ± 0.81 to 8.43 ± 0.27 U/mL. Fermented
Additional Information and beverage shows antibacterial activity against four pathogens. The minimum inhibitory
Declarations can be found on concentration (MIC) values of Escherichia coli ATCC 25922 and Staphylococcus aureus
page 17 ATCC 6538 were 0.05 mL/mL and 0.025 mL/mL, respectively, and the minimum bac-
DOI 10.7717/peerj.11974 tericidal concentration (MBC) were both 0.2 mL/mL. It was observed by fluorescence
microscope that the green fluorescence area of the two biofilms is greatly reduced after
Copyright
2021 Zhao et al. being treated with fermented beverage. Sensory analysis results show that the average
scores of fermented beverage in color, appearance and taste were increased. The overall
Distributed under
Creative Commons CC-BY 4.0
impression and flavor were decreased.

OPEN ACCESS

How to cite this article Zhao Z, Wu X, Chen H, Liu Y, Xiao Y, Chen H, Tang Z, Li Q, Yao H. 2021. Evaluation of a strawberry fermented
beverage with potential health benefits. PeerJ 9:e11974 http://doi.org/10.7717/peerj.11974
 Conclusion. These results demonstrated that strawberry fermented beverage has
potential benefits such as an antioxidant, antibacterial, and anti-biofilm formation,
providing the potential for the fermented beverage to become promising candidates
for natural antioxidants, antibacterial agents and anti-biofilm agents.

Subjects Biochemistry, Food Science and Technology, Microbiology

Keywords Fermentation, Fruit fermented beverage, Antioxidant Capacity, Pathogenic bacteria,
Anti-biofilm formation, Sensory analysis, Strawberry, Probiotics, Fourier transform infrared
(FTIR) analysis, Fluorescence microscopy

INTRODUCTION
Strawberry (Fragaria x ananassa Duch) is one of the most popular berries around the
world and is rich in a variety of nutrients such as minerals, vitamin C, vitamin E,
phenolics, flavonoids, β-carotene, folate and potassium (Zhang et al., 2012; Peretto et
al., 2014; Skrovankova et al., 2015). However, strawberry is an extremely perishable fruit
because of its high respiration rate and high level of water content, being vulnerable to
mechanical damage, water loss, and microbial infections during storage (Sanz et al., 1999;
Perkins-Veazie, 2010). Selection of ideal processing technology to prolong the shelf life of
strawberries and increase the added value are important problems to be solved. With the rise
of vegetarianism and the discovery of the negative effects of probiotic dairy products, such as
lactose intolerance, an increase in cholesterol content, and a milk protein allergy (Camargo
Prado et al., 2015), consumers are focusing on non-dairy probiotic products that are high
in nutrition and free of cholesterol and lactose (Pereira et al., 2017). In the development
of non-dairy probiotics, the development of functional fermented beverages, use fruit and
vegetable juice as substrate, has attracted people’s interest (Pereira, Maciel & Rodrigues,
2011), because fruit and vegetable juice is a good source of nutritional compounds (such
as carbohydrates, dietary fibers, vitamins, minerals), can be used as a carrier of probiotics
(Septembre-malaterre, Remize & Poucheret, 2018). This has been proven with cucumber
juice (Tamang & Tamang, 2010) pomegranates juice (Mantzourani et al., 2019), orange
juice (Escudero-López et al., 2015), pineapple juice (Chanprasartsuk et al., 2010) as well as
others (Dias et al., 2018), and this type of fermented beverages tastes appeal to people of
all ages as they are considered healthy and refreshing (Pereira, Maciel & Rodrigues, 2011).
Fermentation is a biotransformation process that is affected by two main factors: starter
cultures or native microorganisms and substrates, and other inherent factors such as
temperature and pH conditions (Yunita & Dodd, 2018). Therefore, the appropriate starter
cultures and substrate should be selected before the preparation of fermented beverage.
Considering the nutritional value and sensory characteristics of the fruit and vegetable
fermented beverage, LAB and yeast were combined as starter cultures because effect of
multistrain or multispecies probiotic beverages may provide greater beneficial effects than
monostrain culture (Marsh et al., 2014). When yeast and LAB are co-cultured, it can not
only promote the growth of either group of microbes but also the fermented product has
flavor and good organoleptic properties (Mukisa et al., 2017). In this context, strawberry

Zhao et al. (2021), PeerJ, DOI 10.7717/peerj.11974 2/23

 juice rich in a variety of nutrients molecules can be considered fermented into functional
fermented beverages to extend the shelf life.
During the process of cell metabolism, excessive production of free radicals can cause
a series of diseases, such as aging, obesity, arteriosclerosis, etc. (Hassan, Ghareb & Azhari,
2017). Therefore, the balance and stability of free radicals is very important for maintaining
the body. At present, the extensive use of antibiotics poses a great threat to environmental
safety (Gao et al., 2012), and the resistance of bacteria to antibiotics has become very obvious
(Jankauskaite et al., 2016). Some of the common multidrug-resistant microorganisms
are Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, Bacillus subtilis
(Alekshun & Levy, 2007). Staphylococcus aureus is a Gram-positive bacterium with a variety
of virulence factors that can cause some symptoms such as pulmonary, osteoarticular
and skin and soft tissue infections, as well as Bacillus subtilis, which can cause food
spoilage (Tong et al., 2015; Wu et al., 2020). Escherichia coli and Pseudomonas aeruginosa
are Gram-negative microorganisms, related to diseases in humans and animals, such as
gastroenteritis, urinary infections, peritonitis and meningitis (Picoli et al., 2017). Biofilm is
one of the main reasons for the development of multidrug resistance in most bacteria. In
addition, the appearance of biofilm on kinds of medical equipment and implants such as
contact lenses, heart valves, artificial joints and among others usually causing both systemic
and local infections (Vishwakarma & Vavilala, 2019). The selection and development of
novel antioxidants, antibacterial agents or anti-biofilm agents is of great significance in
this context. Fruit and vegetable functional fermented beverages can be used as a new
nature candidate because of their potential health benefits. Hashemi et al. (2017) showed
that fermentation improves the chemical properties such as antioxidant activity as well as
antibacterial properties compared with unfermented sweet lemon juice, and the fermented
juice could be used as a nondairy functional food product. This was consistent with the
results obtained after fermentation of immature pear fruits (Lee et al., 2016). Ayed, Ben
Abid & Hamdi (2017) also observed the same result, that is, the antioxidant capacity and
antibacterial capacity against the tested microorganism of red grape juice after fermentation
with kombucha consortium were enhanced. In addition, other fermented beverages such as
pomegranates (Mantzourani et al., 2019), cupuassu pulps (Pereira et al., 2017), red beetroot
(Vanajakshi et al., 2015), and other fruits as fermentation carriers also have potential health
benefits such as anti-oxidant, antibacterial and other health effects. However, to the best
of our knowledge, no studies have considered the use of fermented fruit and vegetable
beverages as anti-biofilm formation inhibitor, and research on fermented strawberry
beverages has focused on the physiochemical properties (Ordóñez et al., 2015; Hornedo-
Ortega et al., 2016; Park et al., 2017; Yang et al., 2021), and the research on potential health
benefits of strawberry fermented beverage is relatively poor.
Therefore, the current study aims to evaluate the potential health effects (antioxidant,
antibacterial and anti-biofilm potential) of strawberry fermented beverages. In addition, its
sensory analysis has also been studied. Our study will help pave the way to develop novel
natural antioxidants, antibacterial agents, and anti-biofilm properties.

Zhao et al. (2021), PeerJ, DOI 10.7717/peerj.11974 3/23

 Figure 1 Flow diagram.
Full-size DOI: 10.7717/peerj.11974/fig-1

MATERIALS & METHODS

Schematic overview of the experimental program
This study mainly used the non-spontaneous fermentation method to obtain strawberry
fermented beverage, and then explored the potential health promotion benefits of
strawberry fermented beverage. The specific scheme is as follows.
The fruit (strawberry) fermentation conducted in this study was performed in the
laboratory. Starter cultures (LAB and yeasts) are inoculated into strawberry juice, 30 days
for the main fermentation at room temperature (25 ◦ C), 5 months for the maturation is
at 16 ◦ C. The whole process is usually slow requiring 6 months. The production process is
shown in Fig. 1. Unfermented fresh strawberry juice was used as a control. The fermented
strawberry beverage and fresh juice were centrifuged, and the supernatant was used as
test sample for research on FTIR, antioxidant capacity (including antioxidant content,
free radical scavenging capacity, T-SOD activity, T-AOC), and antibacterial capacity. The
fermented beverage-treated biofilms of Escherichia coli ATCC 25922 and Staphylococcus
aureus ATCC 6538 were observed by fluorescence microscopy. Finally, sensory analysis
was conducted on the fermentation samples and strawberry juice.

Raw materials
Fresh strawberries and sugar were procured from a local supermarket in Ya’an City, Sichuan
Province, China.

Starter strains and pathogenic microorganisms

Starter strains of LAB (Lactobacillus plantarum and Lactobacillus bulgaricus) and yeast
(Saccharomyces cerevisiae) were obtained from the Sichuan Food Fermentation Industry
Research and Design Institute, Chengdu City, Sichuan Province, China. Strains of LAB

Zhao et al. (2021), PeerJ, DOI 10.7717/peerj.11974 4/23

 were incubated in de Man, Rogosa, and Sharpe Medium (MRS), yeast was incubated in
extract Extract Peptone Dextrose Medium (YPD) medium.
Four pathogenic microorganisms (Escherichia coli ATCC 25922, Staphylococcus aureus
ATCC 6538, Pseudomonas aeruginosa ATCC 9027, and Bacillus subtilis ATCC6633) from
the College of Life Science, Sichuan Agricultural University, Yaan City, Sichuan Province,
China. The bacteria were cultured in Luria-Bertani (LB).

Preparation of the fermented beverage

Fresh strawberries (1.43 kg) were cleaned, cut and put into the juicer (JYL-C16D/C16V,
Joyoung Company Limited), then sterile water (2.86 L) was supplemented to squeeze the
strawberries to obtain strawberry juice. Then fruit juice (4 L) was taken, and white sugar
(0.64 kg) was added then stirred. Thereafter, the juice sample was pasteurized at 80 ◦ C
for 5 min, after pasteurization, the juice sample was immediately cooled with ice water.
Subsequently, the starter culture was inoculated into the juice (initial cell density of 5 log
CFU/mL for the yeasts and 7 log CFU/ mL for the LAB). Fermentation was conducted in
a glass bottle with a total volume of 5 L, of which the substrate occupies 4 L. The main
fermentation of the juice was performed for 30 days at 25 ◦ C, and then mature fermentation
was conducted for 5 months at 16 ◦ C for a total of 6 months. Finally, the fermented beverage
was centrifuged (8000 r/min, for 20 min, 4 ◦ C) by a centrifuge: (Thermo Scientific Sorvall
ST 16, Shanghai Fuze Trading Co., Ltd) to obtain supernatants for analysis.

Fourier transform infrared (FTIR) analysis

The FTIR spectra of the strawberry juice and fermented beverage was examined by an FTIR
spectrophotometer (Nicolet IS10, Shanghai Precision Instrument Co., Ltd).

Antioxidant activity
Antioxidant
The total phenol content of the sample was determined by the Folin-Ciocalteu colorimetric
method according to previous research, with slight modifications (Abirami, Nagarani &
Siddhuraju, 2014). Firstly, 1 mL of Folin-Ciocalteu reagent was taken and then added to the
1 mL of diluted samples. After 3 min, 3.0 mL of the solution of sodium carbonate (20%,
w/v) was added. Further, the mixture solution was allowed to incubate for 30 min at 25 ◦ C.
Finally, the absorbance of the mixture was measured at 760 nm by a Microplate Reader.
The calibration curve was prepared from a standard solution of gallic acid (ranging from0
to 0.035 mg/mL). The total phenol content was expressed by the equivalent of gallic acid,
which was calculated according to the calibration curve of gallic acid. The calibration curve
equation was A = 15.4867C−0.0036 (R2 = 0.9970).
The total flavonoids content of the sample was determined by the Aluminum chloride
colorimetric method according to previous research, with slight modifications (Abirami,
Nagarani & Siddhuraju, 2014). Firstly, 0.15 mL of sodium nitrite (5%, m/v) was taken and
then added to the 1 mL of diluted samples. After standing for 6 min, 0.3 mL of the solution of
aluminum chloride (10%, w/v) was added. After the mixture was allowed to stand for 5 min,
1.00 mL of 1 mol/mL sodium hydroxide was added. Finally, the absorbance of the mixture
was measured at 510 nm by a Microplate Reader. The calibration curve was prepared from

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 a standard solution of rutin (ranging from 0 to 0.1mg/mL). The total flavonoids content
was expressed by the equivalent of rutin, which was calculated according to the calibration
curve of rutin. The calibration curve equation was A = 3.0219C+0.0105 (R2 = 0.9951).
T-SOD activity of the sample was determined by the T-SOD assay kit (Jiancheng, Nanjing,
China) following the manufacture’s instructions of the kit, respectively.

Scavenging free radical capacity

Samples were diluted with distilled water to different concentration (0.08, 0.16, 0. 24, 0.
32, 0.40, 0.48, 0.56, 0.64 mL/mL), use the 0.1 mg/ mL ascorbic acid (Vc) as the control.
DPPH radical-scavenging activity of the sample was determined following the previous
study (Yang et al., 2018), with slight modifications. In brief, 0.1 mL of 0.2 mmol/L freshly
prepared DPPH solution were added to 0.1 mL of sample and homogenized, then the
mixture was incubated at room temperature for 30 min in the dark. The absorbance of the
mixture was measured at 517 nm. DPPH radical-scavenging activity is expressed as% and
determined as follows the formula:
A1 − A2
DPPH radical-scavenging activity (%) = [1 − ] × 100%
A0
where A1 is the absorbance of the sample, A2 is the absorbance of absolute ethanol instead
of DPPH solution, A0 is the absorbance of solvent instead of the sample solution.
The Hydroxyl radical-scavenging activity of the sample was determined following the
previous study, with slight modifications (Xiao et al., 2015). Briefly, 0.2 mL solution of 6
mmol/L FeSO4 , 0.2 mL solution of 6 mmol/L salicylic acid, and 0.2 mL solution of six
mmol/L H2 O2 was taken and added to the 0.2 mL of sample, then make up to 0.5 mL with
ethanol. The mixture was reacted at 37 ◦ C for 30 min and its absorbance was measured at
510 nm. Hydroxyl radical-scavenging activity is expressed as% and determined as follows
the formula:
A1 − A2
Hydroxyl radical-scavenging activity (%) = [1 − ] × 100%
A0
where A1 is the absorbance of the sample, A2 is the absorbance measured by using ethanol
instead of the sample and H2 02 solution, adding ferrous sulfate and salicylic acid-ethanol
solution, A0 is the absorbance of ethanol instead of sample.
The Superoxide anion radical-scavenging activity of the sample was determined
following the previous study (Leong et al., 2008). Firstly, 1 mL of the sample was added to 3
mL of 0.05 mon/L Tris–HCl (pH 8.2) and homogenized, then the mixture was incubated at
25 ◦ C for 30 min. Next, 0.4 mL of 25 mmon/L pyrogallols, which was also pre-warmed to 25
◦ C, was added to the mixture and incubated for 4 min. Finally, add 0.5 mL of concentrated

hydrochloric acid to stop the reaction. The absorbance of the mixture was measured at
325 nm. Superoxide anion radical-scavenging activity is expressed as% and determined as
follows the formula:
A1 − A2
Superoxide anion radical-scavenging activity (%) = × 100%
A1
where A1 is the absorbance of the sample, A2 is the absorbance of distilled water instead of
the sample.

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 ABTS radical-scavenging activity of the sample was determined following the protocol
previously described by Tang et al. (2020).

T-AOC
The T-AOC of the sample was determined by the Ferric ion reducing antioxidant power
method with (T-AOC) assay kit (Jiancheng, Nanjing, China) according to the manufacture’s
instructions of the kit.

Antimicrobial activity
Inhibitory zone assay
In vitro antimicrobial activity of the sample was determined by the Oxford plate method
previously described by Ye, Dai & Hu (2013), with slight modifications. Firstly, 0.2 mL of
bacterial suspension of the tested were taken and then was coated on the LB culture plates.
Further, three sterile Oxford cups (a stainless cylinder of inner diameter 6.00 mm, outer
diameter 7.80 mm, height 10.00 mm) were gently placed on the surface of the culture
medium with a tweezer, then 0.2 mL of the sample being sterilized by filtration through
0.22 µm Millipore filter, sterile water (negative control) and 10 mg/mL ampicillin (positive
control) were transferred into respectively the Oxford cup and incubated at 37 ◦ C for 12
h. Finally, measurement of the diameter of the inhibition zone (with a micrometer) of the
tested pathogenic microorganisms was evaluated for antimicrobial activity.

Growth curve
50 µL of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 6538 suspensions
with an optical density of 0.6 at 600 nm were added to separate 10 mL sterile LB broth.
The treatment group was added 150 µL of fermented beverages filtered through a 0.22 µm
Millipore filter, and the control group was added 150 µL of normal saline. The sample was
cultured on a shaker at 37 ◦ C and 130 r/min. Sample were taken every 2 h and cultured
continuously for 12 h. Then, the OD value was measured at 600 nm with an ultraviolet
spectrophotometer. Finally, the growth curves of the treatment group and the normal
growth control group were drawn respectively.

Determination of MIC and MBC

The test of MIC and MBC of Escherichia coli ATCC 25922 and Staphylococcus aureus
ATCC 6538 was conducted according to the method of Jardak et al. (2017), with slight
modifications. The final concentration of the fermented beverage is 0.2, 0.1, 0.05, 0.025,
0.0125, 0.00625, 0.003125, 0.00156, 0.00078, 0.00039 mL/mL.

Fluorescence microscopy of biofilm

The effect of fermentation on biofilm inhibition was observed by fluorescence microscopy
(OLYMPUS BX53, Tianjin Leike Optical Instruments Co., Ltd). This test was conducted
according to Jardak et al. (2017).

Sensory analysis
Sensory analysis of the fermented beverage and strawberry juice was conducted according
to Di Cagno et al. (2011). 15 untrained panelists (7 men and 8 women), ranging from 22 to

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 Figure 2 Fourier transform infrared spectra for the strawberry juice (A) and fermented beverage (B)
and second-order derivative spectra with strawberry juice (C) and fermented beverage (D).
Full-size DOI: 10.7717/peerj.11974/fig-2

43 years old from the College of Life Sciences in Sichuan Agricultural University conducted
a sensory analysis based on the color, taste, flavor, appearance, and overall impression of
the samples. Samples were scored from 1 to 7 (1: dislike extremely; 2: dislike moderately;
3: dislike slightly; 4: neither like nor dislike; 5: like slightly; 6: like moderately; 7: like
extremely). The sample is stored in a glass cup at 4 ◦ C with a volume of 15 mL. The sensory
evaluation was conducted from 9:00 to noon under white light. The members were given
drinking water to rinse their mouths before testing the samples. The sensory evaluation
score of the color, taste, flavor, appearance, and overall impression is the average score.

Data analysis
The results are presented as the mean ± standard deviation of three replicates of each
experiment performed. Statistical analysis was carried out using the SPSS 22 software (SAS
Institute Inc., USA). Analysis of variance (ANOVA) and Independent-sample t -test were
used to evaluate the significant difference among various treatments.

RESULTS
FTIR analysis
FTIR spectrophotometric analysis, which is based on the vibration of functional groups
present in the sample when exposed to infrared radiation and was shown in (Figs. 2A–2C).

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 The normalized FTIR spectra were preprocessed second-order derivatives (Figs. 2B–2D).
The characteristic absorption peaks at 3,428, 2,937, 1,640, 1,419, 1,054, 594 cm−1 were
observed for strawberry juice. The broad peak at 3428 cm−1 was attributed to stretching
vibration of the -OH functional group. The absorption peaks at 2,937 cm−1 were found in
the FTIR spectra indicates strong C–H stretching vibration of aliphatic compounds, with
methyl (–CH3 ) and methylene(–CH2 ) symmetric and asymmetric stretching vibration.
The bands of peaks at 1960–1500 cm−1 were assigned to C=C C=N N=N N=O stretching
vibration and skeleton vibration of the benzene ring. The absorption peak at 1,419 cm−1
can be attributed to the asymmetric stretching vibration of carboxylation (COO-). The
absorption peaks at ∼1,040 cm−1 represent–C–O alcohols, with a typical ethanol peak.

Antioxidant activity
Antioxidant
The content of the total phenolics, total flavonoids, and the T-SOD activity in the fermented
beverage is presented in Table 1. It can be seen from the table that the total phenols and
total flavonoids content decreased by 91.1% (p < 0.01) and 97.5% (p < 0.01), respectively,
and T-SOD activity increased by 40.48 U/mL (p < 0.01) after six months of fermentation.

Free radical scavenging ability

DPPH is a simple and efficient assay is commonly applied to evaluating antioxidant
activity. Hydroxyl radical is a very active reactive oxygen species, which could damage
the structure of biological macromolecules and cause tissue damage or cell death (Liu
et al., 2010). Superoxide anion is produced during normal cellular respiration and plays
fundamental roles in cellular physiology with its dysregulation being associated with a
variety of diseases (Rahmani, Ghavamipour & Sajedi, 2019). The ABTS radical assay is
among the most abundant assays to evaluate antioxidant capacity (Garcia-Leis et al., 2016).
In the concentration range of 0.08–0.64 mL/mL, the four free radical scavenging activities
of samples are shown in Fig. 3. Samples were found to have the ability to scavenge free
radicals and the scavenging rate increased with the increase of the concentration. Figure 3A
shows that strawberry juice has the strongest scavenging ability on DPPH free radicals,
followed by VC , and fermented beverage was the weakest. The maximum value of DPPH
scavenging rate was 83.70% and 94.87% of strawberry juice and fermented beverage was
found at a concentration of 0.40 mL/mL, respectively. At this concentration, the scavenging
rate of VC on DPPH free radicals was 91.05%. Fig. 3B shows that the scavenging ability
of strawberry juice and fermented beverage on hydroxyl radicals is stronger than that of
VC, which increases from 14.68% to 96.46% and 38.50% to 96.06%, respectively. And VC
increases from 31.90% to 66.81%. When the concentration range is 0.48 mL/mL to 0.64
mL/mL, the scavenging rate of hydroxyl radicals of the two samples is almost the same.
However, the scavenging rate of fermented beverage on superoxide anions free radical
increase from 15.15% to 80.17% is stronger than that of strawberry juice from 4.14% to
71.41%, and also it is stronger than VC in the range from 0.32 mL/mL to 0.64 mL/mL
(Fig. 3C). In the ABTS assay, the scavenging rate of fermented beverage on ABTS 6.99%
to 74.59% lower than strawberry juice 12.91% to 89.68%, and the scavenging rate of VC

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 Table 1 Analysis of antioxidants in the strawberry fermented beverage.

Parameter Non-fermented Fermented

Total phenolic(mg/mL) 3.47 ± 0.02 0.28 ± 0.03**
Total flavonoids(mg/mL) 0.40 ± 0.05 0.01 ± 0.001**
T-SOD(U/mL) 80.99 ± 1.95 121.47 ± 1.02**
Notes.
Values are expressed as mean (n = 3) ±SD; Independent-sample t -test used to evaluate the significant difference among vari-
ous treatments. Differences were considered to be significant at p < 0.01 (**).
Non-fermented corresponds to strawberry juice; Fermented corresponds to fermented beverages. The following occurrences
all mean the same thing.

Figure 3 Evaluation of scavenging free radical capacity. DPPH radical-scavenging activity assay (A);
Hydroxyl free radical scavenging activity assay (B); Superoxide anion radical-scavenging activity assay (C);
ABTS radical-scavenging activity assay (D).
Full-size DOI: 10.7717/peerj.11974/fig-3

has always been higher than that of fermented beverage (Fig. 3D). To further determine
the antioxidant capacity of fermented beverage, we also measured its T-AOC, and the
results showed that the T-AOC of fermented beverage (8.43 U/mL) was higher than that of
strawberry juice (4.15 U/mL), even though they were all lower than the VC (108.90 U/mL)
(Fig. 4).

Antibacterial activity of strawberry fermented beverage

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 Figure 4 Evaluation of T-AOC. Means that do not share the same letter are significantly different at p <
0.05.
Full-size DOI: 10.7717/peerj.11974/fig-4

Table 2 The diameter of the inhibition zone of the strawberry fermented beverage prepared by lactic acid bacteria and yeast on pathogenic mi-
croorganisms.

Microorganisms Inhibition zone diameter(mm)

10 mg/mL ampicillin Sterile water Non-fermented Fermented
a
E scherichia coli ATCC 25922 24.50 ± 1.114 0 0 28.30 ± 1.80a
Staphylococcus aureus ATCC 6538 24.03 ± 0.61a 0 0 26.60 ± 1.65a,b
b
Pseudomonas aeruginosa ATCC 9027 22.53 ± 0.25 0 0 23.13 ± 1.42c
c
Bacillus subtilis ATCC6633 9.80 ± 0.36 0 0 23.87 ± 2.11b,c
Notes.
Values are expressed as mean (n = 3) ±SD; Analysis of variance (ANOVA) was used to evaluate the significant difference among various treatments, with the criterion of p <
0.05.
Means that do not share the same letter are significantly different at p < 0.05.

Inhibitory zone assay

In this study, Bacillus subtilis ATCC6633, Staphylococcus aureus ATCC 6538, Escherichia
coli ATCC 25922 and Pseudomonas aeruginosa ATCC 9027 were used as indicator bacteria.
The inhibitory zone value of strawberry juice and fermented beverage is shown in Table 2
The represented that the fermented beverage exhibited acceptable antibacterial effects on
the four pathogenic microorganisms compared with strawberry juice. Concerning the size
of the inhibition zone, the results also showed that the fermented beverage has a more
pronounced antibacterial effect against Escherichia coli ATCC 25922 (28.30 ± 1.80 mm)
and Staphylococcus aureus ATCC 6538 (26.60 ± 1.65 mm) compared with Bacillus subtilis
ATCC 25922 (23.13 ± 1.42 mm) and Pseudomonas aeruginosa ATCC 9027 (23.87 ± 2.11
mm), thereby, the first two strains were selected for the following research.

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 Figure 5 Effects of fermented beverage on growth curve of Escherichia coli ATCC 25922 (A) and
Staphylococcus aureus ATCC 6538 (B).
Full-size DOI: 10.7717/peerj.11974/fig-5

Growth curve
The bacterial growth curve is an essential representation for characterizing bacteria
metabolism within a variety of media compositions. To further demonstrate the
antibacterial ability of fermented beverage against Escherichia coli ATCC 25922 and
Staphylococcus aureus ATCC 6538, the bacterial growth curves under the influence of
fermented beverage were measured within 12 h. The results showed that the growth rate
of the two pathogenic microorganisms, in the medium supplemented with a fermented
beverage, was significantly slowed down compared with the control group within 12 h.
We speculated fermented beverage has a good antibacterial effect against Escherichia coli
ATCC 25922 (Fig. 5A) and Staphylococcus aureus ATCC 6538 (Fig. 5B), which may be that
the presence of fermented beverage in the medium inhibits the growth of microorganisms,
resulting in a decrease in the total number of microorganisms and a decrease in the optical
density value at OD600.

Determination of MIC and MBC

The MIC and MBC assays determined that that the MIC of fermented beverage against
Escherichia coli ATCC 25922 and the Staphylococcus aureus ATCC 6538 were 0.05 mL/mL
and 0.025 mL/mL, respectively. whereas the MBC increased, both were 0.2 mL/mL.

Fluorescence microscopy
To confirm the ability of fermented beverage to the anti-biofilm formation of Escherichia
coli ATCC 25922 and Staphylococcus aureus ATCC 6538, we use a fluorescence microscope
to visually demonstrate the inhibitory effect of fermented beverages on biofilm. Figure 6
shows four anti-adhesion images obtained from untreated and fermented beverage-treated
biofilm stained with A pyridine orange and observed by a fluorescence microscope. This
image shows the difference in biofilm formation between untreated (Figs. 6A and 6C) and
treated (Figs. 6B and 6D). It can be seen that the green fluorescent area is greatly reduced
after the fermented beverage is treated, and a large number of single colonies appear. It is
preliminarily speculated that the fermented beverage has an antiadhesive effect.

Zhao et al. (2021), PeerJ, DOI 10.7717/peerj.11974 12/23

 Figure 6 Fluorescence microscopy with biofilm coverslips. Biofilms were stained with acridine orange.
Barequals 100 µm. Escherichia coli ATCC 25922 biofilm (A), control coverslip; Escherichia coli ATCC
25922 biofilm treated with 0.15 mL/mL fermented beverage (B); Staphylococcus aureus ATCC 6538 biofilm
(C), control coverslip; Staphylococcus aureus ATCC 6538 biofilm treated with 0.15 mL/mL fermented bev-
erage (D).
Full-size DOI: 10.7717/peerj.11974/fig-6

Sensory analysis
In this study, strawberry juice and fermented beverage were sensory evaluated.
Compared to strawberry juice, the average scores of fermented beverage in color,
appearance and taste increased by 0.26, 0.66 and 0.07, respectively. The overall impression
and flavor decreased by 0.07 and 0.7. Figure 7 shows these results by exhibiting that the
flavor and appearance average scores were more variable between the strawberry juice and
fermented beverage than the average scores from other attributes, such, color, taste, and
overall impression.

DISCUSSION
Fruit fermented beverages have attracted a lot of attention due to their various potential
health benefits. However, research on strawberry fermented beverages is rare and
incomplete. Given this, this research comprehensively studied the functional groups,
antioxidant capacity, antibacterial capacity, and anti-biofilm formation capacity of the
strawberry fermented beverage. In addition, considering the commercial potential of the
fermented beverage, we also conducted a sensory evaluation on it.
FTIR technology can be used for the general classification and comparison of food
materials including wine due to its fast, accurate, and non-destructive advantage. In

Zhao et al. (2021), PeerJ, DOI 10.7717/peerj.11974 13/23

 Figure 7 Sensory evaluation of color, appearance, flavor, taste and over impression of strawberry juice
and fermented beverage.
Full-size DOI: 10.7717/peerj.11974/fig-7

our study, a characteristic peak appeared at 2,937 cm−1 , which means the presence of
aromatic ester (Refer to Figs. 2A–2C). Vaithilingam et al. (2016) studies have shown that
the presence of aromatic primary amine groups is the result of the fermentation of probiotic
microorganisms, which means that there is a bacterial protein in the fermentation product,
which could affect the antibacterial properties of the fermented product. Ayed, Ben Abid
& Hamdi (2017) stated that the deformation vibration of the C-C bond in the phenolic
group absorption peak between 1,500 cm−1 and 1,400 cm−1 ; the band between 1,542 cm−1
and 965 cm−1 is attributed to vibration of the C-O, C-N, and C-N, demonstrating the
presence of organic acids, sugars, and ethanol in this area; the peak bands at 1,098 cm−1
and 995 cm−1 are due to OH deformation and C-O stretching in the phenolic group. The
fermented beverage showed several novel peaks, suggesting that the active molecules are
transformed by the microorganism during the fermentation process.
Many human chronic diseases such as cancer, aging, obesity, etc. are believed to be
related to the oxidative damage of reactive oxygen species (ROS) to cells (Hassan, Ghareb
& Azhari, 2017), the presence of antioxidants (such as total phenol, flavonoids, and SOD)
to maintain a dynamic balance between the generation and removal of free radicals in
vivo is important. A standard method for assessing the antioxidant capacity of fermented
beverages has not been established (Freire et al., 2017). For that reason the antioxidant
activity of fermented beverage was comprehensively conducted to evaluate through
changes in antioxidant content, free radical scavenging ability, T-SOD activity and T-AOC.
An interesting result that was found in our research was that the content of total phenols
and total flavonoids were reduced but the T-SOD was improved after fermentation (Refer
to Table 1). In addition, the scavenging ability of fermented beverage to DPPH free radicals,

Zhao et al. (2021), PeerJ, DOI 10.7717/peerj.11974 14/23

 hydroxyl free radicals, and ABTS free radicals was reduced, and the scavenging ability of
superoxide anion free radicals was enhanced compared with strawberry juice (Refer to
Fig. 3). Although the content of antioxidants and the ability to scavenge free radicals have
changed, to a greater or lesser degree, the T-AOC has been improved (Refer to Fig. 4). This
analysis is consistent with pomegranate juice (Mousavi et al., 2013). The reason why the
result probably is that the conversion of phenolic compounds into other new substances
with antioxidant activity (Oh et al., 2017), and flavonoid glycosides were converted into
corresponding glycosides with strong free radical scavenging ability by specific bacterial
glycosyl hydrolases during the fermentation, therefore, the antioxidant properties was
increased at the end of fermentation (Marsh et al., 2014; Mousavi et al., 2013). In addition,
SOD is an intracellular enzyme, and the increase in its activity may also be related to the
lysis of yeast during fermentation (Yang et al., 2018), and it also plays an important role
in the increase in antioxidant capacity. Different methods of evaluating antioxidants have
been widely used because antioxidant compounds can pass through different mechanisms,
each with its specific target in the reaction matrix (Fernández-Moriano et al., 2016). The
structure of phenolic compounds has different sites with different free radical scavenging
activities (Kwaw et al., 2018). Therefore, different chemical reactivity may lead to different
degrees of antioxidant capacity in various chemical tests. In the study of the antibacterial
ability of strawberry fermented beverage to pathogenic bacteria, we evaluated the inhibitory
band value (Refer to Table 2), MIC, MBC and growth curve (Refer to Figs. 5A and 5B), and
the results showed that strawberry fermented beverage showed ideal inhibitory activity.
Our results are consistent with Hashemi et al. (2017), who showed that the antibacterial
properties of sweet lemon juice through fermentation were improved compared to the
control (fresh sweet lemon juice). In another study, fermented dragon fruit juice had
a good antibacterial effect on Escherichia coli, Salmonella Typhimurium, Pseudomonas
aeruginosa, and Staphylococcus aureus (Muhialdin et al., 2020). Indeed, a large number
of previous studies have shown that fermentation process can improve the antibacterial
ability of some fruit juices (Hashemi et al., 2017; Vanajakshi et al., 2015; Zubaidah et al.,
2018). The antibacterial property of fermented beverages is affected by many factors.
The antibacterial potential of phenols mainly lies in the inhibition of bacterial cell wall
synthesis, protein synthesis and nucleic acid synthesis (Reygaert, 2014). Flavonoids can
destroy the interaction between acyl-homoserine lactone and their receptor, and can
also inhibit the surface adhesion by Gram-positive bacteria (Cushnie & Lamb, 2011).
However, in this study, the total phenol content and total flavonoid content decreased
after fermentation, so the antibacterial activity may be related to organic acids and other
metabolites with antibacterial properties produced during the fermentation process. The
antibacterial activity of organic acids including un-dissociated acid enters the cell to release
protons in the cytoplasm, thereby reducing the pH in the cytoplasm (Zubaidah et al.,
2018) and un-dissociated acids lead to the destruction of the substrate transport system
by destroying the electrochemical proton gradient or changing the permeability of the cell
membrane (Snijders et al., 1985). In addition, metabolites with antibacterial properties such
as hydrogen peroxide or diacetyl and bacteriocin peptidoglycan hydrolase are synthesized

Zhao et al. (2021), PeerJ, DOI 10.7717/peerj.11974 15/23

 to play an antimicrobial role during fermentation (Cushnie & Lamb, 2011). Therefore, the
antibacterial activity was improved.
Biofilm is a microbial structure that can cause many problems in the medical field,
food industry, and other environments. Therefore, the choice of substances inhibiting
biofilm formation is very important. However, there is almost no research on the effect
of fermented beverages on the formation of biofilms. In this study, it can be seen that
the green fluorescent area is greatly reduced after the fermented beverage is treated (Refer
to Fig. 6). Our work is of great significance to the effect of fermented fruit and vegetable
beverages on the formation of biofilms. Regarding the strategies of inhibiting the formation
of biofilms, a study showed that many plants containing active compounds anti-biofilm
formation can inhibit bacterial growth and achieve the purpose of preventing bacterial
adhesion (Husain et al., 2015). Furthermore, antibacterial peptides, a type of polypeptide
or protein with antibacterial activity encoded by genes and synthesized by ribosomes in
the metabolic process of LAB, have the effect of inhibiting the formation of biofilms (De la
Fuente-Núñez et al., 2013). Therefore, we speculate that the anti-biofilm formation ability
of fermented beverages may be related to strawberry as a fermentation carrier and lactic
acid bacteria as a starter.
Finally, the newly developed strawberry fermented beverage was submitted to sensorial
analysis to its evaluate consumer acceptance in the future. The result showed that
improvement of some sensory attributes such as color, appearance and taste in fermented
beverage (Refer to Fig. 7) refer to. However, the score of overall impression and flavor
were dropped. In consideration development and consumption of the beverage, adding
pulp, flavoring and sweetening agents to the beverage to enhance its taste and flavor
thus may increase consumer acceptance in the future (Menezes et al., 2018). In addition,
Maldonado et al. (2017) reported that the fermentation substrate affects the development
of microorganisms and thus the sensory evaluation of fermented products, so the change
of the sensory evaluation of strawberry fermented beverage may be related to changes in
the composition of strawberry juice before and after fermentation. The large accumulation
of lactic acid produced by lactic acid fermentation forms unpleasant flavor, whereas these
low acid formation levels are beneficial to food processing and preservation (Galle et
al., 2010). Fermentation causes chemical modification of phenolic substances, especially
anthocyanins, which results in a lighter color of fermented beverages than strawberry juice,
and the color is closely related to appearance (Ayed, Ben Abid & Hamdi, 2017). The results
of this study are consistent with this point, and the appearance score is also improved while
the color is improved.

CONCLUSION
This study analyzed the potential health benefits of strawberry fermented beverages
obtained with LAB and yeast as starters for the first time. Fresh strawberry juice was used as
a control, although the content of total phenols and total flavonoids in fermented beverages
has decreased, the ability to scavenge DPPH free radicals, hydroxyl free radicals and ABTS
free radicals has also decreased, but its T-AOC is still improved. In addition, the fermented

Zhao et al. (2021), PeerJ, DOI 10.7717/peerj.11974 16/23

 beverage also has superior antimicrobial activity against four pathogenic microorganisms,
and anti-biofilm formation ability for Escherichia coli ATCC 25922, Staphylococcus aureus
ATCC 6538. This study shows the fermentation with LAB and yeast were combined as
starter culture is a promising tool for obtaining fermented beverages with health benefits.
The results of this study can provide certain theoretical support for the development and
promotion of fermented fruit and vegetable beverages in the future. This fruit fermented
beverage may bring good news to the lactose intolerant public.

ADDITIONAL INFORMATION AND DECLARATIONS

Funding
This study was supported by the Department of Science and Technology of Sichuan
Province Key Research and Development Program of China (Project No 2019YFG0154),
the Department of Science and Technology of Sichuan Province Application Fundamentals
(Key) Research and Development Program of China (Project No 2019YJ0549) and Sichuan
Sharing and Service Platform of Scientific and Technological Resource (Enzyme Resource)
of China (Project No 2020JDPT0018). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors:
The Department of Science and Technology of Sichuan Province Key Research and
Development Program of China: 2019YFG0154.
The Department of Science and Technology of Sichuan Province Application Fundamentals
(Key) Research and Development Program of China: 2019YJ0549.
Sichuan Sharing and Service Platform of Scientific and Technological Resource (Enzyme
Resource) of China: 2020JDPT0018.

Competing Interests
The authors declare there are no competing interests.

Author Contributions
• Zhiqiao Zhao and Zizhong Tang conceived and designed the experiments, prepared
figures and/or tables, and approved the final draft.
• Xulong Wu, Yuntao Liu, Yirong Xiao and Hui Chen performed the experiments,
authored or reviewed drafts of the paper, and approved the final draft.
• Hong Chen performed the experiments, prepared figures and/or tables, and approved
the final draft.
• Qingfeng Li and Huipeng Yao analyzed the data, authored or reviewed drafts of the
paper, and approved the final draft.

Data Availability
The following information was supplied regarding data availability:
The raw data are available in the Supplemental Files.

Zhao et al. (2021), PeerJ, DOI 10.7717/peerj.11974 17/23

 Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.11974#supplemental-information.

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