jmb

J. Microbiol. Biotechnol. (2019), 29(8), 1177–1183

https://doi.org/10.4014/jmb.1905.05022 Research Article

Special Topic - Biofilm

Anti-Biofilm Activity of Grapefruit Seed Extract against Staphylococcus

aureus and Escherichia coli
Ye Ji Song, Hwan Hee Yu, Yeon Jin Kim, Na-Kyoung Lee, and Hyun-Dong Paik*
Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul 05029, Republic of Korea

Received: May 10, 2019

Revised: July 17, 2019
Accepted: July 29, 2019
First published online
August 1, 2019
*Corresponding author
Phone: +82-2-2049-6011;
Fax: +82-2-455-3082;
E-mail: hdpaik@konkuk.ac.kr
pISSN 1017-7825, eISSN 1738-8872
Copyright © 2019 by
The Korean Society for Microbiology
and Biotechnology
Grapefruit seed extract (GSE) is a safe and effective preservative that is used widely in the
food industry. However, there are few studies addressing the anti-biofilm effect of GSE. In this
study, the anti-biofilm effect of GSE was investigated against biofilm-forming strains of
Staphylococcus aureus and Escherichia coli. The GSE minimum inhibitory concentration (MIC)
for S. aureus and E. coli were 25 µg/ml and 250 µg/ml, respectively. To investigate biofilm
inhibition and degradation effect, crystal violet assay and stainless steel were used. Biofilm
formation rates of four strains (S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli O157:H4
FRIK 125) were 55.8%, 70.2%, 55.4%, and 20.6% at 1/2 × MIC of GSE, respectively. The
degradation effect of GSE on biofilms attached to stainless steel coupons was observed
(≥ 1 log CFU/coupon) after exposure to concentrations above the MIC for all strains and 1/2
× MIC for S. aureus 7. In addition, the specific mechanisms of this anti-biofilm effect were
investigated by evaluating hydrophobicity, auto-aggregation, exopolysaccharide (EPS)
production rate, and motility. Significant changes in EPS production rate and motility were
observed in both S. aureus and E. coli in the presence of GSE, while changes in hydrophobicity
were observed only in E. coli. No relationship was seen between auto-aggregation and biofilm
formation. Therefore, our results suggest that GSE might be used as an anti-biofilm agent that
is effective against S. aureus and E. coli.
Keywords: Anti-biofilm, grapefruit seed extract, biofilm, Staphylococcus aureus, Escherichia coli

Introduction biofilms on a variety of surfaces [3]. In addition, many

The World Health Organization (WHO) estimated that
the consumption of contaminated food causes illness in 1 in
10 people every year, and as a result, leads to 420,000
deaths per year [1]. According to the Centers for Disease
Control and Prevention (CDC), Escherichia coli, Listeria spp.,
Salmonella spp., Campylobacter spp., and Staphylococcus
aureus are some of the most common causes of food
poisoning [2]. The most common symptoms of food
poisoning are diarrhea, fever, vomiting, nausea, stomach
cramps, and stomachache. Food poisoning pathogens can
contaminate food products at any time during processing,
distribution, or storage. It is very important to limit the
growth and development of food poisoning pathogens
such as S. aureus and E. coli; however, elimination of these
organisms is difficult because of their ability to form
outbreaks of food poisoning have been found to be
associated with biofilm-forming pathogens in the dairy,
poultry, meat, and ready-to-eat (RTE) food industries [4].
Biofilms are a survival and protection strategy for
pathogens, and the cycle of biofilm formation, maturation,
and dispersal is the main cause of surface-to-food cross-
contamination [5, 6]. After bacterial cells attach to a surface,
they produce a polymer matrix, composed of exopoly-
saccharides (EPS), extracellular proteins, and extracellular
DNA, to defend themselves against antibiotics and other
chemical compounds [7]. It is well recognized that bacteria
within biofilms show increased antimicrobial resistance
compared to planktonic cells grown in suspension [8]. It is
important to use appropriate sanitization procedures in the
processing environment to both ensure the removal of
pathogens and to prevent contamination of the final

August 2019 ⎪ Vol. 29 ⎪ No. 8

1178 Song et al.
products [9, 10].
In order to prevent biofilm formation, several studies
were conducted to discover natural antimicrobial agents
that affect the viability of bacteria in biofilms [11-13].
Furthermore, a shift in consumer preferences towards food
containing safe preservatives has led to the emergence of a
demand for natural antimicrobial agents [14]. Plant extracts
from leaves, roots, and seeds have been reported to
demonstrate anti-biofilm activity against S. aureus, E. coli,
Listeria spp., and Campylobacter spp. [3, 15-18].
In this study, we chose to evaluate the anti-biofilm
activity of grapefruit seed extract (GSE), as it is already
known to be a safe and effective preservative. Previous
studies have demonstrated that GSE has an antimicrobial
effect on gram-positive bacteria, gram-negative bacteria,
and yeasts [19-21]. However, there have been no specific
reports on the effect of GSE on biofilms. Therefore, the
purpose of this study was to investigate the anti-biofilm
effect of GSE on two food poisoning pathogens (S. aureus
and E. coli) to determine if it may be effective for biofilm
removal in the food industry.
Materials and Methods
Bacterial Strains and Growth Conditions
S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli O157:H4
FRIK 125, were used in this study through biofilm-forming
screening. S. aureus strains isolated from bovine were supplied by
Seoul National University (Korea). E. coli ATCC 25922 and E. coli
O157:H4 FRIK 125 were supplied from Korean Culture Center of
Microorganisms (KCCM; Korea) and IOWA State University,
respectively. These strains were cultured in the Tryptic Soy Broth
(TSB; Difco Laboratories, USA) for 24 h at 37°C and stocked at
-80°C with 20% glycerol. In biofilm assay, the strains were
cultured in TSB containing 0.25% glucose (D-(+)-Glucose; Sigma,
Germany) to promote biofilm formation.
Minimum Inhibitory Concentration (MIC) of GSE
Grapefruit seed extract (GSE; ES Food, Korea) was solubilized
in TSB with 0.05% Tween 80. A double broth dilution method was
used to determine the GSE MIC in 96-well plates [11]. GSE was
serially diluted two-fold and 50 µl of each concentration was
transferred into 96-well plates. Each well was then inoculated
with the same volume of bacterial suspension at a concentration
of 1.0 × 105 colony-forming units (CFU)/ml and the microplate
was incubated for 24 h at 37°C. The MIC was measured as the
lowest concentration of GSE that led to complete inhibition of
visible growth.
Biofilm Assay
Biofilm inhibition and degradation by GSE were assessed using
J. Microbiol. Biotechnol.
a crystal violet assay, as previously described [22]. To investigate
the inhibitory effect of GSE on initial stages of biofilm formation,
bacterial cultures were diluted to a final concentration of 1.0 ×
105 CFU/ml and 50 µl was transferred to a 96-well polystyrene
microtiter plate (SPL, Korea) with 50 µl GSE at a range of
concentrations from 3.125 to 50 µg/ml for S. aureus and 31.25 to
500 µg/ml for E. coli (1/8 × MIC – 2 × MIC) and incubated for 24 h
at 37°C. Control was prepared in TSB without GSE for each strain.
The culture medium was removed, and the wells were washed
twice with distilled water to remove planktonic cells. Plates were
incubated at 37°C for 15 min to completely dry, and then 100 µl
1% crystal violet solution was added to each well to stain the
attached biofilm cells. After 30 min, the wells were washed gently
with tap water followed by distilled water. Then, 100 µl
dissolving solution (30% methanol and 10% acetic acid) was
added to each well to dissolve the crystal violet, and the optical
density (OD) was measured at 570 nm using a microplate reader
(Emax, Molecular Devices, USA).
To investigate the degradation effect of GSE on mature
biofilms, 100 µl cultured cell suspension was transferred to 96-
well plates, without GSE. After biofilm maturation (24 h at 37°C),
the cell suspension was replaced with 100 µl of each concentration
of GSE solution (1/8 × MIC – 2 × MIC) and plates were incubated
for a further 24 h at 37°C. The biofilm was quantified as described
above. The biofilm formation rate (%) was calculated using the
following equation:
Biofilm formation rate (%) = ODtreatment/ODcontrol × 100
where ODtreatment and ODcontrol refer to the absorbance at 570 nm in
each well with and without GSE, respectively, after the addition
of dissolving solution.
Hydrophobicity and Auto-Aggregation
The cell surface hydrophobicity of biofilm-forming strains was
determined in the presence of GSE as previously described, with
some modifications [23]. Cells were treated with a sub-inhibitory
concentration of GSE (12.5 µg/ml for S. aureus and 125 µg/ml for
E. coli) for 4 h at 37°C; nontreated cells were used as a control.
Cells were centrifuged (14,240 ×g for 5 min at 4°C) and the
resulting pellets were washed twice and resuspended in
phosphate buffered saline (PBS, pH 7.4; Hyclone, USA) to an OD
of 0.5 ± 0.05 at 600 nm (ODinitial). Toluene (0.5 ml) was added to
each suspension (2 ml), followed by vortexing for 2 min. After
15 min, the OD of the lower aqueous layer was measured at
600 nm (ODtreatment) and hydrophobicity (%) was calculated using
the following equation:
Hydrophobicity (%) = (1 − ODtreatment / ODinitial) × 100
After washing the incubated cells, the cells were resuspended in
4 ml PBS to an OD of 0.5 ± 0.05 at 600 nm (ODinitial). The suspensions
were incubated for 6 h at 37°C and then the OD at 600 nm was

measured for each suspension (ODtreatment). Auto-aggregation (%)
was calculated using the following equation:
Auto-aggregation (%) = (1 − ODtreatment/ODinitial) × 100
Total EPS Production Rate
Total EPS production rate of biofilm-forming strains was
determined as previously described, with some modifications
[24]. Bacterial cultures were treated with 1/2 × MIC of GSE
(S. aureus, 12.5 µg/ml; E. coli, 125 µg/ml) in TSB with 2% sucrose
for 12 h at 37°C; nontreated cells were used as a control. Cultures
were then centrifuged at 8,000 ×g for 10 min at 4°C and 1 ml
supernatant was combined with 2 ml 99% ethyl alcohol and
incubated overnight at 4°C. After incubation, the suspension was
centrifuged (14,240 ×g at 4°C for 5 min) and resuspended in 500 µl
distilled water. Then, 1 ml 5% phenol and 5 ml 95% sulfuric acid
were added to 100 µl cell suspension, which was mixed by
vortexing and left to stand for 10 min at 30°C. Absorbance was
measured at 490 nm and total EPS production rate (%) was
calculated using the following equation:
Total EPS production rate (%) = ODtreatment / ODcontrol × 100
where ODtreatment and ODcontrol refer to the absorbance of the
phenol-sulfuric acid solution at 490 nm with and without GSE,
respectively.
Motility of GSE-Treated Pathogens
Ten microliters S. aureus cultures were stab-inoculated to the
center of tryptic soy agar (TSA) containing 0.3% agar (colony
spreading) and GSE at 1/2 × MIC (treatment) or no GSE (control)
and incubated without lids for up to 20 h at 37°C [25]. For E. coli,
5 µl cultures were stab-inoculated to the center of TSA containing
0.3% and 0.6% agar for swimming and swarming motility,
respectively, and GSE at 1/2 × MIC (treatment) or no GSE
(control) and incubated without lids for up to 12 h at 37°C [25].
After incubation, the diameter of the zone traveled by the bacteria
from the point of inoculation was measured.
Biofilm Degradation Effect of GSE on Stainless Steel
S. aureus and E. coli were incubated for 24 h at 37°C in TSB and
then diluted to a final concentration of 105 CFU/ml, and 2.4 ml of
these suspensions were loaded into each well of 24-well culture
plates (SPL) containing stainless steel coupons (type 304, 10 mm ×
15 mm × 2 mm; i-Nexus Inc., Korea). Plates were then incubated
for 24 h at 37°C. After biofilm formation, the coupons were rinsed
with 0.1% peptone water to remove planktonic cells and
transferred to another 24-well plate. GSE solution was added
(from 1/4 × MIC to 4 × MIC), and plates were incubated for 24 h
at 37°C. After treatment, the coupons were washed again with
0.1% peptone water and vortexed for 1 min with 4-5 autoclaved
glass beads in 10 ml 0.1% peptone water. The suspensions were
diluted 10-fold with 0.1% peptone water and spread on TSA. TSA
Anti-Biofilm Effect of Grapefruit Seed Extract 1179
plates were incubated for 24 h at 37°C, and the number of CFU
were determined and expressed as log CFU/coupon.
Scanning Electron Microscopy (SEM) Analysis
SEM was carried out to evaluate bacterial adhesion to stainless
steel in the presence of GSE [11]. Biofilms formed on the stainless
steel coupons were fixed in 2.5% glutaraldehyde for 1 h at 4°C.
The fixed samples were washed three times with PBS and
dehydrated in 50%, 70%, 80%, 90%, and 100% ethanol (15 min
each), then the ethanol was replaced with isoamyl acetate and the
samples were freeze-dried. Stainless steel samples were sputter
coated with gold (15 mV for 1.5 min) and observed using a field-
emission scanning electron microscope (FESEM; SU8010; Hitachi
High-Technologies Co., Japan).
Statistical Analysis
Statistical analysis was performed using SPSS version 18.0
(SPSS Inc., USA). All experiments were repeated three times. The
results are stated as the mean ± standard deviation. Significant
differences among the means were evaluated using one-way
analysis of variance (ANOVA).
Results and Discussion
Determination of Minimum Inhibitory Concentration
(MIC)
The antimicrobial effect of GSE was assessed by
determining the MIC against selected food poisoning
pathogens. GSE inhibited the growth of S. aureus and E. coli
at 25 µg/ml and 250 µg/ml, respectively. Several studies
have shown that GSE has an antimicrobial effect on various
pathogens including gram-positive bacteria, gram-negative
bacteria, and yeasts [19-21]. For example, the mean MIC of
GSE against Salmonella sp. and L. monocytogenes was 15 ±
0.62 µg/ml and 64 ± 0.24 µg/ml, respectively [19]. In
another study, the MIC of GSE against S. aureus and E. coli
(O:157 and O:128) was 8.25% (w/v) and 4.13% (w/v),
respectively [20]. The same study also confirmed that GSE
contains high levels of polyphenol compounds, which are
produced by plants in self-defense against plant pathogens
[20]. Furthermore, Heggers et al. [21] demonstrated that the
antimicrobial mechanism of GSE involves destruction of
the cell membrane, leading to bacterial cell death.
Biofilm Inhibition and Degradation Effects of GSE
The inhibitory effect of GSE on biofilms formed by food
poisoning pathogens is shown in Fig. 1A. Biofilm inhibition
effect represented inhibitory effects in the initial stage of
biofilm formation [22]. In the crystal violet staining assay,
S. aureus and E. coli biofilms were significantly inhibited at
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1180 Song et al.
Fig. 1. Effect of grapefruit seed extract (GSE) on the biofilm of
food poisoning pathogens (S. aureus 7, S. aureus 8, E. coli
ATCC 25922, and E. coli O157:H4 FRIK 125) in 96-well plates.
(A) Inhibitory effect of GSE on biofilms when co-incubated for 24 h
with different concentrations of GSE. (B) Degradation effect of 24 h
GSE treatment on mature biofilms. GSE concentrations are given
relative to the minimum inhibitory concentration (MIC) for each
organism. The MIC values of S. aureus and E. coli strains are 25 µg/ml
and 250 µg/ml, respectively. a-eValues with different letters in the
same bar indicate significant differences (p < 0.05).
concentrations above 1/4 × MIC (6.25 µg/ml) and 1/8 ×
MIC (31.25 µg/ml), respectively (p < 0.05). Biofilm formation
levels for S. aureus 7, S. aureus 8, E. coli ATCC 25922, and
E. coli O157:H4 FRIK 125 at 1/2 × MIC were 55.8%, 70.2%,
55.4%, and 20.6%, respectively. Interestingly, GSE inhibited
the formation of biofilms at a concentration below the MIC.
These results suggested that GSE can be used as an
antibiofilm candidate at lower concentration than MIC,
especially against the formation of biofilms by pathogens.
Biofilm degradation effects refer to the effects on mature
biofilms of pathogens [22]. GSE had a significant degradation
effect on mature S. aureus and E. coli biofilms at 1/2 × MIC
(12.5 µg/ml) and 1/8 × MIC (31.25 µg/ml), respectively (p <
0.05; Fig. 1B). In biofilm degradation effect, biofilm
J. Microbiol. Biotechnol.
formation rates at 1/2 × MIC were 64.8%, 63.1%, 35.4%,
and 17.6% for S. aureus 7, S. aureus 8, E. coli ATCC 25922,
and E. coli O157:H4 FRIK 125, respectively.
Compared to inhibition of biofilm formation (Fig. 1A)
and degradation of matured biofilm (Fig. 1B), biofilm
formation rate at the initial stage was higher than matured
biofilm. This is because once formed, biofilm becomes
difficult to remove, and inhibition of biofilm formation has
been reported at the initial stage.
A number of studies have demonstrated the anti-biofilm
effects of plant extracts and phytochemicals; for example,
one group found that grapefruit juice has an anti-biofilm
effect against E. coli O157:H7 and S. Typhimurium by
interfering with quorum sensing [26]. However, the anti-
biofilm effect of GSE had not been reported yet.
Mode of GSE Anti-Biofilm Effect
The changes in biofilm forming properties of each
pathogen, in response to GSE, are shown in Table 1.
Hydrophobicity and auto-aggregation are important for
colonization, adhesion, and biofilm growth and these differ
depending on strain type [27]. According to Choi et al. [27],
cell surface hydrophobicity varied according to the
hydrocarbons used but generally S. aureus rather than
E. coli appeared high. We investigated hydrophobicity by
assessing pathogen adhesion to toluene in the presence of
GSE at 1/2 × MIC. GSE reduced cell surface hydrophobicity
significantly in E. coli (p < 0.05) but did not have a
significant reduction in S. aureus (Table 1). Thus, GSE
concentration of 1/2 × MIC did not affect the cell surface
hydrophobicity of S. aureus, the gram-positive bacteria.
Auto-aggregation of E. coli and S. aureus was not
significantly affected by GSE.
Biofilm forming bacteria can overcome external stress
following colonization and biofilm maturation. Cells
within biofilms secrete EPS, which helps to trap nutrients
[6]. In this study, EPS production was significantly reduced
in all pathogens treated with GSE at 1/2 × MIC (p < 0.05).
Compared to a control, EPS production in S. aureus 7 and
S. aureus 8 decreased by 54.87% and 60.82%, respectively;
whereas, in E. coli ATCC 25922 and E. coli O157:H4 FRIK
125, it decreased by 28.85% and 2.64%, respectively.
Bacterial motility has been shown to play a role in
biofilm formation and attachment of cells to a surface [28].
Specifically, flagella-mediated motility of L. monocytogenes
was shown to be essential in the initial stage of biofilm
formation and the motility of E. coli is thought to be
essential for enabling cells to reach to the surface and to
facilitate biofilm growth and spread [29, 30]. In this study,
 Anti-Biofilm Effect of Grapefruit Seed Extract 1181

Table 1. Changes in cell surface hydrophobicity, auto-aggregation, EPS production, and motility in food poisoning pathogens
treated with 1/2 × MIC of GSE.
Pathogens
S. aureus 7 S. aureus 8 E. coli ATCC 25922 E. coli O157:H4 FRIK 125
Hydrophobicity (%)
Control 97.72 ± 1.97 98.53 ± 0.34 59.47 ± 0.34 66.85 ± 2.34
Treated 91.98 ± 3.64 98.53 ± 0.87 30.55 ± 9.96* 62.12 ± 1.44*
Auto-aggregation (%)
Control 34.33 ± 2.27 41.53 ± 1.21 24.39 ± 2.60 23.63 ± 2.30
Treated 34.23 ± 2.09 41.04 ± 0.60 27.96 ± 1.55 20.13 ± 1.63
EPS (%)
Control 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00 100.00 ± 0.00
Treated 45.13 ± 0.21*** 39.18 ± 0.12*** 71.15 ± 0.43*** 97.36 ± 0.63**
Motility
Colony spreading (mm)
Control 3.25 ± 0.75 4.75 ± 0.25 - -
Treated 2.5.0 ± 0.76** 3.50 ± 1.00** - -
Swimming (mm)
Control - - 34.50 ± 0.50 5.00±0.50
Treated - - 26.67 ± 0.83*** 2.33±0.17**
Swarming (mm)
Control - - 5.17 ± 0.17 5.50 ± 0.29
Treated - - 3.33 ± 0.33** 2.67 ± 0.33**
GSE, grapefruit seed extract; MIC, minimum inhibitory concentration; Control, nontreated GSE; Treated, 1/2 × MIC of GSE (S. aureus, 12.5 µg/ml; E. coli, 125 µg/ml);
EPS, exopolysaccharide.
All values are mean ± standard error (* p < 0.05, ** p < 0.01, *** p < 0.001).

the GSE-treated group had significantly reduced motility

compared to the control (p < 0.01).
These results provide insight into how GSE inhibits and
degrades biofilms (Fig. 1). Our results suggest that GSE
influences EPS production and motility in both S. aureus
and E. coli and hydrophobicity only in E. coli. The main
composition of the cell wall of gram-positive strains is
known as peptidoglycan, which is highly influenced by
EPS production. Meanwhile gram-negative strains have cell
walls composed of lipopolysaccharides, and are therefore
influenced by hydrophobicity as shown in these results.

SEM Analysis of Biofilm Degradation on Stainless Steel

GSE has a significant degradation effect on biofilms on
stainless steel at concentrations above the MIC (p < 0.05;
Fig. 2). The SEM image of biofilm cells attached to stainless
steel (Fig. 3) demonstrate that as the concentration of GSE
increases, the attached cells gradually decrease, and the
cells are destroyed.
In the absence of GSE (control), S. aureus cells were
Fig. 2. Anti-biofilm effect of 24 h treatment with grapefruit
seed extract (GSE) on the biofilm of food poisoning pathogens
(S. aureus 7, S. aureus 8, E. coli ATCC 25922, and E. coli
O157:H4 FRIK 125) on stainless steel coupons.
The MIC values of S. aureus and E. coli strains are 25 µg/ml and
250 µg/ml, respectively. a-fValues with different letters in the same
bar showed significant differences (p < 0.05).

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1182 Song et al.

Fig. 3. Scanning electron micrographs of S. aureus 7 (A) and E. coli O157:H4 FRIK 125 (B) biofilms on stainless steel-type 304 after
treatment with grapefruit seed extract (GSE) (× 5,000 magnification).
GSE concentrations are given relative to the minimum inhibitory concentration (MIC) for each strain.

observed as cocci, where several cells were assembled in a
layer; whereas in the presence of GSE at 4 × MIC (S. aureus,
100 µg/ml; E. coli, 1,000 µg/ml), individual cells were
contracted and damaged (Fig. 3A). Similarly, E. coli cells
were observed as bacilli in a biofilm layer in the control
experiments, and as the GSE concentration increased, the
number of cells attached to the stainless steel surface
decreased (Figs. 2 and 3B).
This study is important because, to date, there has been
no investigation of the anti-biofilm effects of the natural
preservative GSE against S. aureus and E. coli. These results
demonstrate that GSE has an anti-biofilm effect on both
gram-positive and gram-negative bacteria, by reducing
EPS production and motility in S. aureus and E. coli.
Therefore, GSE has the potential to be used to safely
prevent problems caused by biofilms in the food industry,
while avoiding the use of the chemical compounds that are
causing concern to consumers.
Conflict of Interest
The authors have no financial conflicts of interest to
declare.
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August 2019 ⎪ Vol. 29 ⎪ No. 8