J Nutr Sci Vitaminol, 52, 157–160, 2006

Note
Antibacterial Activity of Citrus Fruit Juices Against Vibrio Species

Hiroyuki TOMOTAKE1, Tetsuro KOGA2, Masayuki YAMATO2, Afework KASSU2 and Fusao OTA2
1
Iida Women’s Junior College, 610 Matsuo, Iida, Nagano 395–0812, Japan
2
Division of Preventive Environment and Nutrition, Department of Nutritional Science,
Graduate School of Health Biosciences, The University of Tokushima, Tokushima 770–8503, Japan
(Received July 25, 2005)

Summary Lemon, lime and sudachi juices were tested for antibacterial activity against
seven strains of Vibrio species. All juices were effective in inhibiting the growth of the Vibrio
strains. Citric acid, the major organic acid in these juices, was found to be responsible for
inhibiting the growth of Vibrio parahaemolyticus. Sauce prepared from sudachi juice showed
a strong bactericidal activity against Vibrio parahaemolyticus, whereas the sauce adjusted to
higher pH values had no bacterial activity. Diluted sudachi juice or citric acid solution also
had antibacterial activity independently. These results suggest that citrus fruit juices are
effective in preventing infection with Vibrio species.
Key Words citrus, juices, citric acid, Vibrio species

Lemon (Citrus limon Burm. f.) and lime (Citrus auran-
tifolia swingle) are popular citrus fruits and are food
ingredients for flavoring and adding acidity. Sudachi
(Citrus sudachi Hort. ex Shirai) is a small citrus and in
some areas of Japan there is a dietary habit of putting
sudachi juice on raw or grilled fish. Lemon or lime
juices have been reported to exhibit antibacterial activ-
ity against Vibrio cholerae (1, 2). To our knowledge, how-
ever, there is no report on antibacterial activity of citrus
fruit juices against V. parahaemolyticus, an important
food-borne enteropathogen, or other pathogenic Vibrio
species. Therefore, we studied antibacterial effects of
these citrus fruit juices on five Vibrio species. Moreover,
we prepared a fruit sauce from sudachi juice and exam-
ined its bactericidal activity against V. parahaemolyticus
by determining the viable count.
Materials and Methods
Lemon and lime were purchased from a supermarket.
Sudachi was supplied by a cultivator. Citrus fruits were
squeezed in a juicer. The pH values of lemon, lime and
sudachi juices were 2.2, 2.1 and 2.2, respectively.
Organic acid content in citrus fruit juices was analyzed
by high performance liquid chromatography (HPLC)
using a Crestpak C185 column (4.6
150 mm, JASCO)
at 25˚C. The mobile phase consisted of 2% KH2PO4
(pH 2.0) at a flow rate of 0.5 mL/min, and the UV detec-
tor (JASCO 857-U) was set at 210 nm to monitor the
elution profiles. To investigate the effect of organic acids
in sudachi juice on the growth of Vibrio strains, each
solution of citric (52 mg/mL, pH 1.8) and malic acid
(3 mg/mL, pH 2.6) and mixed solution of the both acids
(pH 1.8) were prepared. Seven Vibrio strains, viz. three
strains of V. cholerae non-O1 (RIMD 2214034, 95-5M-
E-mail: tomotake@iidawjc.ac.jp
4P, and 95-5M-1P), V. alginolyticus 6624, V. mimicus
E31, V. anguillalum NCMB 829, and V. parahaemolyticus
Na2 were used as test organisms. The Na2 strain is a
clinical isolate exhibiting a positive Kanagawa phenom-
enon (3). The bacterial strains were grown at 37˚C in
nutrient broth and agar containing 3% NaCl. V. cholerae
were grown in the same medium containing 1% NaCl.
For the detection of antibacterial activity, a well-diffu-
sion assay was performed (4). An overnight culture
(100 L) of Vibrio strains was spread on nutrient agar.
Wells (3.5 mm in diameter) were cut into plates and
15 L of the citrus fruit juice or the organic acid solu-
tion was poured into each well. The plates were incu-
bated at 37˚C for 12 h and the radii of clear inhibition
zones around the wells were measured. A fruit sauce
was prepared by mixing sudachi juice, soy sauce and
sugar (56% sudachi juice, 33% soy sauce, 11% sugar,
w/v). The pH value of the sauce was 3.7. To investigate
the effect of pH on bactericidal activity, pH of the sauce
was adjusted to 5.0 or 6.0 with 10% NaOH. For the
assessment of bactericidal activity of the sauce, V. para-
haemolyticus Na2 was grown to an exponential phase in
nutrient broth at 37˚C for 3 h. The cells were collected
by centrifugation at 3,000
g for 20 min and sus-
pended in 0.1 M phosphate buffer (pH 7.0) containing
3% NaCl (3% NaCl-PB) by the absorbance of 0.4 at
660 nm. Fifty microliters of cell suspension containing
about 2.7
108 colony-forming units (CFU) was mixed
with 4.95 mL of the sauce at room temperature. After
20, 40 and 60 s, 50 L of the mixture was sampled in
9.95 mL of 3% NaCl-PB, serially diluted in the same
buffer and plated on nutrition agar to determine viable
counts (5). The effect of diluted sudachi juice (56%,
pH 2.4) or citric acid solution (29 mg/mL, pH 1.9) on
bactericidal activity was also investigated as described
above.

157
158 TOMOTAKE H et al.

Table 1. Inhibitory effects of lemon, lime and sudachi

juices on the growth of Vibrio species.

Lemon Lime Sudachi

(pH 2.2) (pH 2.1) (pH 2.2)

V. cholerae non-O1 RIMD 2214034   

V. cholerae non-O1 95–5M-4P   
V. cholerae non-O1 95–5M-1P   
V. vulnificus ATCC 27562   
V. alginolyticus 6624   
V. mimicus E31   
V. anguillarum NCMB 829   
V. parahaemolyticus Na2   

All determinations were done in triplicate.

, large inhibition zone (5 mm); , zone 3–
4 mm; , zone 1–2 mm.

Table 2. Contents of organic acids in lemon, lime and

sudachi juices.

Lemon Lime Sudachi

mg/mL
Citric acid 50.80.6 43.10.8 52.10.9
Malic acid 2.60.1 4.30.1 3.00.1

Values are meansSD, n3.

Results and Discussion

The three juices tested showed strong inhibitory
activities against Vibrio strains (Table 1). All juices were
considerably effective in inhibiting the growth of V. algi- Fig. 1. Inhibitory effects of sudachi juice or organic
acid solutions on the growth of Vibrio parahaemolyticus
nolyticus 6624 and V. anguillarum NCMB829. Inhibi-
Na2 (A) or V. anguillalum NCMB 829 (B) by the agar
tory effects of the three juices against V. parahaemolyti-
well diffusion assay. Wells: (1) citric acid solution
cus Na2 were less than those against the other Vibrio (52 mg/mL, pH 1.8); (2) malic acid solution (3 mg/mL,
strains. There was no difference in inhibitory activity pH 2.6); (3) mixed solution of both acids (pH 1.8); (4)
among the three juices. The contents of organic acids in sudachi juice (pH 2.2).
lemon, lime and sudachi juices are shown in Table 2.
Citric acid was the major organic acid in these juices,
malic acid being the minor. As shown in Fig. 1, citric
acid solution (52 mg/mL) was as effective as sudachi
juice in inhibiting the growth of V. parahaemolyticus or V.
anguillarum, whereas malic acid (3 mg/mL) did not
exhibit the inhibitory activity. It is probable that the cit-
ric acid contributes to the antibacterial activity of suda-
chi and other juices.
The bactericidal activity of the prepared sauce
against V. parahaemolyticus Na2 was determined (Fig.
2). The sauce from sudachi juice showed strong bacteri-
cidal activity against V. parahaemolyticus Na2. After
treatment of bacterial cells with the sauce, very low sur-
vival rates were observed. For example, the viability of
cells was less than 1% after treatment for 20 s. On the Fig. 2. Survival of Vibrio parahaemolyticus Na2 treated
other hand, the sauce of which pH was adjusted to 5.0 with a prepared sauce using sudachi juice. Symbols:
,
or 6.0, had no bactericidal activity. Diluted sudachi prepared sauce (pH 3.7) (56% sudachi juice, 33% soy
juice (56%) or citric acid solution (29 mg/mL) also had sauce, 11% sugar, w/v); , prepared sauce adjusted to
bacterial activity independently. After treatment of bac- pH 5.0; , prepared sauce adjusted to pH 6.0. Data are
terial cells for 20 s with either of the two solutions, no means of duplicate determinations.
surviving cell was observed.
 Antibacterial Activity of Citrus Fruit Juices
V. parahaemolyticus is a halophilic bacterium that
commonly inhabits marine environments. At present,
this is one of the most important food-borne pathogens
in Taiwan, Japan and other coastal regions (6). The
high incidence of food-borne diseases caused by this
pathogen results from frequent consumption of marine
foods in these regions. Most food-borne pathogens are
usually sterilized by heat treatment before eating. How-
ever, marine foods are often consumed raw and this is
the reason why the incidence of infection with V. para-
haemolyticus is so high in the region. If we can sterilize
V. parahaemolyticus not by heat treatment but by the
addition of some acidic ingredients before eating, it
would be one of the safest and most convenient ways to
avoid infection with V. parahaemolyticus.
In general, Vibrio strains are very sensitive to acids.
Koga et al. suggested that the acid sensitivity of Vibrio
species was higher than any other enteric bacteria and
that the acid sensitivity of Vibrio varied from species to
species (4). In this study, it was demonstrated that V. alg-
inolyticus 6624 showed a high sensitivity, while V. para-
haemolyticus Na2 was resistant to the antibacterial
activity of three juices.
Citric acid was the major organic acid (52 mg/mL) in
sudachi juice with malic acid as the minor (3 mg/mL),
and their contents were similar to those reported by Nii
et al. (7). In this study, we prepared the organic acid
solution at those concentrations, tested for antibacterial
activity by a well-diffusion assay and found that citric
acid in sudachi juice was responsible for inhibiting the
growth of V. parahaemolyticus. Hasegawa et al. also
reported the bactericidal effects of acetic, citric and
hydrochloric acids against V. parahaemolyticus O3:K6
strains (8). It has been proposed that antibacterial
activity of the organic acid in low pH is due to its undis-
sociated structure, which is hydrophobic and has cyto-
toxicity as well. Because the concentrations of citric
acid in lemon, lime and sudachi juices are higher than
in any other fruit juices, these juices may be antimicro-
bially active against a variety of bacteria.
Vinegar is also a food ingredient for adding acidity.
Although vinegar has been reported to exhibit antimi-
crobial activity against a variety of Gram-positive and
negative bacteria (9), the effects of its high acidity in
food-handling environments on the survival of Vibrio
species have not been well characterized. In this study,
we have also demonstrated the antibacterial activity of
vinegar against many Vibrio species by a well-diffusion
assay (data not shown).
The antimicrobial activity of lime juice against V.
cholerae has been reported (10, 11). De Castillo et al.
also reported that freshly squeezed lemon juice inhib-
ited the growth of V. cholerae O1 (12). Our results with
the sauce agreed with their findings, indicating that cit-
rus fruit juices have the bactericidal activity against all
Vibrio species. When the fruit sauce was prepared, the
pH was an important factor. The sauce adjusted to
higher pH values had no bactericidal activity. These
results may suggest that bactericidal activity of citric
acid is effective only in low pH. It is possible that V. para-
159
haemolyticus Na2 die readily at pH less than 4.5 and
can grow at pH higher than 4.5.
In this study, diluted sudachi juice (56%) or citric
acid solution (29 mg/mL), the concentrations of which
were similar to those of fruit sauce, had also bacterial
activity independently and these activities were stron-
ger than that of fruit sauce. These results suggest that
the bactericidal activity of sudachi juice against V. para-
haemolyticus Na2 is slightly decreased by mixing with
soy sauce or sugar. Soy sauce or sugar may influence
the activity of the undissociated structure. Further
investigation is required to explain the relationship
between the bactericidal activity and the seasoning.
Mata et al. reported that the addition of lime juice to
cabbage, lettuce or fish eliminated V. cholerae O1 experi-
mental contamination in these foods (2, 11). The sur-
face of raw fish can readily be contaminated by V. para-
haemolyticus, but large numbers of V. parahaemolyticus
can be rapidly eliminated after the contact with sudachi
juice or that fruit sauce. In this case, however, we must
take into consideration the protection of bacteria or
buffer action by ingredients of raw fishes.
In conclusion, this study demonstrated the antimi-
crobial activity of citrus fruit juice against Vibrio strains.
The fruit sauce prepared from sudachi juice showed a
strong bactericidal activity against V. parahaemoliticus,
indicating that the sauce could be valuable for minimiz-
ing chances of being infected with Vibrio species when
people appreciate raw fishes.
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