The Inhibitory Effect of Lemon Juice (Citrus limon) on Vibrio parahaemolyticus in Raw Oyster (Crassostrea virginica)

Abstract

Introduction: Serving raw oysters with lemon juice is a delicacy in many restaurants in Malaysia. Oysters (Crassostrea virginica) live in the seacoast and they share the same environment as Vibrio parahaemolyticus. Consumption of raw oysters contaminated with V. parahaemolyticus can lead to severe gastroenteritis. A study was performed to determine whether lemon (Citrus limon) juice is able to inhibit the growth of V. parahaemolyticus after being inoculated in raw oysters. Methods: Frozen oysters bought from a local supplier weighing 6 g each were minced and placed in two bottles using sterile technique. Approximately 1 ml of 107 CFU of V. parahaemolyticus (ATCC strain 17802) was added and mixed in both bottles. The mixture was treated with 1 ml of lemon juice in only one of the bottles and the other bottle served as a control. At every 30 s intervals for 2 min, 1 g of the sample was taken for enumeration of viable cells onto thiosulphate citrate bile salt sucrose (TCBS). Results: After 30 s of treatment with the lemon juice, it was observed that the number of colonies in the treated samples reduced from 7 Log to 3 Log. Subsequently, no viable V. parahaemolyticus was seen. It was also observed that there were 3 Log reductions of V. parahaemolyticus after 30 s in untreated samples, however the number of colonies remained stable until the end of the experiment. Conclusion: This study therefore shows that lemon juice has some antimicrobial effect on V. parahaemolyticus in raw oysters.

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Vol 2 (2) (2017) 31-33 | jchs-medicine.uitm.edu.my | eISSN 0127-984X 31
INTRODUCTION
Oyster (Crassostrea virginica) is a type of bivalve
mollusk and belongs to the family Ostreidae. The
habitat of oyster is in briny water such as the sea coast
or river banks. Oysters are filter feeders as they
consume plankton and organic materials by filtering
the water from the surroundings. Oyster’s meat is rich
in nutrient especially protein (16.8%) followed by
vitamin A and B complex. The meat can be eaten raw,
fried, or marinated.
Vibrio parahaemolyticus is a halophilic gram
negative curved rod which needs brackish salt water to
live. The clinical manifestations of infection with this
organism are gastroenteritis such as watery diarrhoea,
abdominal cramps, nausea and vomiting due to the
toxin produced by the organism. Since vibrio shares
the same environment as oysters, it has the potential to
be present in oysters and hence there is a possibility of
gastrointestinal infection in consumers. A recent report
by Paydar et al (2013), 29% of raw seafood including
oysters in Malaysia was found to have V.
parahaemolyticus [1].
In Malaysia and many other countries, eating
raw oysters with squeezed lemon juice (Citrus limon)
has been considered as an established delicacy. Not
only rich in vitamin C, lemon juice also has the
beneficial chemical content known as bioflavonoids.
Miyake et al. (1998) reported that bioflavonoids in
citrus have many properties such an antioxidant, anti-
inflammatory and anticancer [2]. In addition, Tassou et
al. (1996) found that bioflavonoids also play an
important role as broad spectrum antimicrobial agents
to many virulent organisms such as Staphylococcus
aureus and Salmonella enteritidis [3]. In another study,
Tomotake et al. (2006) applied lemon juice directly on
V. parahemolyticus and found that it inhibited the
growth of the bacterium [4]. To date, there have not
been any reports on whether lemon juice squeezed into
raw oysters may kill V. parahaemolyticus in oysters.
This study aims to determine the effect of lemon juice
ABSTRACT
Introduction: Serving raw oysters with lemon juice is a delicacy in many restaurants in
Malaysia. Oysters (Crassostrea virginica) live in the seacoast and they share the same
environment as Vibrio parahaemolyticus. Consumption of raw oysters contaminated with V.
parahaemolyticus can lead to severe gastroenteritis. A study was performed to determine
whether lemon (Citrus limon) juice is able to inhibit the growth of V. parahaemolyticus after
being inoculated in raw oysters. Methods: Frozen oysters bought from a local supplier
weighing 6 g each were minced and placed in two bottles using sterile technique.
Approximately 1 ml of 107 CFU of V. parahaemolyticus (ATCC strain 17802) was added and
mixed in both bottles. The mixture was treated with 1 ml of lemon juice in only one of the
bottles and the other bottle served as a control. At every 30 s intervals for 2 min, 1 g of the
sample was taken for enumeration of viable cells onto thiosulphate citrate bile salt sucrose
(TCBS). Results: After 30 s of treatment with the lemon juice, it was observed that the
number of colonies in the treated samples reduced from 7 Log to 3 Log. Subsequently, no
viable V. parahaemolyticus was seen. It was also observed that there were 3 Log reductions
of V. parahaemolyticus after 30 s in untreated samples, however the number of colonies
remained stable until the end of the experiment. Conclusion: This study therefore shows
that lemon juice has some antimicrobial effect on V. parahaemolyticus in raw oysters.
KEYWORDS: Vibrio parahaemolyticus, lemon juice, Crassostrea virginica
Received
15th November 2016
Received in revised form
17th May 2017
Accepted
27th November 2017
Corresponding author:
Siti Farah Alwani Mohd Nawi,
Cluster of Laboratory Medical
Sciences,
Faculty of Medicine,
Universiti Teknologi MARA (UiTM),
Sungai Buloh Campus,
Sungai Buloh, Selangor,
Malaysia.
Tel: +60126391413
Email: sitifarah@salam.uitm.edu.my
The Inhibitory Effect of Lemon Juice (Citrus limon) on Vibrio
parahaemolyticus in Raw Oyster (Crassostrea virginica)
Siti Farah Alwani Mohd Nawi, Zaini Mohd Zain, Muhammad Zarif Zahari, Anis Amalina Abdul
Hamid, Nur Fakhriah Ahmad Afandi, Nur Zahirah Haris Fadzilah, Siti Nur Fathihah Azmi
Faculty of Medicine, Universiti Teknologi MARA, Selangor, Malaysia

Lemon juice vs V. parahaemolyticus in oyster
Vol 2(2) (2017) 31-33 | jchs-medicine.uitm.edu.my | eISSN 0127-984X
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on viable V. parahaemolyticus in raw oysters by
spiking the oysters with infective dose of V.
parahaemolyticus.
METHODS
Bacterial Strain
Vibrio parahaemolyticus ATCC strain 17802 was used
in this study. A suspension of 107 colony forming unit
per ml (CFU/ml) of overnight culture was prepared in
phosphate buffer saline (PBS, pH 7.2) according to the
standard method of the Clinical Laboratory Standard
Institute [5].
Preparation of Lemon Juice
A medium sized lemon was surface-sterilized with
70% alcohol before being aseptically cut and
squeezed. The pH of the lemon juice was determined
using litmus pH indicator strip (Merck, USA).
Preparation of Oyster Samples
Frozen oysters of average weight of 6 g each, were
bought from a seafood supplier in Batu Caves, Kuala
Lumpur. They were transported in ice to our laboratory
and kept frozen at -20ºC until used. Prior to the
experiments, two oysters were thawed and checked for
the presence of Vibrio spp. or other bacteria by
culturing on thiosulphate citrate bile sucrose (TCBS)
agar, blood agar and MacConkey agar. The oyster
meat was minced by using scalpel in an aseptic
technique for the following experiments. No growth of
microorganisms was detected on the three types of
culture media used.
Determining Inhibitory Effect of Lemon Juice
on Vibrio parahaemolyticus in Raw Oysters
Two universal bottles containing 6 g of minced
oyster’s meat each were added with approximately 107
CFU of V. parahaemolyticus. Subsequently, 1 ml of
lemon juice (average pH 2.1) was added to one of the
bottles (treated sample) while the other bottle (control)
was not treated with the lemon juice. Both the treated
and control samples were left at room temperature for
2 min. At every interval of 30 s up to 2 min, a scoop of
the meat was taken and weighed to get 1 g. It was then
added to 100 µl of PBS and serial dilutions were
performed for enumeration of viable V.
parahaemolyticus on TCBS agar. Following 24 h
incubation, the number of viable V. parahaemolyticus
was counted using a colony counter. The whole
experiment was repeated once and the viable count
was averaged out.
RESULTS
From this study, it was observed that after 30 s of
treatment with lemon juice, the number of viable V.
parahaemolyticus was reduced by 4 Log compared to
3 Log in the control. When the oysters were treated for
60 s, none of the organisms were detected by culture
method in comparison to untreated samples in which
the bacterium was found to be viable even at the end of
the experiment. It was also observed that the number
of vibrio in the untreated sample reduced to 3.5 Log
CFU, 30 s after spiking the oysters with the bacterium
(see Figure 1).
Figure 1 Average number of viable Vibrio parahaemolyticus in oyster’s
meat after treatment with lemon juice. Each error bar represents standard
error means
DISCUSSION
The reduction in the viability of the bacterium seen
this study can be attributed to the acidity of the lemon
juice. A study by Tomotake et al. (2006) reported that
Vibrio strains, particularly V. parahemolyticus, are
very sensitive and dies readily at pH less than 4.5 but
become tolerant to acidity and can grow at pH higher
than 4.5 [4].
This finding supports the inhibitory effects of
lemon juice on V. parahamolyticus. Being a halophilic
and mesophilic organism, some of V.
parahaemolyticus do not survive long because to
survive, the bacterium requires brackish water which is

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usually equivalent to 1-9% NaCl and temperature of
20˚C for optimum growth [6]. We postulate that there
are other factors which may have contributed to the
inhibition of the growth of this organism such as the
antimicrobial properties contained in oysters. Oysters
contain defensive blood cells (haemocytes) which can
kill Vibrio sp. in addition to histone H2B protein,
which provides defense against gram-negative bacteria
[7].
This experiment was conducted to simulate the
condition where oysters are served in many restaurants
in Malaysia. In most restaurants and hotels, oysters are
usually served with the upper shell removed and the
flesh in the lower shell placed on ice cubes or flakes.
Slices of lemon are also served to complement the
oysters. Not only the lemon juice adds flavor to the
oyster, it also acts in a way to ‘cook’ the oyster. The
inoculation with 107 CFU of V. parahaemolyticus into
the oysters was to simulate the infective dose of this
organism in causing gastroenteritis in food poisoning
[8].
CONCLUSION
This study showed that lemon juice has some
inhibitory effect on V. parahaemolyticus in raw
oysters. Since there was also some reduction of V.
parahaemolyticus count in untreated samples, it is
likely that other intrinsic properties of the oysters also
contribute to the inhibition of V. parahaemolyticus in
raw oysters.
Conflict of Interest
Authors declare none.
Acknowledgements
We would like to express our sincere thanks to the
Director and staffs of the Institute of Medical
Molecular Biotechnology for the permission to use
facilities, and also to the staff of the Microbiology Unit
of the Centre for Pathology Diagnostic and Research
Laboratory, UiTM for their technical advices.
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