Article Type : Research Article
Authors : Sultana S, Kundu D, Islam A, Kamruzzaman Md and Mridha D
Keywords : Antimicrobial activity; Antimicrobial agents; E .coli; MIC; Pathogenic bacteria and Spectrophotometer
This study was conducted aiming at determining the antimicrobial
activity of some citrus and non-citrus fruits juices against some pathogenic
bacterial species. The antimicrobial activity of these fruit extracts were
examined against six bacterial strains including Staphylococcus aureus, E.
coli, enter pathogenic E. coli (EPEC), Enter toxigenic E. coli (ETEC), Vibrio
parahaemolyticus, Salmonella species using agar well diffusion method. The MIC
values of the juices against these bacterial species were determined by
measuring the OD via spectrophotometer as well. The lime (Citrus aurantiifolia)
fruit juice at 100% concentration showed the highest effect against test
organism EPEC, ETEC, E. coli, V. parahaemolyticus, S. aureus, Salmonella with
maximum zone of inhibition (mm) 18.333±0.577, 17.667±0.577, 18.00±1.00,
20.33±0.577, 21.33±1.155, 14.00±1.00 respectively. At the same concentration
the Kaffir lime (Citrus hystrix) had a higher antimicrobial activity on EPEC,
ETEC, E. coli, V. parahaemolyticus, S. aureus, with 23.00±1.00, 16.667±0.577,
16.667±1.528, 19.33±1.154, and 19.67±0.577 zone of inhibition (mm)
respectively. Pomelo (Citrus maxima) showed antimicrobial activity against EPEC
only. Pineapple (Ananascomosus) had a higher antimicrobial activity on EPEC,
ETEC, E. coli with zone of inhibition (mm) 18.00±1.00, 11.33±1.527 and 16.33±0.577
respectively. The Salmonella spp. was less sensitive to all fruit juices. 75%
concentration of lime juice showed as MIC against E .coli, EPEC, V.
parahaemolyticus, S. aureus and Salmonella spp. and 50% against ETEC. Same
concentration of kaffir lime juice observed as MIC against EPEC and V.
parahaemolyticus, 50% concentration against E .coli and S. aureus, and 25%
concentration against ETEC. Pineapple had MIC at 75%, 50%, 25% concentration against E .coli, EPEC and ETEC
respectively. Among four fruit juices lime showed most potential antimicrobial
activity even than penicillin and erythromycin against E .coli and EPEC
respectively. The present study suggested that, the citrus fruits and pineapple
fruit juices have great potential as antimicrobial agents against different
pathogenic bacteria.
In late time, the interest for the restorative plants is expanding in both creating and
created nations because of developing acknowledgment of common items as being non-poisonous, without reactions,
effortlessly accessible, and of reasonable
costs. Conventional medication, being a huge component in the social patrimony,
still remains the fundamental plan of action
for a vast dominant part of individuals for treating medical issues like cancer and so on [1]. Roughly 80% of the populace utilizes it as a
The study was designed with
the attention to antimicrobial activity of some citrus & non citrus
fruit juices against on different
pathogenic microbes.
Samples were collected from different districts of Bangladesh such as Tangail (Modhupur
Bazar), Mymensingh and Gazipur. Used distilled water for sample cleaning.
Fresh fruit were purchased from market of Tangail,
Mymensingh and Gazipur. The outer hard portion of these fruits was removed by
using autoclaved knife and squeezing by autoclaved percolator and fresh juice
was collected by using a sterile sample bottle following filtration.
Six strains of pathogenic
bacterial cultures were used in this study. These are among one is Gram-positive
S. aureus and other is Gram-negative E.
coli, Enteropathogenic E. coli
(EPEC) , Enterotoxigenic E. coli
(ETEC), V. parahaemolyticus, and Salmonella spp. The pure pathogenic
bacterial strains were collected from Department of Microbiology and Hygiene, Bangladesh Agricultural University,
Mymensingh and Department of Microbiology,
Jashore University of Science and Technology, Jashore. The cultures were
maintained on apendrop tube at 4°C and subcultures for 24 hours
before use.
Cultures of six pathogenic bacteria namely- Gram-positive S. aureus and other is Gram-negative E. coli, Enteropathogenic E. Coli (EPEC), Enterotoxigenic E. Coli (ETEC), V. parahaemolyticus, Salmonella spp. were obtained from the institutional microbiology laboratory and were stored in a refrigerator. Antimicrobial properties of fresh fruit juices were evaluated by agar well diffusion method. Briefly, petriplates containing nutrient medium composed of peptone, beef extract, sodium chloride, agar and distilled water were prepared for the evaluation of antimicrobial activities of fruit juices. 100µl of inoculum of each bacterium under our study was uniformly spreaded on the nutrient agar lawn in separate petriplates with the help of a sterilized glass spreader and the plates were then incubated for 30 minutes in the room temperature inside a laminar hood. Following this, with the help of a sterile cork, wells having the diameter of 6mm were bored in the plates. 50 µl of fresh crude and diluted fruit juice samples, distilled water and antibiotic disc erythromycin & penicillin were poured separately into the wells. Distilled water and antibiotic acted as negative and positive controls respectively. After 10 minutes of incubation at room temperature all the plates were transferred to an incubator and kept inside it at 37°C for 24 hours. Following the incubation, plates were checked for the presence of zones of inhibition (ZOI). Diameters of the observed ZOIs were measured in mm and the results were recorded by taking photographs of the ZOIs on the plates.
Minimum Inhibitory Concentration (MIC) is the lowest concentration of an antimicrobial that inhibits or kills the visible growth of microorganisms. MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against microorganisms. Four different concentrations i.e. 100%, 75%, 50%, 25% of these juices were prepared by diluting with normal saline. The nutrient broth was prepared .From this broth 9ml broth was added in each of four test tubes labelled as 100%, 75%, 50%, 25% fruit juices concentrations. Six sets of four test tube containing 9ml nutrient broth were prepared for six test microorganisms. Then 1ml of each concentration of fruit juices was added into the respective test tube. After this step 0.1ml test pathogen suspension were inoculated into respective labelled test tube. After inoculation, the test tubes were kept in a shaker incubator for overnight at 37°C and results were observed in the form of turbidity and O.D. were observed at 600nm on U.V. Spectrophotometer. However, as some shortcomings attached to the use of disc, agar well diffusion methods, the use of spectrophotometer to detect the Minimum Inhibitory Concentration (MIC) has some advantages over the others.
The yields of
extracts were calculated in percentage and the mean and standard deviation of
the three replication of antibacterial susceptibility assay was calculated
using statistical analysis software Statistix 10 (USA), version 10.
Fruit juices are valuable and readily available resources for primary health care and complementary health care system, undoubtedly fruit juices containing substances of medicinal value that have yet to be discovered, though large number of fruit juices are constantly being screened for their antimicrobial effect, these fruits may prove to be a rich source of compounds with possible antimicrobial activities. In the present study agar well diffusion method was used here in order to get antimicrobial activity of fresh citrus and non-citrus fruit juice against six bacterial strains among one is Gram-positive S. aureus and others are Gram-negative E. coli, Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli (ETEC), V. parahaemolyticus, Salmonella spp. Also MIC measurement was done to know the minimum inhibitory concentration of fruit juices by OD measurement using UV. Spectrophotometer. The non-citrus fruit include banana (Musa acuminate), pomegranate (Punicagranatum), sapodilla (Manilkarazapota), apple (Malusdomestica), pineapple (Ananascomosus), cucumber (Cucumissativus), and guava (Psidiumguajava) whereas, only pineapple can show antimicrobial activity. The citrus fruit include lime (Citrus aurantiifolia), pomelo (Citrus maxima), kaffir lime (Citrus hystrix) whereas, all showed antimicrobial activity. Results of our study showed that juices of four fruits exhibited inhibitory actions against the Gram-positive S. aureus and other is Gram-negative E. coli, Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli (ETEC), V. parahaemolyticus, Salmonella spp. Table no.1-4 represented the antimicrobial activity of various concentrations (100%, 75%, 50%, and 25 %.) of fresh Lime, Kaffir lime, Pineapple and Pomelo fruit juices respectively. The minimum inhibitory concentration (MIC) of various concentration of Lime, Kaffir lime, Pineapple and Pomelo juices based on OD measurement (mean±sd) represented in Table 5-8. The zones of inhibition against various selected pathogens were measured in mm (mean±sd). Zone of inhibition were seen against all species confirming the antimicrobial activity of fresh Lime, Kaffir lime, Pineapple and Pomelo juices (Figures 1-4). The fresh crude lime fruit juice produced the highest antimicrobial activity against S. aureus (21.33±1.155mm) followed by V. parahaemolyticus (20.33±0.577mm), E. coli (18.00±1.00mm), EPEC (18.333±0.577 mm), ETEC (17.667±0.577mm), & Salmonella spp. (14.00±1.00 mm) (Table 1). The fresh crude kaffir lime fruit juice produced the highest antimicrobial activity against EPEC (23.00±1.00mm) followed by V. parahaemolyticus (19.33±1.154mm), ETEC (16.667±0.577mm), S. aureus (19.67±0.577mm) & E.coli (16.667±1.528 mm) (Table 2).
Figure 1: Growth inhibition of some pathogenic bacterial
strains caused by lime juice.
(The
antimicrobial activity of lime juices on selected bacterial pathogen such as S.
aureus, E. coli, Enteropathogenic E. coli (EPEC), Enterotoxigenic E. coli
(ETEC), V. parahaemolyticus, Salmonella.)
Figure 2: Growth inhibition of some pathogenic bacterial
strains caused by Kaffir lime juice.
(The antimicrobial activity of kaffir lime juices on selected bacterial pathogen such as S. aureus, E. coli, EnteropathogenicE. coli (EPEC), EnterotoxigenicE. coli (ETEC), and V. parahaemolyticus.)
Figure 3: Growth inhibition of some pathogenic bacterial
strains caused by pineapple juice.
(The
antimicrobial analysis of pineapple juices on selected bacterial pathogen such
as E. coli, Enteropathogenic E. coli (EPEC) and Enterotoxigenic E. coli
(ETEC).)
Figure 4: Growth inhibition of pathogenic bacterial strains
caused by Pomelo juice.
(The antimicrobial activity of pomelo juices on selected bacterial pathogen such as Enteropathogenic E. coli (EPEC).)
Figure 5: Growth
inhibition of pathogenic bacterial strains (E. coli. and EPEC) caused by lime
juice and standard antibiotic (Penicillin and Erythromycin). (The lime juice
shows maximum antimicrobial activity than antibiotic, here the negative control
is distilled water that is not showed antimicrobial activity.)
Figure 6: Optimum microbial activity (Zone of inhibition) of four fruit juices against different pathogenic bacteria.
The fresh crude pineapple fruit juice produced the highest antimicrobial activity against EPEC (18.00±1.00 mm) followed by E. coli (16.33±0.577 mm), & ETEC (11.33±1.527 mm) (Table 3). The fresh crude pomelo fruit juice produced the highest antimicrobial activity against EPEC (15.667±0.577mm) (Table- 4). Table 1-4 show the diameters (in mm) of zones of inhibitions (ZOI) created in the agar plate by crude (100%) and diluted (75%), (50%), (25%) juices of three Citrus and one non Citrus fruits respectively. It appeared from our study that one Citrus fruit namely –lime exhibited inhibitory actions against all the bacteria tested in our study. Pineapple also exhibited inhibitory action against as many as three bacterial species except for S. aureus, V. parahaemolyticus, Salmonella spp. Kaffir lime, another Citrus fruit exhibited its inhibitory activities against as many as five bacterial species except against Salmonella. The fresh citrus fruit juices & pineapple juices were subjected to get the MIC against selected pathogens
The MIC observed by OD measurement, whereas 75% concentration of lime juice showed as minimum inhibitory concentration (MIC) against E. coli (0.083±0.0159), EPEC (0.0447±0.0136), V. parahaemolyticus (0.181±0.0108), S. aureus (0.169±0.0145), Salmonella spp. (0.2530±0.0135), and 50% concentration of lime juice against ETEC (0.0553±0.0138) respectively (Table 5). The MIC observed at 75% concentration of pomelo juice against EPEC (0.2410±0.0105) (Table 8). The MIC observed at 25% concentration of kaffir lime juice against ETEC (0.0303±0.0182), 50% concentration of kaffir lime juice against E. coli (0.064±0.0125), and S. aureus (0.0760±0.0207), 75% concentration of kaffir lime juice against EPEC (0.0957±0.0156), and V. parahaemolyticus (0.1570±0.0125) (Table 6). The MIC observed at 25% concentration of pineapple juice against EPEC (0.2453±0.0145), 50% concentration of pineapple juice against ETEC (0.2503±0.0165), 75% concentration of pineapple juice against E. coli (0.0247±0.0181) (Tables 7,8). From the above observation we can see that, Pineapple juice, showed appreciable inhibitory potential both in crude and in diluted form against E. coli, EPEC & ETEC but failed to inhibit other three bacteria such as V. parahaemolyticus, Salmonella spp. & S. aureus. Lime juice, however, showed appreciable inhibitory potential both in crude and in diluted form against E. coli, EPEC, ETEC, V. parahaemolyticus, Salmonella spp. & S. aureus. Whereas, Pomelo juice, showed appreciable inhibitory potential both in crude and in diluted form against EPEC only. Kaffir lime juice; however, showed appreciable inhibitory potential both in crude and in diluted form against E. coli, EPEC, ETEC, V. parahaemolyticus & S. aureus but failed to inhibit Salmonella spp. (Figures 5-6).
Herbal medicines are a valuable and readily available resources for primary health care and complementary health care system, undoubtedly the plant kingdom still holds many species of plants containing substances of medicinal value that have yet to be discovered, though large number of plants and their fruits are constantly being screened for their antimicrobial effect, these may prove to be a rich source of compounds with possible antimicrobial activities, but more pharmacological investigations are necessary. Overall, in terms of extent of the increasing ZOIs, it appears that Citrus fruits exhibited better inhibitory activities against pathogens than non-Citrus fruits. The non-citrus fruit include banana (Musa acuminate), pomegranate (Punicagranatum), sapodilla (Manilkarazapota), apple (Malusdomestica), pineapple (Ananascomosus), cucumber (Cucumissativus), guava (Psidiumguajava) whereas, only pineapple showed antimicrobial activity [14]. The citrus fruit include lime (Citrus aurantiifolia), pomelo (Citrus maxima), kaffir lime (Citrus hystrix) whereas, all showed antimicrobial activity. Enteropathogenic E. coli (EPEC) is only organism that shows sensitive activity against four fruit juices such as three citrus fruits including lime, kaffir lime, pomelo & one non citrus fruit juice including pineapple. The fresh crude lime fruit juice produced the highest antimicrobial activity against S. aureus (21.33±1.155 mm), followed by V. parahaemolyticus (20.33±0.577 mm), E. coli (18.00±1.00 mm), EPEC (18.333±0.577mm), ETEC (17.667±0.577mm), & Salmonella spp. (14.00±1.00mm) [15]. The fresh crude kaffir lime fruit juice produced the highest antimicrobial activity against EPEC (23.00±1.00mm) followed by
V. parahaemolyticus (19.33±1.154mm)ETEC (16.667±0.577mm), S. aureus (19.67±0.577mm) & E. coli (16.667±1.528mm) [16,17]. The fresh crude pineapple fruit juice produced
the highest antimicrobial activity against different pathogenic bacteria
including EPEC (18.00±1.00mm) followed by
E. coli (16.33±0.577mm) & ETEC
(11.33±1.527mm). The fresh crude pomelo fruit juice produced the highest
antimicrobial activity against EPEC (15.667±0.577mm) only. This study supports
the use of these fresh fruit juices
of Pineapple, Pomelo, Lime, and Kaffir lime not only as dietary supplements but
also as the agent to prevent or control the bacterial infections.
Investigation of the antimicrobial
activity of limejuice has been investigated and limejuice has been found to have high antimicrobial
activity [18]. In this study it is
observed that the potency of lime fruit
is enhanced by the type of
solvent used
The present study suggested that, the citrus fruits and
pineapple fruit juices have great potential as antimicrobial agents against
selected pathogenic bacteria and it also indicate that by intake these type of
common fruits, mass people of developing country like Bangladesh might protect
and cure them from different kinds of pathogen raised diseases.
The authors declare no conflict of interest, financial or otherwise.
The authors acknowledge the department of Microbiology and Hygiene, Bangladesh
Agricultural University, Mymensingh 2202, Bangladesh; Department of Microbiology, Jashore University of
Science and Technology, Jashore 7408. and co-authors for their help during the research work and manuscript writing.