Antibacterial properties of Limau kasturi (C. microcarpa) peels extract.
Citrus microcarpa or Citrus mitis, also known as limaukasturi is one of citrus fruits from Rutaceae family which has skin orange colour with a very thin green coloured peel . It can be processed and used as limaukasturi juice, cordial and cosmetic product which based limaukasturi extracts. In Malaysia, limaukasturi is one of the spices that have high production rates in each year and if compared with Citrus hystrix (limaupurut) and Citrus aurantifolia (limaumpis), limaukasturi production rate is much higher at 25% of the main spices in Malaysia. In fact, the rate of limaukasturi production is expected to increase until 2020 [2, 3]. Due to the high production rates, expected by-products including peels will also be increased by year, so that this research is one of the alternatives to reduce the waste. Fungi and bacteria cause diseases to human and animals, especially in tropical and subtropical regions, and commonly occur on immunocompromised patients. This could results the body of the patients unable to produce enough substances or cells that are normally produce to fight those infections, due to the weakness or absent of the immune inside body system [4, 5, 6]. Thus, the public is becoming increasingly aware of this problem. Moreover, the plants contain many biologically active compounds which have potential to be applied as medicinal agents , therefore, recently, many researchers get interest on traditional plant derived medicines study especially to figure out the effectiveness of the plant extracts in fighting microbial diseases [8, 9]. The potentials of citrus peels are it can possess high amounts of flavonoids, compared to other parts of the fruit . The skin or peels usually contain bioactive compounds in order to protect the inner materials from insects and microorganisms deterioration . So, possibility the potential usage of the peels it can be used as antimicrobial agent. Flavonoids are phenolic groups in plants that responsible on antimicrobial activity  where it can inhibit nucleic acid synthesis, cytoplasmic membrane function, and energy metabolism . The bioactive compound that was isolated from C. microcarpacan possess antimicrobial property against fish pathogenic bacteria such as E. coli, P. aeruginosa and S. agalatine . Thus, for this study aimed to evaluate the antibacterial properties of limaukasturi peels extract against the growth of pathogenic bacteria.
METHODS AND MATERIALS
Limaukasturi were purchased from local market in Kangar, Perlis. The fruits were washed with tap water and peeled manually. The peels were heated at 45[degrees]C for 24 h in a convection oven and the dried peels were grinded to fine powder using a grinder. The ground powders then were packed in a tight plastic sealer and stored at room temperature until use.
About 20 g of sample were packed in a nylon fabric before be placed in an extraction chamber, which is suspended above a flask that containing of solvent and below a condenser. After that, the flask containing samples is heated using heating mantle until the solvent is evaporates and move up into the condenser until it is converted into a liquid form that trickles into the extraction chamber containing the sample. The time taken for the solvent to evaporate will be regulated. At the end of the process, the round bottom flask is taken out to undergo separation process using rotary evaporator. This process aims to separate the solvent from the extract.
Antibacterial Activities Test:
Antibacterial activities were carried out according to method that suggested by Archana  with some modification, where bacteria were obtained from School of Bioprocess, Universiti Malaysia Perlis (UniMAP). Bacterial cultures used are such Escherichia coli, Bacillus subtilis, Salmonella spp., and Streptococcus spp.
Culture media and inoculum:
The culture media and inoculum were prepared according to the method as suggested by Archana  with slight modification. Before use, bacteria cultures were revived in nutrient broth (NB). For maintenance, bacteria cultures were maintained in nutrient agar (NA) at 4[degrees]C. The inoculum of bacteria cultures were incubated at 37[degrees]C. The inoculated broth was incubated overnight for 24 h. Then, a freshly grown microbial cultures were appropriately diluted in sterile both media to obtain cell suspension of [10.sup.6] cfu/ml.
For antibacterial assay, agar well-diffusion method has been modified slightly from Sen and Batra . Nutrient agar (NA) was swabbed (sterile cotton swabs) with 8 hour old-broth culture of respective bacteria. Then, wells (7mm diameter and 20mm a part) were made in each plates using tips 1 ml. About 100mg/ml of peels extracts concentration were used for each solvent. 100 [micro]l of peels extracts will be pipetted into the wells. An antibiotic, named ciprofloxacin was used as positive control while distilled water as negative control. Next, the plates were incubated at 37[degrees]C for 18-24 h. The diameter of the inhibition zone (mm) was measured. The tests were performed in triplicate.
RESULTS AND DISCUSSIONS
Results of antibacterial activity of limaukasturi peel extracts against Gram-positive and Gram-negative bacteria by the agar-well diffusion method were shown on Table (1), (2), (3), (4) and (5), respectively. From overall result, the inhibition zone of extracts against Gram-negative bacteria which are E. coli and Salmonella spp. is higher than Gram-positive bacteria (B. subtilis and Streptococcus spp.). This is because of the cell wall of Gram-negative bacteria is thinner than Gram-positive bacteria , so that the extract can penetrate the wall of Gram-negative bacteria more and inhibit the growth of bacteria. In this test, ciprofloxacin was used as positive control. It is an antibiotic in a group of drugs called fluoroquinolones that have been commercially used to treat bacterial infections on humans . From observations, ciprofloxacin is very effective antibiotic against bacteria.
The bacterial susceptibility was presented in Table (1) where the highest activity of methanol extracts was observed against E. coli with zone of diameters of 19 mm. For Salmonella spp. methanol extracts has inhibited about 17 mm of bacterial growth while for B. subtilis and Streptococcus spp., it's inhibited about 13 mm and 12 mm, respectively. The interpretation of susceptibility of microorganism is divided into three (Resistant: 13 mm or less, Intermediate: 14-16 mm, Susceptible: 17 mm or more) .From this result, it can emphasized that Gram-negative bacteria are susceptible to methanolic extracts of limaukasturi peels while Gram-positive bacteria are moderately susceptible to the extracts. From previous study, Gopal  infer that C. aurantium leaves extracts was significantly effective against both Gram-positive and Gram-negative bacteria with inhibition zone 12-14 mm.
Next, inhibition zone of ethanol extracts of limaukasturi peels was shown in Table (2). A very little zone was inhibited by ethanol extracts on E. coli and Salmonella spp. with zone of diameter 8 mm, respectively. While for Streptococcus spp. the extracts only covered 7 mm and no inhibition on B. subtilis. Thus, from the observation, it can be concluded that ethanol extracts has a low inhibition towards bacterial pathogens, except B. subtilis which is resistant to the extracts. Unnisa et al.  has reported that lowest antibacterial effect of ethanolic extracts from sweet lime was observed on E. coli with diameter of zone is 8 mm. From the previous study, Kumar et al.  considered that the aqueous extract of C. sinensis peel has a moderate percentage with inhibition zone 9 mm against E. coli. For acetone extracts, the zone of inhibition was summarized in Table (3). From the result, only E. coli has resistant to acetone extracts. While the other three bacteria shows low inhibition on the plates. Salmonella spp. has recorded about 7 mm of diameter zone, while B. subtilis and Streptococcus spp. are inhibited only 6 mm, respectively. Jwanny et al.  concluded that aqueous extract of orange peels has antimicrobial activity against microbes with zone of inhibition 7-12 mm.
The last two results of inhibition zone were presented in Table (4) for hexane extracts and Table (5) covered for water extracts. From the appearance result, no inhibitions are detected in each bacterial plate, therefore, all the bacteria are resistant towards both hexane and water extracts. It's possibly that there are no or less phenolic compound are presence in the extracts which responsible for antibacterial activity.
As the conclusion, limaukasturi peel extracts has possessed antibacterial activity towards all tested bacterial pathogens. However, only certain extracts shows a good inhibition effects on agar plates. The most goods appearance of inhibition zone was found using methanolic extracts where it's inhibit 19 mm on E. coli, 17 mm on Salmonella spp, 13 mm on B. subtilis and and 12 mm on Streptococcus spp. For overall result, Gram-positive bacteria are susceptible to methanol extracts and Gram-negative bacteria are moderately susceptible to the extracts. While forethanol extracts, it gave low inhibition on all bacteria except B. subtilis. For acetone extracts, it's covered low inhibition on all bacteria except E. coli, while hexane and water extracts showed no inhibition for all bacteria. Thus, the results obtained in the present study suggest that methanol extracts of limaukasturi peels can be used for further study in treating diseases caused by the test organisms.
The authors would like to express their appreciations to Ministry of Education Malaysia for funding this project under Fundamental Research Grant Scheme (FRGS) Grant (Grant Number: 9003-00356). The authors also like to acknowledge Universiti Malaysia Perlis (UniMAP) for facilitating this project.
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(1) Rubiatul, A.S., (2) Nor Helya I. K., (3) Zarina Z., (4) Dachyar A., (5) NurulAin H.A.
(1,4) School of Bioprocess Engineering, Universiti Malaysia Perlis, Jejawi 3 Engineering Complex, 02600 Arau, Perlis, Malaysia.
(2,3,5) Faculty of Engineering Technology, Universiti Malaysia Perlis, P.O Box 77, D/A Pejabat Pos Besar Kangar, 01000 Kangar, Perlis, Malaysia.
Received 23 June 20I5
Accepted 25 July 2015
Available online 30 August 20I5
Corresponding Author: Nor HelyaIman Kamaludin, Faculty of Engineering Technology, University Malaysia Perlis, P. O Box 77, D/A Pejabat Pos Besar Kangar, 01000 Kangar, Perlis, Malaysia.
Table 1: Zone of inhibition (mm) of methanol extracts of limaukasturi peels Zone of Bacteria inhibition (mm) E. coli Salmonella spp. Ciprofloxacin 26 20 Methanol extracts 19 17 Appearance [ILLUSTRATION [ILLUSTRATION OMITTED] OMITTED] Zone of Bacteria inhibition (mm) B. subtilis Streptococcus spp. Ciprofloxacin 17 17 Methanol extracts 13 12 Appearance [ILLUSTRATION [ILLUSTRATION OMITTED] OMITTED] Table 2: Zone of inhibition (mm) of ethanol extracts of limaukasturi peels Zone of inhibition Bacteria (mm) E. coli Salmonella spp. Ciprofloxacin 24 20 Ethanol extracts 8 8 Appearance [ILLUSTRATION [ILLUSTRATION OMITTED] OMITTED] Zone of inhibition Bacteria (mm) B. subtilis Streptococcus spp. Ciprofloxacin 12 15 Ethanol extracts NI 7 Appearance [ILLUSTRATION [ILLUSTRATION OMITTED] OMITTED] * NI--no inhibition Table 3: Zone of inhibition (mm) of acetone extracts of limaukasturi peels. Zone of Bacteria inhibition (mm) E. coli Salmonella spp. Ciprofloxacin 24 21 Acetone extracts NI 7 Appearance [ILLUSTRATION [ILLUSTRATION OMITTED] OMITTED] Zone of Bacteria inhibition (mm) B. subtilis Streptococcus spp. Ciprofloxacin 10 17 Acetone extracts 6 6 Appearance [ILLUSTRATION [ILLUSTRATION OMITTED] OMITTED] * NI--no inhibition Table 4: Zone of inhibition (mm) of hexane extracts of limaukasturi peels Zone of Bacteria inhibition (mm) E. coli Salmonella spp. Ciprofloxacin 24 19 Hexane extracts NI NI Appearance [ILLUSTRATION [ILLUSTRATION OMITTED] OMITTED] Zone of Bacteria inhibition (mm) B. subtilis Streptococcus spp. Ciprofloxacin NI 17 Hexane extracts NI NI Appearance [ILLUSTRATION [ILLUSTRATION OMITTED] OMITTED] * NI--no inhibition Table 5: Zone of inhibition (mm) of water extracts of limaukasturi peels. Zone of Bacteria inhibition (mm) E. coli Salmonella spp. Ciprofloxacin 26 19 Water extracts NI NI Appearance [ILLUSTRATION [ILLUSTRATION OMITTED] OMITTED] Zone of Bacteria inhibition (mm) B. subtilis Streptococcus spp. Ciprofloxacin NI 17 Water extracts NI NI Appearance [ILLUSTRATION [ILLUSTRATION OMITTED] OMITTED] * NI--no inhibition
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|Author:||Rubiatul, A.S.; Nor Helya, I.K.; Zarina, Z.; Dachyar, A.; NurulAin, H.A.|
|Publication:||Advances in Environmental Biology|
|Date:||Aug 1, 2015|
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