stability study of antibacterial activity of mixed lime juice and

Int. J. Pure Appl. Sci. Technol., 21(2) (2014), pp. 1-7

International Journal of Pure and Applied Sciences and Technology ISSN 2229 - 6107 Available online at www.ijopaasat.in

Research Paper

Stability Study of Antibacterial Activity of Mixed Lime Juice and Honey of Heating Temperature on Staphylococcusaureus and Streptococcuspyogenes

Hardoko1, * and Fonny Yuliana2

1 Faculty of Fisheries and Marine Sciences, Brawijaya University and Non-Permanent Lecturer

Department of Food Technology, University of Pelita Harapan 2 Department of Food Technology, University of Pelita Harapan, Jl. M.H. Thamrin Boulevard 1100,

Tangerang 15811, Indonesia

* Corresponding author, e-mail: ([email protected])

(Received: 29-1-14; Accepted: 24-3-14)

Abstract: Traditionally in Indonesian, lime juice (Citrusaurantifolia Swingle) added honey is often used as a cough medicine. The study aimed to determine the effect of the process of heating the mixture of lime - honey to the antibacterial activity against the bacteria S. aureus and S. pyogenes as bacterial causes of cough have been done. At first the lime juice is made by splitting and squeeze an lime, then mixed with honey (1:1 ratio) and heated for 15 minutes. The results show that lime juice contains compounds phytochemicalssaponins, alkaloids, tannins, phenolics, flavonoids, and terpenoids which is antibacterial. Antibacterial activity of lime juice is characterized by damage to the cell wall resulting in changes in cell morphology analysis of bacteria. Mixing lime juice with honey and heating processto100°Cdid not decrease the antibacterial activity of lime juice. Keywords: Antibacterial, Heating Honey, Lemonjuice.

1. Introduction: Orange is a plant that has many benefits for humans. The wood can be used for firewood. The leaves can be used for medicinal plant pests and diseases. The fruit can be used as medicine and food (Ghafar et al., 2010). One type of citrus is widely used as a spice in cooking and traditional medicine

Int. J. Pure Appl. Sci. Technol., 21(2) (2014), pp. 1-7 2

in Indonesia is lime (Citrus aurantifolia). The lime is generally small and sour taste (Razak et al., 2013, Jayana et al., 2010). For food, lime is often used as a acidifier, remover fishy odour (Poernomo et al., 2004), or mixed with the spices, and can also be used as a food preservative. The lemon is often used as a food is juice or peel. For example, George-Okafor et al (2010) using lemon juice in the fermentation of corn are rich in probiotic Lactobacillus. Jafari et al (2011) make use of lemon peel oil extracts to inhibit spoilage and pathogens bacteria in fillet cream cakes and pastries. The ability to inhibit the bacteria associated with the content of limonene, α- terpeniol, and γ-terpinen that dominant in orange peel. Dabbah et al (1970) reported that gram positive bacteria were more sensitive to essential oils than gram negative bacteria. Terpineol extended the shelf life of commercially pasteurized skim milk, low- fat milk, and whole milk for more than 56 days at 4oC. Orange oil extended the shelf life of skim milk and low- fat milk for the same period. For drugs, lime is used as an appetite enhancer, fever (antipireutic), diarrhea, lose weight, anti-inflammatory, antibacterial (Razak et al, 2010), and others. Part of lime that can be used for the medicine treatment is juice or peel. Tomotake et al (2006), Aibinu et al (2007), and Jayana et al (2010) reported that lemon juice effectively prevent Vibrio infections. Ayoola et al (2008), Jafari et al (2011), Taiwo et al (2007), Mahmud et al (2009), and Matan (2012) stated that the lime peel oil can inhibit pathogenic bacteria (such as Staphylococcus aureus, Salmonella paratyphi, flexneriiShigella, Streptococcus faecalis, Citrobacterspp, Serratiaspp, Klebsiellapneumoniae, Pseudomonas aeruginosa, Escherichia coli ATCC 25922, and Escherichia coli) and pathogenic fungi (such as Aspergillusniger and Candida albicans,and anaerobes bacteria which includes Bacteroidesspp, Porphyromonasspp, and Clostridium spp.). Munawaroh (2012) states that ethyl acetate extracts of lime peel could inhibit the bacteria that cause acnes. Owhe-Ureghe et al (2010) reported that a mixture of garlic with lime juice can inhibit tartar - causing bacteria. Kristina et al (2008) that the citrate acid in lemon juice can inhibit the formation of kidney stones. Traditionally in Indonesia, anti-bacterial properties of the lime used in treating cough disease. Cough treatment using lime is usually done using lime juice added honey and then heated to boil. A mixture of lime-honey after cold drunk on cough sufferers. Therefore it is necessary to learn the effect of the heating process on anti bacterial activity of a mixture of lemon juice-honey on bacteria that causes a cough, such as S. aureus and S. pyogenes.

2. Materials and Methods:

Materials Material under study is a mixture of lime juice and honey against bacteria that causes orethroatchannel. Lime (Citrus aurantifolia Swingle) used were obtained from "Hypermart Karawaci" Tangerang- Indonesia, round-oval shape with a diameter of 3-4cmandgreen. The honey that is used is the brand" Madu Rasa" production of “Air Mancur Ltd”, Solo - Indonesia. The bacteria used were Staphylococcusaureus and Streptococcuspyogenes which is a collection of the Department of Microbiology, University of Indonesia. Other ingredients such as Nutrient Agar media "Merck", Nutrient Brothmedia "Merck", Blood Agar Basemedia "Oxoid", defibrinationsheep blood coming from the UI Department of Microbiology, and distilled water.


Research Methods: Preparation Juice Lime-Honey Fresh lime juice that has been cleaned, halved, and squeezed with a manual juicer utility in order to obtain juice and then filtered usinga tea strainer. Lemon honey were then added at a ratio of 1:1 (v /v) and heated in a water bath at 65 °C, 70°C, 75°C, 80°C, 85°C, 100°C for15min and control (without heating). This study was designed using a completely randomized design (CRD) with a factor of three replications with a mathematical model Yij=µ+Di+εij.. Antibacterial Activity Analysis of Mixed Juice Lime-Honey Analysis of the antibacterial activity of a mixture of lime juice-honey made by the well diffusion method (Bloomfield, 1991). A total of 1mL of a suspension of bacteria (S. aureus and S. pyogenes) were inoculated into the agar medium is then poured into a petri dish and allowed to solidify and made wells with a diameter of 0.6 cmin aseptic. A mixture of lemon and honey (1:1) 60 mL inserted into wells made in aseptic. Petri dishes were incubated at 37°C for 24 hours. The diameter of inhibition zone is characterized by a clear area was measured with a ruler. 3. Results and Discussion: Phytochemicals Components Qualitatively Lime Juice Table 1 shows that in the lime juice, there are many types of phytochemical components that can act as bioactive. Some components have been reported to have antibacterial activity that will play a role in traditional medicine, for example, flavonoids, triterpenoids, alkaloids and phenolic.

Table1: Phytochemical components in lime juice

Phytochemical components

Content

Saponins +

Alcaloids +

Tannin +

Phenolic +

Flavonoids +

Triterpenoids +

Steroids +

Glycoside +

Some of these phytochemical antibacterial component supported by Sohn et al., (2004) that the phenolic components, flavonoids, alkaloids, and triterpenoids have antibacterial activity against B. subtilis, Salmonella, E. coli, and S. aureus. It is also supported by the statement Soetan et al., (2006) that the saponins, tannins, steroids and glycosides also have antibacterial activity against gram positive and negative bacteria such as S. aureus, E. coli, and B. cereus. Further added Basri et al. (2012) that the phenolics, flavonoids, and tannins can act as an antibacterial against pathogenic bacteria, such as E. faecalis, S. pyogenes, and B. cereus. Min et al. (2008), tannin compounds have


antibacterial activity against E. coli, Klebsiellapneumoniae, and S. aureus. Steroid compounds have antibacterial activity against gram positive and negative bacteria, such as E. coli, S. aureus, S. mutans, and E. faecalis (Taleb-Contini et al., 2003). Glycoside compounds have antibacterial activity against gram positive and negative bacteria, such as B. cereus, S. aureus, and E. coli (Molnar et al., 2011). Phenolic components which have bactericidal activity, such as carvacrol, eugenol and thymol. Phenolic components may interfere with the function of the cytoplasmic membrane proton included in the force and active transport (Davidson and Naidu, 2000). Triterpenoid compounds, such asborneol, pinene, kamfene, linalool, indole and kadien can inhibit the growth of B. subtilis, E. coli, and S. aureus (Naufalin et al., 2005). Lime Juice Effects on Bacterial Cells Effects of exposure of lime juice in bacterial cells of S. aureus and S. pyogenes can be observed using the SEM results is shown inFigure1.

Staphylococcus aureus

Normal cell (5000x) Expose lime (7500x)

Streptococcus pyogenes

Normal cell (3000x) Expose lime (10000x)

Figure 1: Morphology normal cell and expose lime juice of S. aureus and S. pyogenes

Figure 1 shows the changes in cell morphology of S. aureus and S. pyogenes after being exposed to lime juice. Morphological damage to the cells of S. aureus and S. pyogenesseen from the shape of the cells that are not whole damaged cell walls and formed holes that indicate damage to the cell wall analysis. This phenomenon is believed to result in the death of bacterial cells exposed to lime. Changes in cell morphology by lime was supported by Hartman et al., (2010) that the cell damage that occurs in bacteria with the addition of antimicrobials is a form of bacterial cells that do not form whole (dent), perforated, analysis.


Effect of Heating Temperature on Antibacterial Activity Lime-Honey The addition of honey in the treatment of cough is traditionally intended to reduce the sourer and to provide nutrients for cough sufferers. USDA (2011) reported that per 100 grams of honey contains energy 1.272 kJ (304 kcal) 1.272 kJ (304 kcal), carbohydrates 82.4g (82.12g sugar, 0.2g dietary fiber), protein 0.3g, 0g fat, 17.10g water, Riboflavin (vitamin B2) 0.038Mg0, 038mg, Niacin (vitamin B3) 0.121mg, pantothenic acid (vitamin B5) 0.068mg, 0.024mg vitamin B6, folic acid (vitamin B9) 2mg, 0.5mg of ascorbic acid (vitamin C), Calcium 6mg, 0.42mg of iron, Magnesium 2mg, 4mg Phosphorus, Potassium 52mg, Sodium 4mg, 0.22mg Zinc. In addition, honey has a Awof 0.6 resulted in the majority of microorganisms cannot grow (Prescott et al., 1999), containing anti oxidant compounds, namelychrysin, pinobanksin, ascorbic acid, catalase, and pinocembrin (Gheldof et al., 2002) and also compounds are compounds of hydrogen peroxide antibacterial active case of glucose oxidation reaction (Molan, 2001). In addition because of the presence of hydrogen peroxide compounds, honey can prevent the growth of bacteria due to low pH, which ranges between 3.2 to 4.5. According Randerson (2007), honey can be used to treat a sore throat and cough. Traditionally, the process of heating a mixture of lemon juice-honey before it is used in cough remedies are intended to kill bacteria microbial contaminants and also to get the flavor and aroma are preferred. Belitz et al., (2004) claim that if the sugar is heated with acid will be formed with a caramel color and aroma are appealing. Results of one-way ANOVA on the data of bacterial inhibition mixture of lemon juice-honey (1:1 v/v) showed that the heating temperature had no significant effect (p>0.05) inhibition zone against Staphylococcus aureus and Streptococcuspyogenes, as shown in Figure2.

Figure 2: The influence of temperature on the inhibition zone on S. aureus and S. pyogenes (Description: Notation behind letters indicate significantly differentat α 0.05.)

Figure 2 shows that the antibacterial activity of a mixture of lemon juice - honey that is indicated by the zone of inhibition did not change during heating to a temperature of 100oC. In other words, the antibacterial activity remained stable by heat. This is presumably due to the heating process in a short time can accelerate the reaction of the active components to inhibit the growth of bacteria, such as the formation of new compounds, namely terpene, diterpene, saponin, and glycoside of triterpen compounds (Adawiyah, 1996).


4. Conclusions: Lime juice contains compounds active phytochemical saponins, alkaloids, tannins, phenolics, flavonoids and terpenoids which is antibacterial. The antibacterial activity of lime juice resulted in changes in cell morphology in the form of bacterial cell wall broken and hollow to be able to inhibit the growth of deadly bacteria. Heating a mixture of lime juice-honey to 100°C causes node crease antibacterial activity against Staphylococcusaureus cough or Streptococcuspyogenes.

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stability study of antibacterial activity of mixed lime juice and

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