Antimicrobial Activity of Clove (Syzygium aromaticum) against Pathogenic Bacteria

DEEJA KAPOOR1, TARNNUM2, QAYYUM KHAN3

Assistant Proffesor1, Research scholar2, Research scholar3

1Himachal Institute of Life Sciences,Rampur Ghat Road, Paonta Sahib 173025, Himachal Pradesh

deejakapoor73@gmail.com, tarnnum4586@gmail.com, abdulkhan4586@gmail.com

Abstract: The present study investigated the antimicrobial potential of Indian spice viz., Clove (Syzygium aromaticum) against Bacteria isolated from food samples viz., Salmonella sp., Pseudomonas sp., Klebsiella sp., Proteus sp. using Aqueous, 100% Methanol, 75% Chloroform extracts. The antibacterial activity of spices extract was determined by agar well diffusion method and the antimicrobial activity of antibiotics (Ofloxacin, Ciprofloxacin, Gentamycin, Tetracycline, Amikacin, Chloramphenicol, Cefotaxime, Imipenem, etc.) was measured by disc diffusion method. The result of the agar well diffusion method showed that the aqueous extract of Syzygium aromaticum had antimicrobial potential followed by the methanol extracts of Syzygium aromaticum and Chloroformic extracts. Phytochemical screening was carried out on Methanol, chloroform and distilled water extracts of spices for its chemical composition. Qualitative phytochemical analysis of these spice extracts confirms the presence of various phytochemicals like alkaloids, terpenoids, flavonoids, saponins, steroids and tannins. The spices exhibited effective antimicrobial potential.

Index Terms: Antimicrobial activity, Clove, Phytochemicals, Food Poisoning.

1. INTRODUCTION

Clove (Syzygium aromaticum), belonging to family Myrtaceae, is widely used in medicine as antiseptic against infectious diseases like periodontal disease due to the antimicrobial activities against oral bacteria [1]. Clove is also commonly applied in food industry as a natural additive or antiseptic to increase shelf-life due to the effective antimicrobial activities against some food borne pathogens [2]. The main active component of clove oil and extract was found, i.e., eugenol [1, 3]. The spices used in Indian cooking have been used since ages for adding flavor and also for house-hold treatment of infectious diseases. It is imperative to study their antimicrobial activity against the common human pathogens so that the best spices can be further exploited to determine their active component which can be used for developing drugs. The present study was aimed at studying the antimicrobial activity of clove (Syzygium aromaticum) against the pathogens isolated from food samples viz., Salmonella sp., Pseudomonas sp., Klebsiella sp., Proteus sp.. Clove could destroy cell walls and membranes of microorganisms, and permeate the cytoplasmic membranes or enter the cells, then inhibit the normal synthesis of DNA and proteins [2]. Eugenol, the major component of clove, could inhibit the production of amylase and proteases in Bacillus cereus (B. cereus) and has the ability of cell wall deterioration and cell lysis [4]

2. MATERIALS AND METHODS The present study will be carried out at Department of Microbiology, Himachal Institute of Life Sciences (PG), Paonta sahib (Himachal Pradesh).

2.1 Sterilization of materials: All materials used were adequately and appropriately sterilized before and after use. Glass wares such as test tubes, conical flasks, pipettes, etc. were thoroughly washed with detergents, rinsed properly with water and drained. They were wrapped in aluminium foil and sterilized in hot air oven at 170°C for 1 hour. All media prepared according to the manufacturer’s instruction. Prepared media and distilled water were prepared according to the 15 minutes. Metal equipment’s like the inoculating loop were heated to redness in an open flame before and after use. The laboratory bench was always swabbed using 70% alcohol for disinfection before analysis was made. Every isolation and inoculation was done near the flame to reduce contamination of the agar plates tubes.

2.2 Sample Collection: Various samples (Food samples) were collected from hotels of Paonta sahib and nearby places in sterile containers, without disinfectant or detergent residue and tight-fitting leak-proof lids.

2.3 Sample processing/ enrichment:

The samples were inoculated in nutrient broth (NB) for 24hrs for enrichment, 1ml each sample was added in 9ml of nutrient broth (NB) for sufficient enrichment and incubated at 37°C for 24hrs.

2.4 Identification of bacteria: The enriched samples were spread on Nutrient agar media with the help of calibrated loop and incubated at 37°C for 24hrs for recovery of bacteria.

2.5 Isolation of Bacteria: The single isolated colony from the NA plate is picked up by inoculation loop and streaked on the selective media plates- King’s medium, Urea agar base, MSA agar (Mannitol Salt agar), PIA (Pseudomonas Isolation agar), BSA (Bismuth Sulphite Agar) for the selective isolation of different types of bacterial species. After streaking plates, plates were incubated at 37°C for 24hrs.

2.6 Gram staining: Gram staining (Gram’s Method) is empirical method of differentiating bacterial species into two large groups (Gram positive and Gram negative) based on the chemical, primarily the presence of high levels of peptidoglycan and physical properties of their cell walls.

2.7 Kirby-Bauer Disc Diffusion method – Culture will be inoculated by spread plate over Muller-Hinton Agar surface dried after 1-2 minutes antibiotic discs will be

applied using sterile forceps. Different antibiotics disc were placed equidistantly on surface of the plates with the sterile forceps. The plates are incubated for 24hrs. at 37°C. Observe the plates for zone of inhibition in mm and measure the zone with the help

of scale.

2.7 Collection of Spices powder material: Clove (Syzygium aromaticum) powder are collected from the surrounding area of Paonta sahib (H.P).

2.8 Instruments and Reagents used: Grinding machine, beakers, conical flasks, filter paper, methanol, chloroform, water, china dish, water bath.

2.9 Preparation of spice extracts Three solvents distilled water, 100% methanol and 75% chloroform were used to prepare extract of Clove (Syzygium aromaticum)

2.10 Extraction with distilled water: 10 g of powdered plant material was dissolved in sterile distilled water to make 40 ml of aqueous extract (25% w/v). The mixture was placed undisturbed at room temperature for 24 h in a sterile flask and it was filtered through sterilized Whatman no.1 filter paper. After filtration, the extract was evaporated in water bath until 25 ml extract was left in the container.

2.11 Extraction with 100% methanol: 10 g of powdered plant material was dissolved in enough methanol to make 40 ml of methanolic extract (25% w/v). The extraction procedure followed was the same as that used for aqueous extract.

2.12 Extraction with 75% chloroform: 10 g of powdered plant material was dissolved in chloroform to make 40 ml of chloroformic extract (25% w/v). The extraction procedure was similar to that used for aqueous extract. Extracts thus obtained were evaluated for antibacterial activities.

3. ANTIBIOTIC SENSITIVITY AND RESISTANT ACTIVITY OF RECOVERED ISOLATES

The antibiotic sensitivity of all the isolates against 16 commonly used antibiotics namely, Amoxicillin, Tetracycline, Ofloxacin, Gentamycin, Tigecycline, Ampicillin, Vancomycin, Ceftriaxone, Cefotaxime, Chloramphenicol, Imipenem, Piperacillin/Tazobactam, Amikacin, Linezolid, Ciprofloxacin.

Salmonella sp. Pseudomonas sp.

Klebsiella sp. Proteus sp.

Fig.1. Antibiotic pattern of recovered isolates of Food samples

4. DETERMINATION OF ANTIMICROBIAL ACTIVITY OF SPICE EXTRACT:

In the present study, the antimicrobial activity of Clove (Syzygium aromaticum) spice plant extracts were checked against pathogenic culture (Salmonella sp, Pseudomonas sp, Klebsiella sp, Proteus sp.). For antimicrobial on agar well diffusion method as described by Klaenhammer was used with some modifications. The antimicrobial activity of spice plant extracts against different pathogenic bacteria is given in the Table 1, 2, 3.

Table 1. Antimicrobial activity of different plant extracts (Aqueous) against pathogenic bacteria

Plant used Bacteria used zone of inhibition (in milli meter)Salmonella Pseudomonas Klebsiella Proteus

Syzygium aromaticum

37mm. 36mm. 42mm. 40mm.

Control Against Salmonella sp.

Against Pseudomonas sp. Against Klebsiella sp.

Against Proteus sp.

Fig.2. Antimicrobial activity of Syzygium

aromaticum (Aqueous)

Table 2. Antimicrobial activity of different plant extracts (Methanolic) against pathogenic bacteria

Control Against Salmonella sp.

Plant used Bacteria used zone of inhibition (in milli meter)Salmonella Pseudomonas Klebsiella Proteus

Syzygium aromaticum

37mm. 42mm. 35mm. 34mm.

Against Pseudomonas sp. Against Klebsiella sp.

Against Proteus sp.

Fig.3. Antimicrobial activity of Syzygium aromaticum (Methanolic)

Table 3. Antimicrobial activity of different plant extracts (Chloroformic) against pathogenic bacteria

Plant used Bacteria used zone of inhibition (in milli meter)Salmonella Pseudomonas Klebsiella Proteus

Syzygium aromaticum

22mm. 17mm. 14mm. 13mm.

Control Against Salmonella sp.

Against Pseudomonas sp. Against Klebsiella sp.

Against Proteus sp.

Fig.4. Antimicrobial activity of Syzygium aromaticum (Chloroformic)

5. RESULTS OF PHYTOCHEMICAL ASSAY: Chemical tests of plant extracts of each plant were carried out qualitatively using standard procedures to identify the major phytochemical constituents. The methods described below were used to assess the phytochemical constituents of the plant extracts.

5.1 Test For Tannins:

Ferric chloride test – To the extract, few drops of 1% natural ferric chloride solution was added.Result: formation of blackish blue colour indicates the presence of tannins.

5.2 Test For Saponins:

Mercury chloride test – To 0.5ml plant extract, add 5ml. distilled water and mix. Add 1-3ml. mercury chloride solution.Result: formation of white precipitate indicates the presence of saponins.

5.3 Test For Flavonoids:

Lead acetate test – The plant extract was treated with a few drops of lead acetate solution.Result: Formation of yellow precipitates indicates the presence of flavanoids.

5.5 Test For Terpenoids:

About 0.5g. of plant extract in separate test tube was taken with 2ml. of chloroform; 5ml. of conc. H2SO4 was carefully added to form a layer & observed.Result: Presence of reddish brown colour interface to show positive results for the presence of terpenoids.

5.6 Test For Phenols:

Lead acetate test – To the plant extract, a few drops of 10% lead acetate solution were added.Result: Formation of white precipitate indicated the presence of phenolic compounds.

5.7 Test For Reducing Sugars:

Benedict’s test – Approximately 1ml. of sample is placed in a clean test tube. Add 2ml. of Benedict’s reagent in test tube. The solution is then heated in a boiling water bath for 3-5 minutes.

Result: Green colour indicates the presence of 0.1 to 0.5 percent sugar in solution. Yellow colour indicates the presence of 0.5 to 1 percent sugar in solution. Orange colour indicates the presence of 1 to 1.5 percent sugar in solution. Red colour indicates the presence of 1.5 to 2.0 percent sugar in solution. Brick red colour indicates the presence of more than 2 percent sugar in solution.

5.8 Test For Alkaloids:

Extract were dissolved individually in dil. HCL & filtered. Add few drops of 5% NaOH solution.Result: Positive result indicates the presence of turbidity and yellow precipitate.

Fig.5. Phytochemical analysis of Syzygium aromaticum (Chloroformic)

Fig.6. Phytochemical analysis of Syzygium aromaticum (Methanolic)

Fig.7. Phytochemical analysis of Syzygium aromaticum (Aqueous)

Table 4. Phytochemical analysis of Plant extract

6.DISCUSSION: Food borne illnesses are diseases, usually either infectious or toxic in nature, caused by agents that enter the body through the ingestion of food (WHO, 2007).Microbial agents that cause food borne illness may include, bacteria such as; Salmonella, Pseudomonas, Klebsiella, Proteus. This investigation was aimed at documenting herbal medicines in the preservation of food and reduction of chances of Food borne illness in India and other countries. In the present a total 68 isolates were recovered from 28 samples of Food collected from various hotels of Paonta Sahib and nearby places. These 68 isolates were identified as Salmonella sp., Pseudomonas sp., Klebsiella sp., Proteus sp. So, the prevalence of recovered isolates was (Salmonella sp.-29.4%, Proteus sp.-25%, Pseudomonas sp.-23.6%, Klebsiella sp.-22%).So, the food poisoning is caused by the ingestion of contaminated food and water. The pathogenic bacteria were identified on the basis of morphological and biochemical characters. The direct antimicrobial activities of ultra-fine powders of ball-milled cinnamon and clove were tested by Kuang, X et al., 2011 against E. coli, S. aureus, Brochothrix thermosphacta (B. thermosphacta), Lactobacillus rhamnosus (L. rhamnosus), and Pseudomonas fluorescens (P. fluorescens) from meat, using broth dilution method. Clove powder showed strong inhibitory effects on five microorganisms tested with the MICs ranging from 1.0% w/v (L. rhamnosus and B. thermosphacta) to 2.0% w/v (P. fluorescens), and the inhibitory effects were positively associated with the concentrations of powder, which increased from 0.5% to 2.5% w/v. Bayoub et al., 2010 reported the antimicrobial activities of ethanol extracts of 13 plants including clove against L. monocytogenes, the MICs were determined by agar well diffusion test. The results showed that clove extract was the most effective inhibitor against L. monocytogenes compared with the other 12 selected plant ethanol extracts, with the MIC 0.24 mg/mL. Cui et al., 2016 tested the antimicrobial activities of 90 plant extracts (water and 99.5% ethanol extracts) against Clostridium spp. Clove water extract was found with the greatest antimicrobial activity against Clostridium botulinum in trypticase peptone glucose yeast extract broth (pH = 7.0) among all the water extracts, and the MICs of clove extract ranged from 0.1% to 0.2% against Clostridium spp. Mvuemba et al., 2009 evaluated the inhibitory effects of aqueous extracts of four spices (cinnamon, ginger, nutmeg, and horseradish) on the growth of mycelial of various spoilage pathogens (A . niger, Fusarium sambucinum (F. sambucinum), Pythium sulcatum (P. sulcatum), and Rhizopus stolonifera (R. stolonifera). At the concentration of 0.05 g/mL, cinnamon extract totally inhibited A.niger and P. sulcatum, while at the level of 0.10 and 0.15 g/mL F. sambucinum and R. stolonifer were completely inhibited, respectively. In my study, antimicrobial activity of Spices extracts is done by agar well diffusion method. The result of the agar well diffusion method showed that the aqueous extract of Syzygium aromaticum had antimicrobial potential followed by the methanol extracts of Syzygium aromaticum and Chloroformic extracts. Natural antioxidants such as flavonoids, tannins and phenols are increasingly attracting because they are disease preventing, health promoting and anti-aging substances [9]. Medicinal plants produce bioactive molecules that have both antibacterial and antifungal activities. The results obtained revealed that the aqueous, methanol and chloroformic extracts of all the ten Spice plants investigated have Antimicrobial activity against pathogenic bacteria isolated from food sample and also used as a food preservants.

7. CONCLUSION: Natural spice (clove) possesses effective anti-bacterial activity against micro-organisms and can be used for prevention microbial diseases. The phytochemicals (alkaloids, flavonoids, saponins, terpenoids, tannins and steroids) were present commonly in all the studied spice. The spices have been screened for phytochemical constituents and seemed to have the potential to act as a source of useful drugs and also to improve the health status of the consumers as a result of the presence of various compounds that are vital for good health. This study opens up the possibility for the search of new antimicrobials as an alternative to the antibiotics.

8. ACKNOWLEDGMENT:

Syzygium aromaticum(Chloroformic)

Syzygium aromaticum(Methanolic)

Syzygium aromaticum(Aqueous)

Alkaloids + + +

Flavonoids + + +

Phenols + + +

Tannins + + +

Terpenoids + + +

Saponins - + -

ReducingSugar

+ + +

I have great pleasure in expressing my deep sense of gratitude to the supervisors Miss Deeja Kapoor, Assistant professor, Department of microbiology, HILS Paonta Sahib (H.P.) for their valuable support, continuous and guidance throughout my dissertation work. I am also sincerely thankful to the Lab Attendent Mr. Vijay for help in my project compilation.

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