Evaluation of Antimicrobial Activity of Ethanolic Extracts of Some Indian Spices against Food-related Microorganisms
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1 Article International Journal of Food Nutrition and Safety, 2012, 2(1): International Journal of Food Nutrition and Safety Journal homepage: ISSN: X Florida, USA Evaluation of Antimicrobial Activity of Ethanolic Extracts of Some Indian Spices against Food-related Microorganisms Ram Kumar Pundir 1 * and Pranay Jain 2 1 Department of Biotechnology, Ambala College of Engineering and Applied Research (ACE), Devsthali, Near Mithapur, Sambhalkha , Ambala, Haryana, India 2 Department of Biotechnology, University Institute of Engineering and Technology (UIET), Kurukshetra University, Kurukshetra , Haryana, India * Author to whom correspondence should be addressed; drramkpundir@gmail.com Article history: Received 8 November 2012, Received in revised form 6 December 2012, Accepted 8 December 2012, Published 9 December Abstract: In the present endeavor, thirteen ethanolic extracts of Indian spices were investigated for antibacterial potential against six Gram-positive and Gram-negative foodrelated bacteria using agar well diffusion method and antifungal potential against six fungi using poisoned food technique. Ethanolic extracts of cumin, fenugreek, black pepper, turmeric, clove, garlic and cinnamon were found to be effective against all the tested bacteria, Bacillus subtilis, B. megaterium, B. sphaericus, B. polymyxa, Staphylococcus aureus and Escherichia coli and 4 extracts namely coriander, anise, rye and ginger did not show any antagonistic activity against any of the tested bacterium. Diameter of zone of inhibition was found to be maximum in clove (25 to 32 mm) followed by garlic (20 to 31 mm) extract. Caraway extract exhibited moderate inhibitory activity against the tested bacteria (10 and 26 mm). Interestingly, activity was found to be the highest in kalonji extract only against S. aureus showing (32 mm). Of the thirteen extracts, six ethanolic extracts showed antifungal activity. Clove and ginger ethanolic extracts showed the maximum antifungal activity against all the six tested molds such as Aspergillus luchuensis, A. flavus, Penicillium oxalicum, Rhizopus stolonifer, Mucor sp. and Scopulariopsis sp with percent mycelial growth inhibition between 70% and 100% followed by garlic. Garlic exhibited the growth inhibition of five tested molds (30% and 50%) except P. oxalicum. Turmeric inhibited the growth of two molds (R. stolonifer and Mucor sp.), cumin of P. oxalicum and cinnamon of A. luchuensis. In the present findings, of the tested spice extracts, clove ethanolic extract has been found to be an excellent
2 38 antimicrobial agent with wide spectrum activity which implicates its importance as a natural preservative to reclaim the shelf life of food and may also be used as nutraceuticals. Keywords: antibacterial activity; antifungal activity; food; bacteria; fungi; preservative; spice; food-related microorganisms. 1. Introduction Microorganisms in food products may cause spoilage which contributes to the deterioration in safety, flavour, texture and colour of the products and finally resulting in food-borne diseases (Decker et al., 1996). Prevention of pathogenic and spoilage microorganisms in food is usually achieved by using chemical preservatives but they are responsible for many carcinogenic and teratogenic attributes as well as residual toxicity and with growing concern of microbial resistance towards conventional preservatives, consumers tend to be suspicious of chemical additives and thus the exploration of naturally occurring antimicrobial for food preservations receives increasing attention. As the popularity of botanical biopreservatives continues to increase, consumers, regulatory agencies, and food processors require reliable information on the safety, standardization, and efficacy of these products (Draughon, 2004). The potential sources of natural products and preservatives are spices, herbs, fruits, seeds, leaves, barks and roots. Of these, spices and herbs are an important part of human diet. They have been used for thousands of years to enhance the flavour, colour and aroma of foods. In addition to boosting flavour, spices and herbs are also known for their preservative and medicinal values (Parekh and Chanda, 2007). The potential antimicrobial properties of plants has long been related to their ability to synthesize, by the secondary metabolism, several chemical compounds of relatively complex structures with antimicrobial activity, including alkaloids, flavonoids, isoflavonoids, tannins, cumarins, glycosides, terpenes, phenylpropanes, and organic acids (Cowan, 1999). Attention of the scientific community worldwide is shifting towards spices and herbs to harness their antimicrobial properties for use as natural food preservatives and nutraceuticals (Chattopadhyay and Bhattacharyya, 2007). The objective of this study was to evaluate the antibacterial and antifungal activity of thirteen Indian spices ethanolic extracts against six food-related bacteria and six fungi. 2. Materials and Methods For the present study, the plant parts were collected from the localities of Kurukshetra, Haryana, India followed by the phytochemical extraction and evaluation for their antimicrobial activity
3 39 against six food-associated bacteria and six fungi. Crude phytochemical extraction was performed by using the methods of Chen et al. (1987) and Barreto et al. (2002). Plant parts were collected and washed in sterile distilled water, and then dried in oven at 45 o C for h and grinded into a fine powder. The 25% (w/v) extract was prepared in ethanol and kept at room temperature for 24 h, after that filtered through sterilized Whatmann no. 1 filter paper. Evaporation of the extract in water bath till the total volume reduced to ¼ and screening of concentrated extract against food-associated bacteria by agar well diffusion method and fungi by poisoned food technique Agar Well Diffusion Method for Antibacterial Activity In agar well diffusion, twenty ml of molten cooled (45 o C) PCA poured into each Petri dish and allowed it to solidify and swabbing of the agar plates with 100 µl of each food-associated bacterial inoculum ( CFU/mL) thereafter wells (8 mm in diameter) made into seeded agar plates with sterile cork borer. Wells were loaded with 100 µl plant extract and with ethanol (95%) i.e. solvent (control) and inoculated agar plates were left at room temperature for 10 min for adsorption of plant extract. Incubation of plates was carried out at 37 o C for 24 h, and diameter of zone of growth inhibition (in mm) was measured by zone reader (Hi Antibiotic zone scale) (Ahmad and Beg, 2001) Poisoned Food Technique for Antifungal Activity In poisoned food technique, the fungal inocula were prepared by disc inoculation on PDA plates followed by incubation at 25 o C for 3 to 7days. The 100 µl of plant extract was added in to 15 ml of molten cooled (45 o C) PDA (Ethanol was added to the agar plates which acted as control) poisoned PDA and control plates were inoculated with fungal plugs (6 mm) diameter cut from periphery of 3 to 7 days old cultures. The inoculated plates were incubated at 25 o C for 3 to 7 days and thereafter diameter of fungal colony was measured and expressed as percentage mycelial growth inhibition. 3. Results Control - Sample Percentage mycelial growth inhibition (%) = 100 Control 3.1. Antibacterial Activity Ethanolic extracts of seven spices namely cumin, fenugreek, black pepper, turmeric, clove, garlic and cinnamon were found to give promising results against all the tested bacteria, Bacillus subtilis, B. megaterium, B. sphaericus, B. polymyxa, Staphylococcus aureus and Escherichia coli
4 40 (Table 1 and Fig. 1). Four extracts namely coriander, anise, rye and ginger did not have any inhibitory activity against any of the tested bacterium. Diameter of zone of inhibition was found to be the maximum in clove (25 to 32 mm) (Fig. 2) followed by garlic (20 to 31 mm) extract. Caraway extract exhibited moderate inhibitory activity against the tested bacteria with zone of inhibition varying between 10 and 26 mm. Interestingly, the activity was found to be the highest in kalonji extract against S. aureus showing 32 mm as diameter of inhibition zone. Table 1. Antibacterial activity of thirteen ethanolic spice extracts against six food-associated bacteria by agar well diffusion method S. N. Plant extract Diameter of inhibition zone (mm a ) Bacillus subtilis B. megaterium Note: - = No activity; a -Values, including diameter of well (8 mm), are mean of the three replicate; b mean ± standard deviation. B. sphaericus B. polymyxa Staphylococc us aureus Escherichia 1 Cumin 22±0.81 b 20± ± ± ± ± Fenugreek 10± ± ± ± ± ± Black pepper 15± ± ± ± ± ± Turmeric 15± ± ± ± ± ± Clove 30± ± ± ± ± ± Garlic 31± ± ± ± ± ± Caraway - 20± ± ± ± ± Coriander Anise Kalonji 21± ± ± Cinnamon 14± ± ± ± ± ± Rye Ginger coli 3.2. Antifungal Activity Antifungal activity was observed in six ethanolic extracts, the mode of activity varying from narrow to wide. Clove and ginger ethanolic extracts showed the maximum antifungal activity against all the six tested molds namely Aspergillus luchuensis, A. flavus, Penicillium oxalicum, Rhizopus
5 41 stolonifer, Mucor sp. and Scopulariopsis sp with per cent mycelial growth inhibition between 70% and 100% (Table 2 and Fig. 3). Garlic inhibited the growth of five tested molds except P. oxalicum with percent mycelial growth inhibition varying between 30% and 50% (Fig. 4). Turmeric inhibited the growth of two molds (R. stolonifer and Mucor sp.), cumin of P. oxalicum and cinnamon of A. luchuensis. In the present finding, clove ethanolic extract has been found to be an excellent antimicrobial agent with wide spectrum activity as it not only inhibited Gram-positive and Gramnegative food-associated bacteria but also the molds which implicate its importance as a natural preservative to reclaim the shelf life of food. Table 2. Antifungal activity of thirteen ethanolic spice extracts against six food-related fungi by poisoned food technique S. N. Plant Percent mycelial growth inhibition extract Aspergillus Aspergillus Penicillium Rhizopus Mucor Scopulariopsis luchuensis flavus oxalicum stolonifer sp. sp. 1 Cumin Fenugreek Black pepper Turmeric Clove Garlic Caraway Coriander Anise Kalonji Cinnamon Rye Ginger Note: - = No activity. 4. Discussion Spices are one of the most commonly used natural antimicrobial agents in foods and have been used traditionally for thousands of years by many cultures for preserving foods and as food additives to enhance aroma and flavour (Nevas et al., 2004, Souza et al., 2005). Our results substantiate the findings of Sulieman et al. (2007) who demonstrated the antibacterial activity of clove ethanolic extract against E. coli, S. aureus and B. subtilis and found that the highest
6 42 antibacterial activity was against E. coli. The antibacterial activity of clove is attributed to eugenol (2 methoxy-4-allyl-phenol) (Gupta et al., 2008). High tannin content (10-19%) in clove also provides additional antimicrobial activity (Namasombat and Lohasupthawee, 2005). The antimicrobial activity of garlic has earlier been reported against S. aureus, E. coli and Klebsiella pneumoniae (Jabar and Mossani, 2007) and E. coli and S. aureus (Vuddhakul et al., 2007). Shelef (1983) reported that allicin, the essential oil substance isolated from garlic, inhibited bacteria in culture media and also discovered that most of the antimicrobial substances were phenol compounds such as eugenol, thymol and carvacol. Several workers (Meena and Sethi, 1994, Arora and Kaur, 1999) have earlier reported that clove ethanolic extract showed antimycotic activity against fungal genera such as Aspergillus, Penicillium, Rhizopus, Cladosporium and Saccharomyces which is in harmony with the present study. This activity may be due to the presence of eugenol and caryophyllene. Several studies have reported that garlic extract can inhibit the growth of bacteria, fungi, viruses in culture media and food systems and it has been shown to posses insecticidal, antiparasitic and antitumour properties. Several ajoene compounds, derivative of allicin, obtained from garlic with ethanol extraction have been found to be very inhibitory against A. niger and Candida albicans (Irkin and Korukluoglu, 2007; Mahmoudabadi and Nasery, 2009). Yashida et al. (1987) reported that ajoene compound from garlic have stronger antifungal activity than allicin. Diameter of zone of inhibition Cumin Fenugreek Black pepper Antibacterial activity of thirteen ethanolic plant extracts by agar well diffusion method Turmeric Clove Garlic Caraway Coriander Plants used Anise Kalonji Cinnamon Rye Ginger Bacillus subtilis B. megaterium B. sphaericus B. polymyxa Staphylococcus aureus Escherichia coli Figure 1. Antibacterial activity of thirteen ethanolic spice extracts against six food-associated bacteria by agar well diffusion method.
7 43 Figure 2. Antibacterial activity of clove ethanolic extract against Bacillus subtilis. Percent mycelial growth inhibition Cumin Fenugreek Black pepper Antifungal activity of thirteen ethanolic plant extracts by poisoned food technique Turmeric Clove Garlic Caraway Coriander Anise Plants used Kalonji Cinnamon Rye Ginger Aspergillus luchuensis Aspergillus flavus Penicillium oxalicum Rhizopus stolonifer Mucor sp. Scopulariopsis sp. Figure 3. Antifungal activity of thirteen ethanolic spice extracts against six food- related fungi by poisoned food technique. Figure 4. Antifungal activity of clove ethanolic extract against Scopulariopsis sp. (Left Plate-Control; Right Plate-Experimental)
8 44 5. Conclusions It may be suggested from the findings that the clove and garlic ethanolic extracts can be used as a potential source of natural antimicrobial compound. Further research is needed for the identification of bioactive molecules present in the two extracts and in vivo efficacy against food spoilage and pathogenic microorganisms before it is used for commercialization in the form of natural food preservatives, additives and nutraceutical foods. Acknowledgements The authors are thankful to Directors of their institute for encouraging us to write this research article. References Ahmad, I., and Beg, A. J. (2001). Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multidrug resistant human pathogens. J. Ethnopharmacol., 74: Arora, D. S., and Kaur, J. (1999). Antimicrobial activity of spices. J. Antimicrob. Agent., 12: Barreto, M., Critchley, A. T., and Straker, C. J. (2002). Extracts from seaweeds can promote fungal growth. J. Basic Microbiol., 42: Chattopadhyay, R. R., and Bhattacharyya, S. K. (2007). Herbal spices as alternative antimicrobial food preservatives: An update. Pharm. Rev., 1: Chen, C. P., Lin, C. C., and Namba, T. (1987). Development of natural crude drug resources from Taiwan. In vitro studies of the inhibitory effects on 12 microorganisms. Shouyakugaku Zasshi, 41: Cowan, M. M. (1999). Plant products as antimicrobial agents. Clin. Microbiol. Rev., 12: Decker, E. A., Chan, W. K. M., Livisay, S. A., Butterfield, D. A., and Faustman, C. (1995). J. Food Sci., 23: Draughon, F. A. (2004). Use of botanicals as biopreservatives in foods. Food Technol. Feat., 58: Georgii, A., and Korting, H. C. (1991). Antifungal susceptibility testing with dermatophytes. Mycoses., 21: Gupta, C., Garg, A. P., and Uniyal, R. C. (2008). Antibacterial activity of Amchur (dried pulp of unripe Mangifera indica) extracts on some food borne bacteria. J. Pharm. Res., 1: Irkin, R., and Korukluoglu, M. (2007). Control of Aspergillius niger with garlic, onion and leek extracts. Afr. J. Biotechnol., 6: Jabar, M. A., and Al-Mossawi, A. (2007). Susceptibility of some multiple resistant bacteria to garlic
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