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Antimicrobial Activity of Garlic, Cinnamon, and Clove Against a Complex Mixture of Soil Bacteria

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Antimicrobial Activity of Garlic, Cinnamon, and Clove Against a Complex Mixture of Soil Bacteria RESEARCH ARTICLE Antimicrobial Activity of Garlic, Cinnamon, and Clove Against a Complex Mixture of Soil Bacteria Hind Benkerroum*, Kajal Patel Dawson college 3040 Rue Sherbrooke O, Montreal, QC H3Z 1A4, Canada *Corresponding author: Hind Benkerroum, email: [email protected] Abstract: Open Access Article Citation: Benkerroum H., Patel K. (2018) The antimicrobial effect of garlic, cinnamon, and clove was tested against a Antimicrobial Activity of Garlic, Cinnamon, suspension of soil microorganisms by the disk method. The results showed that and Clove Against a Complex Mixture of garlic was the most inhibitory to soil microorganisms followed by cinnamon, as Soil Bacteria. ESR Journal 3(1) the mean diameters (cm) of their inhibition zones were 4.2 (± 0.6) and 2.6 (± 0.5), respectively. While clove extract did not inhibit the microbial growth. No inhibition was observed with any of the spices or garlic extracts obtained with Received: 19th February 2017 boiling water. These results suggest that the antimicrobial effect of these spices would be significantly reduced when used in cooked foods. The results also Accepted: 22nd January 2018 suggest that garlic would be a good candidate to use in the preservation of foods exposed to contamination with microorganisms from various sources. Published: 29th January 2018 Copyright: © 2018 This is an open Keywords: garlic, clove, cinnamon, antimicrobial, soil microorganisms access article under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Data Availability Statement: It has long been known that spices and herbs, and their extracts inhibit Funding: The authors received no pathogens and spoilage bacteria, and have thus been extensively used since specific funding for this work ancient generations around the world in the preservation of traditional foods (1) Competing Interests: The authors have as well as to cure infectious diseases (2). However, some spices preserve food declared that no competing interests exist. longer or more efficiently than others, and inhibit more effectively one group of microorganisms than another (3). Many studies have related the antimicrobial effects of herb and spice extracts to the presence of an array of active substances (active principles) in their composition. These substances may be volatiles, lipo- soluble, or hydro-soluble compounds (4, 5). Depending on the number and nature of active principles of a given spice or herb, it can have narrow, large spectrum of inhibition, or may even have antiseptic effect (6). The purposes of this this study were: (i) to demonstrate the antimicrobial activity of garlic, cinnamon, and clove against a complex mixture of soil microorganisms with unknown composition for a potential application in food preservation and safety, and (ii) to determine whether the temperature (boiling/cooking) would affect the putative inhibitory activity of the items tested. ESR Journal - 29th January 2018 1 ESR Journal Antimicrobial Activity of Garlic, Cinnamon, and Clove Against a Complex Mixture of Soil Bacteria Materials and Methods Preparation of extracts of spices Garlic and spices were studied for their antimicrobial effects against a complex microbial suspension obtained from soil. Garlic and two spices, cloves and cinnamon, were purchased from the supermarket in Montreal (Canada). The antimicrobial effect of spices was tested on two types of water extracts (10 g in 40 mL water): (i) extracts obtained by boiling each spice for 5 min and allowing it to cool at room temperature for 2 h before testing; this was named “hot extract”, and (ii) by soaking each spice in cold water for 24 h before testing; this was named “cold extract”. An antibiotic solution of 125 mg/mL in tap water was prepared to serve as a positive control. The antibiotic used was Ciprofloxacine (AresTM) known for its inhibitory effect against a large spectrum of bacteria. Preparation of the microbial suspension Soil (10 g) was taken from the garden, suspended in 100 mL of tap water and filtered through a cellulose paper filter. This microbial suspension was supplemented with sucrose (10% w/v) and left at room temperature overnight to be enriched with bacteria. Preparation of microbial lawn on a Trypticase Soy Agar (TSA) plate A volume (300 μl) of the overnight microbial soil suspension was spread evenly onto sterile TSA (OxoidTM, Hampshire, UK) plate with cotton swab, and left to dry at room temperature for about 10 min. This lawn of soil microbiota was used as an indicator of the inhibitory activity. Antimicrobial activity testing To test the antimicrobial activity of garlic, the spices and the antibiotic, blotting paper disks of about 1 cm diameter were soaked in each spice extract or antibiotic solution, and placed onto the lawn of inoculated TSA plates. Preliminary tests showed that water extract of garlic gave no inhibition by this technique. Therefore, garlic was finely crushed in a mortar and a sample (∼ 0.5 g) was directly placed as a spot on the plate instead of the soaked disks. The plate was divided into four quarters on each of which was placed a disk or a spot of garlic. Inoculated plates were then incubated at room temperature (~20°C) and inspected daily the following 7 days for the development of inhibition zones. The diameters of the inhibition zones were measured after 48h of incubation. Statistical analysis Data are means of triplicate determinations for each spice, statistical analysis was done by the analysis of variance (ANOVA) and student t-test for α = 5% using StatPlusTM software. The spices and the antibiotic used were considered as the independent variables whereas the microbial growth was the dependent variable. ESR Journal - 29th January 2018 2 ESR Journal Antimicrobial Activity of Garlic, Cinnamon, and Clove Against a Complex Mixture of Soil Bacteria Results and Discussion Results of the antimicrobial tests showed that the microbial suspension of soil was inhibited to different extents by garlic and cinnamon cold extract as well as by the antibiotic solution used as a control. Garlic was significantly (p<0.05) more inhibitory than cinnamon, while neither clove nor the hot extracts of spices exhibit inhibitory activity (Figures 2A and Table 1). Significant differences (p<0.05) were observed in the extent of inhibition between crushed garlic and cinnamon cold extract with inhibition zone mean diameters of 4.2 (± 0.6) (0.5) and 2.6 (± 0.5) cm, respectively (Table 1). Extension of the incubation for 7 days showed growth of molds within the zone of inhibition of the antibiotic, starting from day 5. No such growth was observed in the zones of inhibition produced by garlic and cinnamon (Figure 2B). The growth of molds within the inhibition zone of the antibiotic and not within those of the garlic and cinnamon indicates that the latter plant extracts exert a potent antifungal activity, as has been demonstrated earlier (7, 8). In contrast, the intensive mold growth in the antibiotic zone of inhibition is not surprising since antibiotic activity is known to be restricted to bacteria. Therefore, these results indicate that garlic has an antiseptic effect and would be worthwhile to investigate as a source for natural food preservatives, which in addition to its large spectrum of action, may possess health promoting effects. Figure 1: TSB plates showing the inhibition of antibiotic (1), clove (2) and cinnamon (3) extracts, and garlic (4) against a lawn of soil microorganisms, after 48 hours (A), and 7 days (B) of incubation at room temperature. The inhibitory activity of spices and garlic against various microorganisms including viruses, bacteria, fungi, and parasitic protozoa has been extensively demonstrated in vitro (6, 9) and in food systems (10, 11). However, the spectrum of inhibition varies greatly among spices, and even for the same spice depending on geographical regions, period of the year, and environmental conditions (12). ESR Journal - 29th January 2018 3 ESR Journal Antimicrobial Activity of Garlic, Cinnamon, and Clove Against a Complex Mixture of Soil Bacteria This study confirms such an inhibitory effect against a complex mixture of soil microorganisms, with garlic being the most effective followed by the cinnamon cold extract (Figures 1A and Table 1). The failure of the hot extracts of spices to inhibit the microbial growth suggests that the heat treatment applied (Boiling for 5 min) during extraction destroys, at least partially, the active principles of these spices. However, the absence of inhibition by clove extracts was unexpected since the antimicrobial activity of this spice against bacteria, molds, and yeasts is well established (8, 13-17). This may be explained by the presence of resistant strains among the mixture soil microorganisms used as an indicator lawn in this study, which may have grown and have hindered the inhibition of the sensitive strains of the mixture. Therefore, the use of such a complex inoculum as an indicator would allow to detect only for amicrobial substances/preparations of the largest possible spectrum of action, and hence the best candidates for the application in food safety and keeping quality. Indeed, contrary to our findings, most of the studies on the antimicrobial activity of plant extracts were conducted on pure cultures (11, 16). The extraction of active principles in cold water may also account for the discrepancies with previous studies where clove extracts were obtained by using other techniques and solvents. This makes a difference in the nature and the inhibitory spectrum of the active principles in each extract. For example, Mau et al. (11) showed that ethanol extract of cinnamon had enhanced antimicrobial activity against various bacterial pathogens, yeasts, and molds and was stable to heat and pH, and one fraction was identified to be the main active. The lack of this fraction in any cinnamon extract may result in the reduction or the absence of antimicrobial activity, especially if it is challenged against a complex microbial culture, as in the present study.
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