THE ANTIBIOTIC ACTIVITY OF VARIOUS NATURAL FOOD ITEMS
Hanan El and Austin Weld
Department of Biological Science
Saddleback College
Mission Viejo, CA, 92692
In recent years, antibiotic resistance in bacteria is on the rise. As a result, alternate methods to combat pathogenic bacteria must be examined. Cinnamon oil, garlic, roasted garlic, lemon oil, habanero, onion, and honey have been shown to have antibiotic activity and were compared in this study. Cinnamon oil was predicted to have the greatest antibiotic activity. The antibiotic activities of these food items were compared using the disk diffusion method with 50.0 µL of Staphylococcus aureus lawn spread onto trypic soy agar plates. Filter paper disks containing 10.0 µL of each item were added to the plates and the zone of inhibition was measured compared to water (explain zone of inhibition). The plates were then incubated for 48 hours at 37°(space)C. A significant difference was shown between the groups (p= 6.54 x 10-41, ANOVA and Bonferoni Correction). Cinnamon oil had a mean zone of inhibition of 16.12 ± 1.08 mm (±SEM, N=10), which was significantly greater than all other groups. Garlic had a mean zone of inhibition of 7.72 ± 0.24 mm (±SEM, N=10), which was significantly greater than all others except cinnamon oil and honey. Honey had a mean zone of inhibition of 9.96 ± 0.52 mm (±SEM, N=10), which was significantly greater than all others except cinnamon oil and garlic. Although previous literature suggested antibacterial activity in lemon oil, habanero, roasted garlic, and onion, no significant antibiotic activity was observed. This suggests a potential use of cinnamon oil, garlic, and honey as antibiotic agents.
Introduction
In recent years, the presence of antibiotic-resistance genes in bacteria is on the rise. Much of this can be attributed to misuse and over(space)prescription of antibiotics and antimicrobial agents, the use of antibiotics in farm animals to increase growth rate, and the ability of antibiotic resistant genes to travel long distances due to mass transportation (Lowy, 2003). Therefore, it is of interest to investigate new compounds and ways of controlling disease-causing bacteria. In addition, with an increasing public interest in natural products, naturally occurring compounds in common food items are of particular interest.
For centuries, essential oils have been used in alternate(alternative) medicine, and many have been shown to have antimicrobial effects. In the study conducted by Ott and Morris (2008), lemon oil was found to be one of the strongest of several antimicrobial oils. The mechanism by whichof citrus oils such as lemon oil acts is incompletely understood; however, it is believed that it is due to the aldehyde citral or lemonel, which has two stereoisomers: neral, the Z-isomer and geranial, the E-isomer(very wordy sentence). These compounds contain a carbonyl group (that) act as alkylating agents to bind cellular nucleophilic groups, rendering them inactive causing damage to bacterial cells (Ott and Morris 2008).
Geranial Neral
Another natural product, garlic (Allium sativum) is the most famous food with antimicrobial activity. In a study by Chikwem et. al. (2008), aqueous raw garlic extract was shown to have significant inhibitory effects against both Staphylococcus aureus and Salmonella typhimurium. However, interestingly, garlic extract which was heated had significantly reduced antimicrobial properties. Garlic’s inhibitory effect was brought about by a thiosulphinate molecule called allicin, which protects the garlic itself from bacterial attack as it grows. Allicin is capable of rapidly penetrating into cells through their cell membrane and attack the sulfur (SH) groups of enzymes and proteins to alter their function (Jonkers et al. 1998).
Allicin
Hot peppers from the Capsciumbb genus also have suggested antimicrobial properties. A study by Koffi-Nevry et. al. (2012) suggests that extracts from the fruits of the bell pepper plants Capsicum annuum and Capsicum frutescens were effective antimicrobials against Vibrio chlorea, Staphylococcus aureus, and Salmonella typhimurium. The chemical responsible for the antimicrobial properties of plants from the Capsicum genus is capsaicin. Furthermore, Wei et al. (2006) found that the concentration of capsaicin in the sample was shown to dramatically affect the antibacterial activity, with concentrations lower than 0.0125 mg/mL having no effect.
Capsaicin
A study by Elnima et al. (1983) tested both garlic and onion extracts against various gram-positive and gram-negative bacteria and found both to have antimicrobial effects. However, garlic was significantly stronger and a 10% garlic mouth wash solution was shown to reduce the number of oral bacteria. In onion, the antimicrobial activity was found to be mainly produced by a flavonoid called quercetin (Lee 2010).
Quercetin
Honey has also been shown to have antimicrobial properties. Interestingly, it was initially thought that the high sugar content may be responsible for these antimicrobial activities; however, when honey was compared against artificially constructed honey produced from a combination of sucrose, maltose, fructose, glucose, and deionized water, honey had a significantly higher antimicrobial affect than the artificial honey, suggesting the presence of a different antimicrobial compound causing the effect (Cooper et. al. 2002). These other compounds are mainly hydrogen peroxide, but the high osmolarity, phytochemicals, such as methylglyoxal were found to play a roll (Mandal and Mandal 2011).
Cinnamon oil and its active ingredient, cinnamaldehyde, have also been shown in several studies to have antimicrobial effects on the bacteria Escherichia coli and on various biofilms (de Oliveira et al. 2012 and Hong et al. 2013). The inhibitory effect of cinnamaldehyde on bacteria has been suggested to be due to its detrimental effect on oxidative phosphorylation, mainly involving the electron transport chain, preventing the production of ATP (de Oliveira et al. 2012).
Cinnamaldehyde
In this study, a variety of common, natural food items that have been shown in past studies to have antimicrobial products, including garlic, habanero (Capsicum chinense), onion, honey, lemon oil, and cinnamon oil, were surveyed by the disk diffusion method for their effectiveness on Staphylococcus aureus to determine which is the most effective. Cinnamon oil was expected to have the largest zone of inhibition due to a study by Chaundhari et al. (2012) which showed cinnamon oil to have the largest zone of inhibition out of nine different essential oils.
Materials and Methods
Garlic (Allium sativum), roasted garlic, habanero (Capsicum chinense), onion (Allium cepa), honey, lemon oil (Citrus Limon), cinnamon oil (Cinnamomum cassia), water, and Staphylococcus aureus were used in this study. Garlic, habanero, onion, honey, lemon oil, and cinnamon oil were purchased at Mothers Market in Irvine, CA. Twenty tryptic soy agar plates were made on Saturday, November 16, 2013 at 10:10 A.M. Extracts of garlic, roasted garlic, habanero, and onion were prepared at 10:00 P.M. in Hanan’s kitchen, the night before the study was conducsted. Two cloves of raw garlic and 2 mL of sterile deionized water were blended until it became a liquid solution. Two cloves of roasted garlic were prepared at 177°(space)C for 20 minutes, and the roasted garlic cloves and 2 mL of sterile deionized water were blended until it became a liquid. Two habanero peppers and 2 mL of sterile deionized water were blended until it became a liquid. A piece of onion and 2 mL of deionized water were blended until it became a liquid solution. Honey, lemon oil and cinnamon oil were already in a liquid state. Garlic extract, habanero extract, onion extract, honey, lemon oil and cinnamon oil were aseptically transferred into sterile test tubes.
The experiment began on Friday(,) November 22, 2013 at 10:30 A.M. Aseptic technique was used throughout the experiment. Each agar plate was divided into four sections. Twenty tryptic soy agar plates were lawn spread with Staphylococcus aureus. Sterile filter disks were placed onto each section of the agar plates for a total of 80 samples, 10 for each group. For the control, 10 µL of sterile deionized water was pipetted onto each sterile filter disk for a total of 10 samples. 10 µL of garlic extract, roasted garlic extract, habanero extract, onion extract, honey, lemon oil, and cinnamon oil were pipetted onto 10 different sterile filter disks each, for a total of 70 samples. All 20 agar plates were placed into a 37˚C incubator for 48 hours. A ruler was used to measure the zone of inhibition produced from each sample in millimeters.
Results
The mean zone of inhibition of cinnamon oil was 16.12 ± 1.08 mm (±SEM, N=10). The mean zone of inhibition of garlic was 7.72 ± 0.24 mm (±SEM, N=10). The mean zone of inhibition of roasted garlic was 0.00 ± 0.00 mm (±SEM, N=10). The mean zone of inhibition of honey was 9.96 ± 0.52 mm (±SEM, N=10). The mean zone of inhibition of lemon oil was 1.03 ± 0.12 mm (±SEM, N=10). The mean zone of inhibition of habanero was 0.40 ± 0.06 mm (±SEM, N=10). The mean zone of inhibition of onion was 0.00 ± 0.00 mm (±SEM, N=10). A significant difference was observed between the eight groups (p= 6.54 x 10-41, ANOVA and Bonferoni Correction). A post-hoc comparison showed cinnamon oil had a significantly larger zone of inhibition than all other groups. Garlic and Honey had a significantly greater zone of inhibition than all other groups except cinnamon oil; however, there was no significant difference observed between garlic and honey’s zone of inhibition. These data are shown in Figure 1.
Figure 1. Mean zone of inhibition radius of the water, cinnamon oil, honey, garlic, roasted garlic, lemon oil, habanero, and onion (n=10). There was a significant difference between the groups (p= 6.54 x 10-41). Cinnamon oil had a significantly larger zone of inhibition than all other groups. Honey had a significantly larger zone of inhibition than all groups except cinnamon oil and was not significantly different from garlic. Garlic had a significantly larger zone of inhibition than all groups except cinnamon oil, but was not significantly different form honey.
Discussion
The results of the experiment support the hypothesis that cinnamon oil will have the greatest zone of inhibition. Cinnamon oil had a significantly greater zone of inhibition than all other food items tested.
The results for cinnamon oil, garlic, and honey were consistent with previous studies that showed significant antibiotic activity in cinnamon (de Oliveira et al. 2012 and Hong et al. 2013), garlic (Chikwem et. al. 2008 and Jonkers et al. 1983), and honey (Cooper et. al. 2002 and Mandal and Mandal 2011). Honey had a greater zone of inhibition than garlic, but this was not significant (why was it not significant? What statistical test supported this result?).
However, contrary to past research, lemon oil, habanero, onion, and roasted garlic did not have significant antibacterial activity. Lemon oil had a slight zone of inhibition, but it was not significant, which is in opposition to the study by Ott and Morris (2008), which showed lemon oil to have significant antibiotic properties, even surpassing various other essential oils.
Surprisingly, habanero showed no significant antibacterial activity. This is inconsistent with Koffi-Nevry et. al. (2012) and Wei et al. (2006)’s studies. Koffi-Nevry et. al. (2012)’s study showed that different peppers have different capsaicin content. It is very possible that habanero may have a lower capsaicin content than other hot peppers. Also, since we added water to the mix, it may have caused the capsaicin content to be diluted below the minimum inhibitory concentration (MIC). Future research could be done using habanero peppers without water and producing a more concentrated solution.
Onion showed no zone of inhibition, which is in contrast to Elnima et al. (1983), which showed that, although onion was less effective than garlic, it still had significant antibacterial activity. The results were also in contrast to the study by Lee (2010) which suggested significant antibacterial activity. It is possible that adding water to the extraction mix also diluted onion past its MIC. Future experiments could use undiluted onion extract.
Our results for roasted garlic agree with the study by Chikwem et. al. (2008) that showed a significant reduction in antibiotic activity of roasted garlic versus raw garlic, however, our results did not show any significant antibiotic activities, which is in contrast to their study. It is possible that the water played a part, or the garlic may have been roasted for a longer period of time or at a different temperature, causing it to lose more of its antibiotic activity.
Because cinnamon oil, garlic, and honey showed significant antibacterial activity, there is a potential application of these items in antibiotics and antimicrobial agents. These compounds may have a use as inexpensive and effective mouthwashes to combat oral bacteria. They could have applications against antibiotic resistant strains of Staphylococcus aureus. For example, topical creams could be produced that contain these compounds to fight bacterial infections on wounds and staph infections. In modern times, with the increasing prevalence of antibiotic resistance, exploring alternative products with antibiotic properties is extremely important.
Literature Cited
Chaundhari, L. K. D., Jawale, B. A., Sharma, S., Sharma, H., Kumar, C.D.M., Kulkarni, P.A. 2012. Antimicrobial Activity of Commercially Available Essential Oils Against Streptococcus mutans. Vol. 13 (No. 1): p 71-74
Chikwem, A.J., Chikwem, J.O., Swinton, D.J. 2008. Aqueous Extraction of Dried and Fresh Garlic, and Comparative Antimicrobial Susceptibility Testing of Garlic Extracts on Selected Bacteria. Bios. Vol. 79 (No. 2): p 56-60.
Cooper, R.A., Molan, P.C., Harding, K.G. 2002. The Sensitivity to Honey of Gram-positive Cocci of Clinical Significance Isolated from Wounds. Journal of Applied Microbiology. Vol. 93. p 857-863.
Hong, Y., Bae, Y., Moon, B., Lee, S. 2013. Inhibitory Effect of Cinnamon Powder on Pathogen Growth in Laboratory Media and Oriental-Style Rice Cakes (Sulgidduk). Journal of food protection. Vol. 76 (No. 1): p 133-138.
Elnima, E.I., Ahmed, S.A., Mekkawi, A.G., Mossa, J.S. 1983. The Antimicrobial Activity of Garlic and Onion Extracts. Die Pharmazie. Vol. 38 (No. 11): p 747-748.
Jonkers, D., van den Broek, E., van Dooren, L., Thijs, C., Dorant, E., Hageman, G., Stobberingh, E. 1998. Antibacterial Effect of Garlic and Omeprazole on Heliobacter pylori. Journal of Antimicrobial Chemotherapy. Vol 43. (No. 6): p 837-839.