Antibacterial Properties of Dry and Fresh Chile Pepper and Garlic

For thousands of years now different cultures across the globe have used a wide array of different spices for numerous reasons. Whether it's for flavoring and preserving food or medicinal purposes, different ethnic groups have incorporated everything from garlic to cinnamon into their diets (Zimet 2002). Within the last half-century, numerous scientists and researchers have been intrigued by the use of certain spices, especially for medicinal purposes. Studies of different cultures have revealed that specific spices were prescribed for nearly everything, from stomachaches to lethargy (Zimet 2002). Scientists hypothesized that certain spices may have antimicrobial properties that inhibit the growth of bacteria, specifically bacteria commonly found in the human gastrointestinal tract, both pathogenic and non-pathogenic. Kang et. al (1992) found that Perilla frustescens (shiso leaves) combined with polygodial, the active ingredient in Polygonum hydropiper (water-pepper), inhibits several different kinds of bacteria, fungi, and mold, especially Candida albicans and Mucor mucedo. Seaton (2007) also yielded similar results, with a combination of both green tea and ginger inhibiting the growth of Enterococcus faecalis, a Gram-positive gastrointestinal bacteria. Synder (1997) also analyzed the bacteria in which the spices were applied and determined that the spices caused inhibited the bacteria colonies.

We decided to design our own experiment using disk diffusion and base it on a question that we all found intriguing: Is bacteria inhibited more by fresh or dry spices? The chosen spices were fresh and dry variations of both garlic (Allium sativum) and chili pepper (Capsicum annuum). We chose these spices based on their use in several different cuisines, including American cuisine. The bacterium being tested is Bacillus megaterium, a nonpathogenic Gram-positive bacterium commonly found in soil. This specific bacterium is large in size and gets its energy from decaying organic matter. Additionally, Bacillus megaterium is particularly resistant to extreme heat and cold (Todor 2009). We hypothesized that the fresh garlic and chili would show a greater inhibiting effect on the bacteria than their dry counterparts.

Methods

Each different spice was applied to a separate section in a bacteria agar plate. Each dry and fresh spice was prepared separately and included fresh garlic, powdered dry garlic, fresh chili pepper, and ground dry cayenne pepper. Due to the methods used in the two previously mentioned studies, we extracted our ingredients using 95 percent ethanol. We mixed 0.1 grams of each spice with 0.5 mL of ethanol. In order to measure 0.1 grams of the fresh ingredients, they were chopped using a knife and cutting board and then powdered using a mortar and pestle. Each measured spice was added to a micro centrifuge tube with a sealable cap. After the spices were added to the containers, 0.5 mL of 95 percent ethanol was added. A control micro centrifuge tube was prepared with only ethanol. After all five containers were sealed, they were placed into a centrifuge and spun at 14,000 RPM for 45 seconds. By centrifuging the containers, the suspended solids from each ingredient separated from the desired liquid. After centrifuging, the five containers were placed in a 0-degree Celsius freezer for a 24-hour period. Following the removal on the capped containers from the freezer, they were placed in the centrifuge again for another 45 seconds at 14,000 RPM.

Five plates containing the Bacillus megaterium agar were prepared prior to beginning the experiment. Each plate was divided into four equally sized sections using a permanent marker on the bottom side of the plate. Each spice/control was tested five times in each separate dish; therefore the sections drawn on the plate was labeled with the corresponding spice or control, as well as a given number 1 through 5 as to keep track of the different samples of the same spice.

In order to apply the different spices to the agar plates, standard-sized hole-punched filter paper disks were cut out and placed into an autoclave. One at a time, 20 paper disks were placed on sterile foil to prepare them for the spice application. Using a pipette and sterile disposable tips, 30 microliters were extracted from each container. All five disks for each corresponding spice were applied with 30 micro-liters each. Each spice was applied with a new disposable tip. After the spices and control were applied to the paper disks, they were allowed to soak in and air dry for roughly 30 minutes. Following the drying, each disk was applied to its corresponding section on one of the agar test plates directly in the middle of one of the plate's sections using sterile tweezers. Once all disks were applied to their appropriate section in each individual plate (see Figure 1), the agar plates were sealed and placed into an incubator for 24 hours that was set at 37-degrees Celsius. After the 24-hour incubation period, the plates were removed from the incubator and were placed into a room that was 22 degrees Celsius. A dissecting microscope was used to examine the results of each spice disk on each plate and the zone of impact on each plate section was to be measured with a standard ruler.

Results

No noticeable or measurable results were yielded in our experiment. We examined our plates under a dissecting microscope every 24 hours for 96 hours. During each examination of the agar plates, the bacteria remained the same, neither overgrown nor inhibited by the presence of the spice-infused disks. Had the presence of the spice disks inhibited the bacteria, there would have been a measurable zone of impact around the disks in which there would be a ring of little or no bacteria present. Likewise, had the spices encouraged the growth of the bacteria, there would be a similar ring around the disks with a larger amount of bacteria present. Either results would have been visible using the dissecting microscope and due to the lack either, there were no results from the application of the spices and the ethanol control.

Discussion

We hypothesized that the fresh spices would have a greater zone of inhibition on the bacteria than their dry counterparts. Our lack of results caused us to reject our hypothesis. Due to our lack of any results, it is important to consider possibilities, changes, and errors that could have possibly impacted the results. We believe that there are several possible reasons why the bacterium neither grew nor was inhibited in the presence of the spices. The first possibility is the type of bacteria used. In both Kang et. al (1992) and Seaton (2007), the spices were applied to bacteria commonly found in the digestive system. However, Bacillus megaterium is a bacterium that is generally found in soil, not the human gastrointestinal tract (Todor 2009). Additionally, we believe that errors or changes in our experimental design could have impacted our lack of results. It's most likely that the step involving the incubation of the disks was the main cause for the lack of results. As a soil bacterium and one that feeds on decaying organic matter, Bacillus megaterium responds particularly well to warm temperatures. Warm temperature increases the rate of decay and amount of decayed material, which in turn impacts the bacteria, which consumes more and grows more (Todor 2009). Due to this property of the bacteria, we believe that incubating the disks at 37 degrees Celsius caused a growth of the Bacillus megaterium, and hence a possible resistance to the spice. This growth was possibly enough to nullify any visible or measurable effect that the spice could have had on the bacteria within the first 24 hours of application because the growth most likely could have covered any zone of inhibition. Even after the disks were taken out of the incubator into a room temperature climate for the remaining 72 hours, the first 24 hours of warm incubation and bacterial growth could have easily affected the results during the additional 72 hours. While these may be the two largest errors associated with the lack of measurable results, it is important to take all other errors into account when evaluating issues in the experiment that caused the results that occurred. We chose to use 95 percent ethanol as the solvent based on Seaton (2007) and Kang et. al (1992) which also used ethanol. Perhaps the ethanol impacted our results and using distilled de-ionized water could have yielded measurable and visible results. Non-sterility was a problem with the paper disks, which were taken out of a sterile environment several times. Many of the sterile instruments (including the tweezers, foil, paper disks) came in contact with non-sterile equipment.

It would be very beneficial to make changes to this experiment, namely changing the bacterium type or the incubation methods, and try to conduct it again. By changing the type of bacteria or the incubation/sterility methods, we believe measurable results would be much more likely to be obtained.

Works Cited

Kang, R., R. Helms, M.J. Stout, H. Jaber, C. Zhengqing, and T. Nakatsu. Antimicrobial activity of the volatile constituents of Perilla frutescens and its synergistic effects with polygodial. 1992. Journal of Agriculture and Food Chemistry 40(11): 2328-2330.

Seaton, G. 2007. The Synergistic and Individual Anti-microbial Impacts of Green Tea and Ginger on Common Gastro-Intestinal Bacteria. Portland Community College. http://www.pcc.edu/library/news/prize/seaton_graham.pdf

Snyder, O. 1997. Antimicrobial Effects of Spices and Herbs. Hospitality Institute of Technology and Management. http://www.hi-tm.com/Documents/Spices.html

Todor, K. The Genus Bacillus. 2009. Online Textbook of Bacteriology. http://www.textbookofbacteriology.net/Bacillus.html

Zimet, I. 2002. Use of Spices as Medicines. UCLA Louise M. Darling Biomedical Library.http://unitproj.library.ucla.edu/biomed/spice/index.cfm?spicefilename=SpicesAsMeds.txt&itemsuppress=yes&displayswitch=0