Antimicrobial Activity of a Blend of Essential Oils Extracted from Oregano and Cinnamon

The antimicrobial property of essential oils extracted from oregano and cinnamon have been documented and widely reviewed. However, most of the in vitro studies determined the inhibitory activity based on a single essential oil and/or purified active compound. The aim of this study was to determine the overall inhibitory activity in terms of minimum inhibitory concentration (MIC) of a blend of oregano and cinnamon essential oils in a commercial product against pathogenic bacteria. The result showed that the blend of the essential oils inhibited Escherichia coli, Escherichia coli O157:H7, Salmonella enteritidis, Salmonella typhimurium, Vibrio cholerae, and Vibrio parahaemolyticus at MICs ranging from 31.25 to 250 mg/L. The MICs of the examined blend against Bacillus cereus, Campylobacter jejuni, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus were greater than 1,000 mg/L. This finding suggests that the examined blend of essential oils could, to some extent, inhibit Escherichia coli, Salmonella, and Vibrio, and could be therefore applicable for use as a bacteriostatic agent.

anti-inflammatory, and antimicrobial that positively affects livestock growth [2].Essential oil of medicinal plants exerts intense antimicrobial activity against pathogenic bacteria [2].In vitro inhibition of bacteria development by the essential oils has been extensively studied, and their antimicrobial activities have been confirmed [2][3][4][5].
Oregano and cinnamon have been used as growth promoting agents in livestock industry as they are among the medicinal plants well-known for their antimicrobial properties against animal pathogenic bacteria [1].Essential oils of these plants are mainly responsible for this activity.Carvacrol and thymol in oregano, and cinnamaldehyde in cinnamon are the major active compounds in the essential oil of these plants [6].Inhibitory activity in terms of minimum inhibitory concentration (MIC) of carvacrol, thymol and cinnamaldehyde against pathogenic bacteria such as Escherichia coli, Salmonella, Staphylococcus, Vibrio, etc., have been extensively reported [3][4][5][6].Other compounds in the extracted essential oils of oregano and cinnamon have also been found to possess antimicrobial activity though these compounds are not the major components of the extract [7][8][9][10][11][12].However, these studies determined the inhibitory activity based on a single essential oil and/or purified active compound, which may not represent the actual inhibitory activity of the essential oils in the commercial products.This study aimed to determine the inhibitory activity of a commercial product containing a blend of essential oils extracted from oregano and cinnamon to examine if the inhibitory profile against pathogenic bacteria is similar to that of the previous literature.

Essential oils blend
The blend of essential oils used in this study was a commercial feed additive product for livestock (Orecim; Zagro Singapore Pte Ltd., Singapore).The product contained 3.75% of oregano and 2.5% of cinnamon essential oils.These two essential oils provided three active compounds; carvacrol 3%, cinnamaldehyde 2% and thymol 0.06%.The working essential

Background
Medicinal plants have been widely used as growth promoting agents in livestock production industry [1] as they possess a wide range of biological activities such as antioxidative,

Journal of Animal Science and Research
Open Access Journal oil solution was prepared by diluting the product 1:1000 (according to manufacturer's recommendation) with sterile deionized water.The working essential oil solution was then used for MIC determination against pathogenic bacteria.

Tested microbes
We determined the inhibitory activity of the examined blend of essential oils against nine strains of Gram negative and two strains of Gram positive bacteria.The bacterial names and their American Type Culture Collection (ATCC) reference numbers are listed in table 1.

MIC determination
The MIC was determined according to the broth micro dilution method described in the Fourth Edition of Clinical and Laboratory Standard Institute (CLSI) VET01-A4 [13].In brief, the working essential oil solution was added to the 96well plate, followed by two-fold dilution using sterile water.Bacterial suspension containing approximately 5 × 10 5 CFU/ mL was prepared and added to the plates.The plates were sealed with plastic tape to prevent drying and then incubated at 37 °C for 24 hours.After the incubation was over, the plates were observed for bacterial growth inhibition.The MIC value was the concentration value of the examined blend of essential oils in the well that completely inhibited bacterial growth.

Results and Discussion
In this study, we determined the MIC of a blend of oregano and cinnamon essential oils against 11 pathogenic bacteria by taking into account major and minor active compounds as well as other substances such as excipients in the commercial product.The MICs of the examined blend of essential oils are shown in table 1.The MIC range varied; 31.25 mg/L for Vibrio parahemolyticus; 125 mg/L for Vibrio cholerae, Salmonella enteritidis and Escherichia coli O157:H7; 250 mg/L for Escherichia coli and Salmonella typhimurium; and >1000 mg/L for Bacillus cereus, Campylobacter jejuni, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus.
The MICs for Escherichia coli, Salmonella and Vibrio found in this study are consistent with previous reports [14].However, high MICs of Bacillus, Campylobacter, Klebsiella, Pseudomonas and Staphylococcus in this study indicated that these bacteria were resistant to the blend of oregano and cinnamon essential oils, finding that is inconsistent with previous reports [15][16][17].We speculate that high MICs of these bacteria were mainly due to the high dilution of working essential oils solution used for MIC determination.After 1:1000 dilution according to manufacturer's recommendation, the working essential oil solution contained only 0.00375% or 37.5 ppm oregano oil and 0.0025% or 25 ppm cinnamon oil concentration that are possibly not sufficient to effectively inhibit growth of these bacteria.
However, high dilution of working essential oils solution did not explain the resistance of Staphylococcus aureus in this study.Staphylococcus aureus is usually used as a representative of Gram positive bacteria, while Escherichia coli is used as a representative of Gram negative bacteria in MIC determination of essential oils [5,18].It is widely accepted that Gram positive are generally more sensitive to essential oils than Gram negative bacteria [19].Nevertheless, the MICs of these two representatives in this study showed the opposite trend since Escherichia coli was inhibited at 125 mg/L while Staphylococcus aureus was not inhibited at 1000 mg/L.This is not uncommon since inconsistent results of the in vitro MIC of essential oils against pathogenic bacteria have been already reported [20].The reported MICs vary among experiment because there are differences in species and parts of plant used for essential oils extraction, and other laboratory test conditions that prevent direct comparison of the MICs and inhibition patterns.However, we presume that these Gram positive and Gram negative bacteria would show similar inhibition patterns as previous reports when the working solution of essential oils blend is used at higher concentration.Mild in vitro inhibitory activity of the essential oils blend suggests that this type of product might be applicable for use as bacteriostatic agent that could be beneficial to livestock and aquaculture industries, where new sources of feed additives have been sought to replace antibiotic growth promoters.Essential oils might contribute in the control of Escherichia coli load in piglet gut during weaning period and alleviate diarrhea severity and mortality rate of the piglet.Essential oils might also help limit colonization of Salmonella in poultry gut, which could reduce shedding of Salmonella to the environment as poultry are considered one of the most important Salmonella reservoirs.Finally, essential oils might help control Vibrio load in shrimp's hepatopancreas and gut, where Vibrio colonizes, disrupts nutrient absorption, and causes cell death and tissue necrosis.Further efficacy trials in these animals would prove the economic benefits of essential oils blend for livestock and aquaculture industries.

Conclusion
This study determined the MIC values of essential oil blend against pathogenic bacteria.The in vitro inhibitory activity observed was the overall activity of carvacrol, thymol,

Table 1 :
The MICs of the examined blend of oregano and cinnamon essential oils extracted.