Study on the Antimicrobial activity and Minimum Inhibitory Concentration of Essential Oils of Spices

Antibacterial activity and minimum inhibitory concentration (MIC) of essential oils of garlic, clove and cinnamon were estimated by using various bacterial pathogens. Among the bacterial pathogens tested against essential oil of garlic, Staphylococcus aureus was found to be highly sensitive followed by E.coli. L.monocytogenes and S.pyogenes were found to be less sensitive. The essential oil of clove was found to be most active against S.aureus followed by E. coli. B.cereus and C. jejuni. The essential oil of cinnamon was also most active against S.aureus followed by E.coli and C.jejuni. Essential oil of cinnamon was found to be active against all the bacterial pathogens tested, when compared to garlic and clove oils. However Staph. aureus, E. coli and C.jejuni were found to be most sensitive to the action of essential oils of garlic, clove and cinnamon. Among the bacterial pathogens tested against essential oils of spices to know the MIC by agar diffusion method, C.jejuni was found to be most sensitive to the essential oil of garlic followed by E.coli, S. typhimurium and Staphylococcus aureus. L. monocytogenes and Methicillin resistant Staph. aureus were found to be comparatively less sensitive. Essential oil of clove was also found to be highly effective against C.jejuni followed by E.coli, S.typhimurium and S.aureus. Again L.monocytogenes and Methicillin resistant S.aureus were comparatively less sensitive to the action of essential oil of clove. All most all the bacterial pathogens tested were found to be sensitive to the essential oil of cinnamon. However C.jejuni and E.coli were found to be most sensitive followed by S.typhimurium, Staph. aureus and Methicillin resistant Staph. aureus .


Introduction
foods rather than extending shelf-life.In recent years antimicrobial properties of spices have been Due to the awareness among the public now a documented and interest continued to the present.days, major importance has been given for meat There is little information available emphasizing the preservation which has been characterized for preservative and antimicrobial role of spices in the microbiologically stable meat.Recently there has prevention of meats.been an increasing interest in discovering new natural antimicrobial substances or bio preservatives for the Materials and methods preservation of meat and meat products.Bio The essential oils of spices viz., garlic, clove and preservatives include a range of natural products from cinnamon were obtained from M/S Plant lipids Ltd, plants, animals and microorganisms which can be Cochin, Kerala.The reference strains of Escherichia used to improve the keeping quality of foods.Being coli (MTCC 452), Staphylococcus aureus (MTCC plant natural food stuffs, spices appear to be an 3103) and Salmonella typhimurium (MTCC 1251) alternative for the chemical antimicrobials to the were obtained from the Institute of Microbial consumers who tend to question their safety.Spices Technology (IMTECH), Chandigarh.

active compounds have been included in class of
The reference strains of Bacillus cereus, naturally occurring food preservatives and their Campylobacter jejuni and Listeria monocytogenes inclusion in foods allowed by food production were procured from the Department of Microbiology, regulatory offices.Although, spices have been well known for their medicinal, preservative and Christian Medical College, Vellore, Tamilnadu.The antioxidant properties, currently they have been used reference strains of Streptococcus pyogenes and Methicillin resistant Staphylococcus aureus were with primary purpose of enhancing the flavour of obtained from the Department of Veterinary Public classified by the diameter of the inhibition zone as per Health, College of Veterinary Science, Rajendranagar, the procedure of Ponce et al (2003)  Remi R8 ) at 8000 rpm for 10 minutes.The essential oils of spices.In to 100 ml of sterile Tryptic supernatant was discarded and the pellet was mixed in 0 soy agar which was maintaining at 45 C, 1 ml of the sterile normal saline and centrifuged at 8000 rpm for prepared bacterial culture was added to give a final 10 minutes.Then the cells were washed twice with 7 concentration of 10 cells / ml in the medium.The normal saline and the concentration of the cells was culture was thoroughly mixed in the appropriate matched to the Nephelometer tube no 4 which gave a 9 artificial medium and the plates were poured, allowed cell concentration of 10 cells / ml of culture.This to solidify and placed in a refrigerator for 10 minutes.culture was used as inoculum for the seeded plates in Holes were then punched into the agar to create wells, determining the antimicrobial activity and minimum into which measured volume of 100µl of the essential inhibitory concentration of essential oils of spices.
oil of spices were placed after sealing the bottom of the Dilution of the essential oils of spices: All the well with sterile agar of 1%.This ensured that radial essential oils of spices were diluted in Diethyl ether.
diffusion from the well gave a clear and easily Dilutions were made from 1: 50 to 1:1300.All measured zone of inhibition.The plates were then concentrations of the essential oils were prepared a 0 0 incubated at 37 C for 24 hours.The inhibition zones day prior to use and stored at 4 C.
were measured after the incubation period.This assay Test to determine the antimicrobial activity: The was carried out in duplicate with two replicates on disc diffusion method was used to determine the each occasion.antimicrobial activity of the essential oils of spices.9 The volume of 0.1ml ( approximately 10 cells / ml ) of

Results and Discussion
the tested microorganisms grown in liquid growth 0 Antibacterial activity of essential oils of garlic, media at 37 C was inoculated on Muller-Hinton clove and cinnamon: The mean + SE values of the growth media and then spread on the entire surface of antibacterial activity of essential oils of garlic, clove the Petri dish using a sterile swab.Then sterile paper and cinnamon are presented in Table 1.discs (Whatman 1.6 mm) with 30 µl absorbed essential The results obtained in this study indicated that oil of spices were placed on to the Muller -Hinton agar the bacterial pathogens tested against the essential oil by pressing gently.The plates were incubated at 35 + 0 of garlic, Staphylococcus aureus was found to be 1 C for 48 hours.After the incubation period the highly sensitive to its action followed by Escherichia inhibition zones around the paper discs were measured in millimeters.The sensitivity of the individual oil was coli, Listeria monocytogenes, Bacillus cereus, Campylobacter jejuni, Methicillin resistant Streptococcus pyogenes, Campylobacter jejuni and Staphylococcus aureus, Salmonella typhimurium and Bacillus cereus.The antibacterial activity of essential Streptococcus pyogenes.The antibacterial activity of oil of clove may be attributed to eugenol, the essential oil of garlic may be attributed to allicin the antimicrobial compound of clove.The microbial antimicrobial component of garlic.The broad inhibition of eugenol might be related to either spectrum antimicrobial activity of allicin and its membrane disruption or according to Wendakoon and Sakaguchi (1995) Among the bacterial pathogens tested against the Escherichia coli and Staphylococcus aureus were essential oil of cinnamon, the results revealed that sensitive to garlic juice.
Staphylococcus aureus was found to be highly Among the bacterial pathogens tested against the sensitive to its action followed by Escherichia coli, essential oil of clove, the results indicated that Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus was found to be highly Salmonella typhimurium, Methicillin resistant sensitive to its action followed by + : 08 -09 mm, ++ : 10 -13 mm, +++ : 14 -16 mm oil of cinnamon may be attributed to cinnamaldehyde, One cluster consists of Campylobacter jejuni, the antimicrobial compound of cinnamon.Escherichia coli, Salmonella typhimurium and Cinnamaldehyde exhibits its antibacterial activity due Staphylococcus aureus, which were more sensitive to the action of essential oil of garlic.The second cluster to its lipophilicity of terpenoids and phenyl comprising of Bacillus cereus, Listeria monocytopropanoids, which can penetrate the membrane and genes, Streptococcus pyogenes and Methicillin reach the inner part of the cell and impair bacterial resistant Staphylococcus aureus which were enzyme system ( Helander et al. 1998 ).
comparatively less sensitive to the action of essential The results of this study were in consistent with oil of garlic.The microbial inhibitory action of the findings of Bowels et al. (1995)

Test to determine the minimum inhibitory
, by inactivation of enzymes and associated molecule ajoene appears to be due to the genetic material.multiple inhibitory effects on various thiol dependant The results of this study were in agreement with enzymatic systems such as, alcohol dehydrogenase, Matan et al. (2006), who reported complete inhibition thioredoxin reductase and RNA polymerase which can of Staphylococcus aureus by application of essential affect essential metabolism of cysteine proteinase oil of clove, Smith-Palmer et al. (2004), who reported activity involved in virulence of the pathogen (Ankri the growth inhibition of Staphylococcus aureus at sub and Mirelman, 1999).inhibitory concentrations of clove oil, Moreira et al.The results obtained from this study were (2005), who observed the significant bactericidal comparable with the results of El-Khatib and Abdel action of clove against Escherichia coli, Blaszyk and

Table - 2: Minimum inhibitory concentration of various concentrations of essential oil of garlic on the growth of bacterial pathogens Bacterial pathogens Various concentrations of essential oil of garlic
Study on the Antimicrobial activity and Minimum Inhibitory Concentration of Essential Oils of Spices

Minimum inhibitory concentration of essential oil of garlic:
who reported that essential oil of garlic may be due to its antimicrobial cinnamon oil inhibited the growth of Staphylococcus compound, allicin and the effect will be brought about aureus, Helander et al. (1998) who observed the by its ability to inactivate proteins (Barone and Tansey, inhibition of Escherichia coli O157:H7 and 1977) and also by inhibition of RNA synthesis Salmonella typhimurium by the essential oil of (Feldberg et al. 1988).cinnamon, Friedman et al. (2002) who found that Many scientists have tested the efficacy of garlic essential oil of cinnamon was active against preparations in inhibiting different bacterial Campylobacter jejuni and Escherichia coli, Kwon et pathogens and reported varying results depending al. (2003) who reported that Bacillus cereus was most upon the microorganisms tested in their study.The sensitive to cinnamic aldehyde and Hoque et al.Escherichia coli, Salmonella salmonella and Escherichia coli O157:H7 counts and typhimurium and Staphylococcus aureus with a a slow inactivation of Listeria monocytogenes in minimum inhibitory concentration of 1:1000.Listeria garlic butter.In another study Yin and Cheng.(2003) monocytogenes is comparatively less sensitive to found that Salmonella typhimurium was much essential oil of garlic with a minimum inhibitory sensitive to the antimicrobial action of garlic derived concentration of 1:150.Among the two strains of organosulfur compounds, similarly Mousumi Banarjee Staphylococcus, Staphylococcus aureus was more and Prabir Sarkar (2004) reported that aqueous extract sensitive (1:1000) than the Methicillin resistant of garlic was bacteriostat against Staphylococcus Staphylococcus aureus (1:700).Depending upon the aureus, Escherichia coli and Salmonella typhimurium.minimum inhibitory concentrations two major In a recent study Noori Al-Waili et al (2007) reported clusters of human bacterial pathogens could be that Escherichia coli and Staphylococcus aureus were observed.more sensitive to garlic juice.Study on the Antimicrobial activity and Minimum Inhibitory Concentration of Essential Oils of Spices www.veterinaryworld.orgVeterinary World, Vol.4 No.7 July 2011 314

Table 3 : Minimum inhibitory concentration of various concentrations of essential oil of clove on the growth of bacterial pathogens Bacterial pathogens Various concentrations of essential oil of garlic
: 08 -09 mm, ++ : 10 -13 mm, +++ : 14 -16 mm +