STUDIES ON THE ANTIMICROBIAL EFFECTIVENESS OF ESSENTIAL OILS OF GARLIC, CLOVE AND CINNAMON ON Staphylococcus aureus IN CHICKEN MEAT PATTIES A. Jagadeesh Babu 1, A. Rupasundari 2, B. Sankar Reddy 3 and M. Sravanthi 4 College of Veterinary Science, Tirupati - 517 502, (A.P.) Chicken meat patties were treated with essential oils of garlic, clove and cinnamon separately and they were inoculated with Staphylococcus aureus (MTCC 3103) and stored at refrigeration temperature (5±10C) to study the effect of essential oils of spices on Staphylococcus aureus (MTCC 3103). The results revealed that essential oil of garlic in and reduced the counts up to day 4 and day 2 of storage and then gradual increase was there in all the three treatments. Chicken meat patties with essential oil of clove revealed that in and even though there was an increase in counts they were less than the counts of day 0 up to day 14 and day 10 of storage respectively. In no significant (p<0.05) difference could be observed up to day 6 of storage and essential oil of cinnamon revealed that in and even though there was an increase in counts up to day 14 of storage, they were less than the counts that were registered on day 0 of storage. In the counts were decreased on day 2 of storage after that gradual increase was observed up to day 14. Key words: Essential oils of garlic, clove and cinnamon, chicken meat patties, Enterotoxin production is generally associated with coagulase positive Staphylococci and nearly all cases of Staphylo coccal food poisoning are caused by World wide each year, food poisoning kills more than one million people. A recap of several notable recent food poisoning outbreaks illustrated the diverse and sometimes convoluted pathways to food borne illness and demonstrated the need for constant vigilance by both individuals and the food industry. Addition of antimicrobial chemical preservatives can better protect the meat from microorganisms. Because of the awareness among the consumers, they are preferring the meat with out any chemical preservatives. Now a it has been proven that the best alternative for the chemical preservatives are the essential oils of spices, which can stabilize the meat from microbial deterioration. Several workers studied the antimicrobial effect of essential oils of garlic, clove and cinnamon on Hence, in the present investigation an attempt was made to study the antimicrobial effectiveness and public health safety of essential oils of garlic, clove and cinnamon on 1, 2 & 3 Department of Veterinary Public Health 4 Department of Biochemistry 45
The birds were procured from the local market, slaughtered and dressed conventionally in the mini slaughter house of the department of Livestock Products Technology by adopting all the hygienic precautions. Chicken meat mince was prepared by following all the aseptic precautions. To test for a particular essential oil of spices, meat mince was divided into four groups, one for the control and the remaining three for the essential oil of particular spice at three different concentrations. The essential oils of garlic, clove and cinnamon were tested at a concentration of 1:250, 1:500 and 1:1000 and were added to meat at 2% level. The reference strain of Staphylococcus aureus (MTCC 3103) was obtained from the Institute of Microbial Technology (IMTECH), Chandigarh. The culture was maintained at 40C in Brain Heart Infusion broth and was tested for purity, morphology and biochemical characters for every 15. The bacterial pathogen Staphylococcus aureus was inoculated into Brain Heart Infusion broth and incubated at 37 0 C overnight. The inoculum was then centrifuged (Remi Model: Remi R8 ) at 8000 rpm for 10 minutes. The supernatant was discarded and the pellet was mixed in sterile normal saline and centrifuged at 8000 rpm for 10 minutes. Then the cells were washed twice with normal saline and the concentration of the cells was matched to the Nephelo met er tube no 4 which gave a cell concentration of 10 9 cells / ml of culture. This culture was used as inoculum for the seeded plates in determining the antimicrobial activity and minimum inhibitory concentration of essential oils of spices. All the essential oils of spices were diluted in Diethyl ether. Dilutions were made from 1: 50 to 1:1300. All concentrations of the essential oils were prepared a day prior to use and stored at 40C. The disc diffusion method was used to determine the antimicrobial activity of the essential oils of spices. The sensitivity of the individual oil was classified by the diameter of the inhibition zone as per the procedure of Ponce et al (2003) and 46 Jagadeesh Babu et.al., Moreira et. Al., (2005). The experiment was repeated in duplicate for all of the test strains. As per the procedure followed by Moreira et al.,( 2005 ) agar diffusion assay was used to determine minimum inhibitory concentration of the essential oils of spices. Challenging studies of the chicken meat patties with essential oils of garlic, clove and cinnamon were carried out with Staphylococcus aureus (MTCC 3103) as per the protocol of Fadia Naim et al (2004) with little modifications. Statistical analysis of the data was carried out as per the SPSS (Version 10.0) software. Staphylococcus aureus count of chicken meat patties treated with essential oil of garlic revealed significant (P<0.05) difference when compared to control. In the counts were decreased up to day 4 of storage after that both and there was a gradual increase in counts was observed but no significant (P<0.05) difference could be found up to day 8 of storage. In the counts were decreased on day 2 of storage after that a gradual increase was found up to day 14 of storage. The decreased Staphylococcus aureus count might be due to allicin the antimicrobial compound of garlic. The result of this study was in accordance with an early study of Fletcher et. al., (1 974) who fou nd reduced growth of Staphylococcus aureus in nutrient broth with garlic extract, Kumar and Berwal (1998) who observed inhibition of Staphylococcus aureus growth by garlic, Mei-Chin Yin and Wen-Shen Cheng (2002) revealed that organosulfur compounds of garlic reduced the growth of Staphylococcus aureus, Tassema et. al., (2003) reported the inhibition of Staphylococcus aureus by crude garlic extract, Shokrazadeh and Ebadi (2006) reported that garlic extract had concentration dependant antibacterial activity against Staphylococcus aureus, Noori Al- Waili (2007) revealed that Staphylococcus aureus was found to be sensitive to garlic juice and the antimicrobial activity was influenced by storage and heating.
Studies on the... With essential oil of clove Staphylococcus aureus counts were significantly (P<0.05) different from control. In and even though there was an increase in counts they were less than the counts of day 0, up to day 14 of storage and day 10 of storage respectively. In no significant (P<0.05) difference could be observed up to day 6 of storage even though there was an increase in counts as the st orage perio d advances. The decreased Staphylococcus aureus count might be due to eugenol, the antimicrobial compound of clove. The results of this study were in agreement with Hoque et al., (2007) who reported the essential oil of clove at 5 percent had highest antimicrobial activity against Staphylococcus aureus and Abdel Monein Sulieman et al., (2007) revealed that essential oil of clove had a potential antimicrobial activity against In chicken meat patties treated with essential oil of cinnamon, the Staphylococcus aureus counts were significantly (P<0.05) different from control. In and even though there was an increase in counts up to day 14 of storage, they were less than the counts that were registered on day 0 of storage. In the counts were decreased on day 2 of storage after that gradual increase was observed up to day 14 of storage but no significant (P<0.05) difference could be found up to day 8 of storage. The decreased Staphylococcus aureus co unt may be due to ci nnamal dehyde t he antimicrobial compound of cinnamon. The result of this study was similar to the findings of Hoque et al., (2007) who found that essential oil of cinnamon at 10 percent concent rati on showed highest antimicrobial effect and Sema agaoglu et al., (2007) reported that diethyl ether treated cinnamon extract was found to be most effecti ve against During the refrigeration storage of the samples there were significant reduction in counts upto 4 in all the treatments and control groups. After that, the count increased in all the groups as the storage period advances, but the counts did not exceed the original count (i.e. count before storage) in some of the treatment groups. Where as in all the control groups the counts exceeded the original counts during the storage period. Among the treatment groups chicken meat patties treated with essential oil of cinnamon at 1:250 and 1:500 co ncent rat ions and oi l o f clove at 1:2 50 concentration recorded significantly (P<0.05) lower counts of Staphylococcus aureus during the storage period. Since the essential oil of cinnamon gave similar results even at lesser concentration (i.e. at 1:500) it is considered as having the best antimicrobial effect in this study. REFERENCES Abdel Monein E Sulieman., Iman M O E L Bashra., El Amin A., and El Khalifa. (2007): Nutritive value of clove (Sazygium aromaticum) and detection of antimicrobial effect of its bud oil. Research Journal of Microbiology 2(3 ): 266-278 Fadia Naim., Serge Messier., Linda Saucier., and Gabriel pitte. (2004): Post processing in vitro digestion challenges to evaluate survival of Escherichia coli O157:H7 in fermented dry sausages. Applied Environmental Microbiology 70(11): 6637-6642. Fletcher, R. D., Parker, B., and Hassett, M. (1974): Inhibition of coagulase activity and growth of Staphylococcus aureus by garlic extract. Folia Microbiologica 19(6): 494-497. Hoque, M. D., Inatsu, M. L., Juneja., Vijay., and Kawamoto, S. (2007): Antimicrobial activity of clove and cinnamon extracts against food borne pathogens and spoilage bacteria and inactivation of Listeria monocytogenes in ground chicken meat with their essential oils. Journal of Food Science & Technology.72: 9-21. Kumar, M., and Berwal, J. S. (1998): Sensitivity of food pathogens to garlic (Allium sativum). Journal of Applied Microbiology 84(2): 213-215. 47
Jagadeesh Babu et.al., Mei-Chin Yi n, and Wen-shen Cheng. (2002): Antioxidant and antimicrobial effects of four garlic derived organosulfur compounds in ground beef. Meat Science 63(1): 23-28. Moreira, M. R., Ponce, A. G., Delvalle, C. E., and Roura, S. I. (2005): Inhibitory parameters of essential oils to reduce food borne pathogens. Food Science and Technology 38(5): 565-570. Noori S Al-Waili., Khelod Y Saloom., M Akmal., Thia N Al-Waili., Ali N Al-Waili., Hamza Al-Waili., Amjed Ali., and Karem A-Sahlani. (2007): Effects of heating, storage and ultraviolet exposure on antimicrobial activity of garlic juice. Journal of Medicinal Food 10(1): 208-212. Ponce, A. G., Fritz, R., Delvalle, C. E., and Roura, S. I. (2003): Antimicrobial activity of essential oils on the native microflora of organic swiss chard. Lebensmittel-wissemschaft und technologie 36: 679-684. Sema agaoglu., Nursel Dostbil., and Suleyman alemdar. (2007): Antimicrobial activity of some spices used in the meat industry. Bull Vet Inst Pulaway 51: 53-57. Shokrzadeh, M., and E badi, A. G. (2 006): Antibacterial effect of garlic (Allium sativum L) on Pakistan Journal of Biological Sciences 9(8): 1577-1579. Tassema, B., Mulu, A., Kassu, A., and Yismaw, G. (2003): An in vitro assessment of the antibacterial effect of garlic (Allium sativum) on bacterial isolates from wound infection. Journal of Egypt Public Health Association 78(5-6): 361-372. Table 1 Mean + SE values of Staphylococcus aureus count of chicken meat patties with essential oil of garlic at refrigeration temperature Treatment with essential oil of garlic at a concentration of 0 4.68 + 0.17 b3 4.02 + 0.06 b1 4.18 + 0.05 a1 4.41 + 0.07 bc2 4.32 + 0.07 b 2 4.09 + 0.06 a3 3.96 + 0.13 ab1 4.20 + 0.06 a2 4.13 + 0.05 a2 4.10 + 0.04 a 4 4.00 + 0.05 a2 3.70 + 0.21 a1 4.22 + 0.09 a3 4.30 + 0.04 b2 4.06 + 0.07 a 6 4.73 + 0.09 b3 3.94 + 0.12 a1 4.13 + 0.05 a2 4.49 + 0.12 c2 4.32 + 0.08 b 8 4.81 + 0.10b c2 4.08 + 0.09 b1 4.25 + 0.06 a1 4.62 + 0.13 d2 4.44 + 0.08 b 10 4.98 + 0.23 cd2 4.25 + 0.09 c1 4.41 + 0.08 b1 4.79 + 0.16 d2 4.61 + 0.09 c 12 5.07 + 0.04 d3 4.38 + 0.14 cd1 4.68 + 0.13 c1 4.86 + 0.10 de2 4.75 + 0.07 cd 14 5.19 + 0.05 e3 4.44 + 0.09 d1 4.75 + 0.03 c1 4.92 + 0.03 e2 4.83 + 0.06 d mean 4.69 + 0.07 D 4.10 + 0.05 A 4.35 + 0.04 B 4.57 + 0.05 C Least count 4 2 -- 4 Less than the count of day 0 Up to 4 Up to 6 -- Up to 4 48
Table 2 Mean + SE values of Staphylococcus aureus count of chicken meat patties with essential oil of clove at refrigeration temperature Treatment with essential oil of clove at a concentration of 0 4.68 + 0.17 b1 4.31 + 0.11 f1 4.52 + 0.09 f1 4.61 + 0.10 c1 4.53 + 0.06 d 2 4.09 + 0.06 a2 3.38 + 0.11 a1 3.34 + 0.10 a1 3.90 + 0.15 a1 3.68 + 0.09 a 4 4.00 + 0.05 a3 3.54 + 0.14 b1 3.69 + 0.11 b1 3.96 + 0.16 a2 3.80 + 0.07 a 6 4.73 + 0.09 b3 3.85 + 0.09 c1 3.98 + 0.08 c1 4.13 + 0.08 b2 4.17 + 0.08 b 8 4.81 + 0.10 c4 3.91 + 0.12 d1 4.15 + 0.09 d2 4.59 + 0.10 c3 4.37 + 0.09 c 10 4.98 + 0.23 d3 3.98 + 0.10 d1 4.25 + 0.15 e2 4.71 + 0.06 d3 4.48 + 0.11 cd 12 5.07 + 0.04 e3 4.03 + 0.08 d1 4.49 + 0.13 f1 4.81 + 0.03 e2 4.60 + 0.09 de 14 5.19 + 0.05 f3 4.15 + 0.12 e1 4.58 + 0.10 g2 4.93 + 0.03 f2 4.71 + 0.09 e mean 4.69 + 0.04 D 3.89 + 0.06 A 4.13 + 0.04 B 4.45 + 0.05 C Least count 4 2 2 2 Less than the count of day 0 Up to 4 Up to 14 Up to 12 Up to 8 Table 3 Mean + SE values of Staphylococcus aureus count of chicken meat patties with essential oil of cinnamon at refrigeration temperature Treatment with essential oil of cinnamon at a concentration of 0 4.68 + 0.17 b1 4.28 + 0.06 f1 4.41 + 0.07 e1 4.38 + 0.05 b1 4.44 + 0.06 d 2 4.09 + 0.06 a4 2.64 + 0.06 a1 3.76 + 0.06 c2 4.10 + 0.08 a3 3.65 + 0.13 a 4 4.00 + 0.05 a4 2.95 + 0.08 b1 3.42 + 0.05 a2 4.18 + 0.08 ab3 3.64 + 0.14 a 6 4.73 + 0.09 bc3 3.09 + 0.08 c1 3.58 + 0.05 b2 4.26 + 0.08 b3 3.92 + 0.14 b 8 4.81 + 0.10 c3 3.29 + 0.06 d1 3.86 + 0.06 c2 4.31 + 0.07 b3 4.07 + 0.12 b 10 4.98 + 0.23 cd2 3.61 + 0.07 e1 3.97 + 0.06 d1 4.54 + 0.06 c2 4.28 + 0.12 c 12 5.07 + 0.04 d4 3.69 + 0.05 e1 4.03 + 0.06 d2 4.72 + 0.06 d3 4.38 + 0.12 cd 14 5.19 + 0.05 e4 3.78 + 0.05 e1 4.09 + 0.08 d2 4.86 + 0.10 e3 4.48 + 0.12 d mean 4.69 + 0.07 D 3.42 + 0.08 A 3.89 + 0.05 B 4.42 + 0.04 C Least count 4 2 4 2 Less than the count of day 0 Up to 4 Studies on the... Up to 14 Up to 14 Up to 8 49