STUDIES ON THE SYNERGISTIC EFFECT OF SOME IRRADIATED ESSENTIAL OILS IN SOME FOOD PRODUCTS
By
MOHAMED ABDELRAZEK ABDELALEEM HANAFY
B.Sc. Agric. Sc. (Food Science and Technology), Ain Shams University, 1998 M. Sc. Agric. Sc. (Food Science), Cairo University, 2008
A thesis submitted in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
in
Agricultural Science (Food Science and Technology)
Department of Food Science Faculty of Agriculture Ain Shams University
2013
Approval Sheet
STUDIES ON THE SYNERGISTIC EFFECT OF SOME IRRADIATED ESSENTIAL OILS IN SOME FOOD PRODUCTS
By MOHAMED ABDELRAZEK ABDELALEEM HANAFY
B.Sc. Agric. Sc. (Food Science and Technology), Ain Shams University, 1998 M. Sc. Agric. Sc. (Food Science), Cairo University, 2008
This thesis for the Ph. D. degree has been approved by:
Dr. Samy Ibrahim El-Syiad…………………………………...... Prof. of Food Science, Faculty of Agriculture, Assiut University
Dr. Hamdy Mostafa Ebeid…………………………………...... Prof. of Food Science and Technology, Faculty of Agriculture, Ain Shams University
Dr. Ahmed Yousef Gibriel…………………………………...... Prof. Emeritus of Food Science and Technology, Faculty of Agriculture, Ain Shams University
Dr. Hanan Mohamed Abdo AL-Sayed………...…...…………...………………...... Associate Prof. of Food Science and Technology, Faculty of Agriculture, Ain Shams University
Date of Examination: 29 /5 /2013
STUDIES ON THE SYNERGISTIC EFFECT OF SOME IRRADIATED ESSENTIAL OILS IN SOME FOOD PRODUCTS
By
MOHAMED ABDELRAZEK ABDELALEEM HANAFY
B.Sc. Agric. Sc. (Food Science and Technology), Ain Shams University, 1998 M. Sc. Agric. Sc. (Food Science), Cairo University, 2008
Under the Supervision of:
Dr. Ahmed Yousef Gibriel Prof. Emeritus of Food Science and Technology, Dep. of Food Science, Faculty of Agriculture, Ain Shams University (Principal supervisor)
Dr. Ali Hassan Mohamed Rady Prof. Emeritus of Food Science and Technology, Nuclear Research Center, Atomic Energy Authority
Dr. Hanan Mohamed Abdo Al-Sayed Associate Prof. of Food Science and Technology, Dep. of Food Science, Faculty of Agriculture, Ain Shams University
ABSTRACT
Mohamed Abdelrazek Abdelaleem Hanafy: Studies on the Synergistic Effect of some Irradiated Essential Oils in some Food Products. Unpublished Ph. D. Thesis, Department of Food Science, Faculty of Agriculture, Ain Shams University, 2013.
Cumin, rosemary and thyme essential oils were gamma irradiated. Then, antibacterial and antioxidant activities were studied to measure the synergistic effect of their essential oils mixtures. 4, 6 and 4 kGy were the recommended doses for cumin, rosemary and thyme, respectively according to antimicrobial activity (agar well-diffusion) against S. typhimurium, S. aureus, B. cereus and E. coli. There were no changes in the physiochemical properties due to irradiation but, some changes occurred in the GC/MS analysis where, the amount of oxygenated compounds increased in cumin and thyme essential oils while, the oxygenated compounds decreased in rosemary essential oil.
The mixture made from non-irradiated cumin (C0) and rosemary (R0) essential oils were showed the highest antimicrobial activity against E. coil and B. cereus at 50 µl. Mixtures made from non-irradiated cumin and thyme
(T0) essential oils showed the highest antimicrobial activity against B. cereus.
Mixtures made form irradiated cumin at dose 4 kGy (C4) and rosemary at dose 6 kGy (R6) essential oils introduced promising antimicrobial activity as well as C0XR0 mixture. Fraction inhibitory concentrations (FIC) were studied against selected four bacterial strains for measuring synergistic activity however, (FIC) represented indifference in all essential oils mixtures but, the
C0 X R0 mixture against B. cereus (0.375) and E. coli (0.375) was synergy
(below0.5). Furthermore, the FIC shows addition in case of R0XT0, C2XR6,
C4XR6 and R6XT4 against B. cereus. And in case of C4XR6 against S. typhimurium. Preliminary experiment represented that 0.2, 0.4 and 0.1% were the acceptable odor in sunflower oil supplemented with rosemary, cumin and thyme essential oils, respectively. Antioxidant activity was measured with by Rancimat test and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity method. Rancimat test represented the highest oxidative stability in sunflower oil supplemented with 0.1% irradiated thyme essential oil at dose 4kGy. While, DPPH radical scavenging activity showed the highest scavenging activity in irradiated cumin and rosemary essential oils at 4 and 6kGy at concentration 500ppm while, irradiated thyme essential oil introduced promising result where, the scavenging activity was higher than that obtained from butylated hydroxytoluene (BHT) at the same concentration. The preferable mixture of all was C0XT0 where the scavenging activity at concentration 500ppm was 93.51%. The percentage 0.2% of mixtures made from both irradiated and non-irradiated cumin, rosemary and thyme essential oils were added to handmade mayonnaise then mayonnaise tested organoleptically (odor and taste). C0XR0 and C0XT0 mixtures reduce the microbial load from 56x103cfu/g to 42x101 and 18x101cfu/g. While, mayonnaise recipe supplemented with 0.2% of different essential oils mixtures inoculated with S. typhimurium, all mixtures completely inhibit the growth of S. typhimurium expect T4 X R6 and C4 X T4 mixtures reduce the inhibition to 3 x 101 and 6 x 101 cfu/g.
Key words: Gamma irradiation; Thymus vulgaris; Cuminum cyminum; Rosmarinus officinalis; Essential oils; Synergy ACKNOWLEDGEMENT
First of all. I wish to express my deep thanks and gratitude for ALLAH who gave me the ability to finish my thesis.
I wish to express my sincere thanks, deepest gratitude and appreciation to Dr. Ahmed Y. Gibriel Professor of Food Sciences and Technology, Faculty of Agriculture, Ain Shams University, for his sincere help, continuous encouragement, keen supervision and giving every possible advice throughout the investigation and during writing of the manuscript.
I am very grateful to Dr. Hanan M. A. AL-Sayed, Associate Professor of Food Sciences and Technology, Faculty of Agriculture, Ain Shams University, for suggesting the problem, kind help and encouragement throughout investigation and supervision.
I would like offer my deep thanks to Dr. Ali H. Rady Professor of Food Sciences and Technology and Ex-Chairman of Nuclear Research Center, Atomic Energy Authority of Egypt, for his encouragement, guidance, and unlimited help throughout this investigation supervision.
My deepest thanks to Dr. Hesham M. Badr Professor of Food Sciences and Technology, Nuclear Research Centre, Atomic Energy Authority of Egypt for his help in laboratory work and preparation of this manuscript.
I want also to thanks all members, of Food Irradiation Unit, Agriculture Research Division, Radio Isotope Application Department, Nuclear Research Centre, Atomic Energy Authority of Egypt.
CONTENTS Page LIST OF TABELS……………………………………………………... iii LIST OF FIGURES……………………………………………………. vii LIST OF ABBREVIATIONS………………………………………….. xiv 1. INTRODUCTION…………………………………………… 1 2. REVIEW OF LITERATURE………………………………. 5 2.1. Essential oils and essential oil formation...... 5 2.2. Spices essential oils...... 7 2.2.1. Cumin essential oil……………………………………………. 7 2.2.2. Rosemary essential oil………..………………………………. 11 2.2.3. Thyme essential oil…………..………………………………. 14 2.3. Irradiation of spices and herbs……………………...... 17 2.4. Effect of gamma irradiation on the chemical composition of essential oils...... 20 2.5. Antimicrobial activity of essential oils...... 25 2.6. Antioxidant activity of essential oils...... 34 2.7. Effects of irradiation on the antimicrobial and antioxidant activity of essential oils...... 43 2.8. Synergistic effect of essential oils as antimicrobials and antioxidants...... 47 2.9. Utilization of essential oils in food industry...... 49 2.10. Application of synergistic essential oil effect on some food products...... 50 3. MATERIALS AND METHODS……………………………. 52 3.1. Materials…...... 52 3.2. Method...... 55 3.2.1. Extraction of essential oils...... 55 ii
3.2.2. Irradiation of essential oils…...... 56 3.2.3. Analysis of the extracted essential oils...... 56 3.2.4. Experiments for antimicrobial activity of the essential oils...... 57 3.2.5. Experiments for antioxidant activity of the essential oils...... 66 3.2.6. Statistical analysis...... 69 4. RESULTS AND DISCUSSION……………………………... 70 4.1. Effect of gamma irradiation on the physiochemical properties of essential oil…………………………………………………. 70 4.2. Chemical composition of essential oil……………………….. 77 4.3. Antimicrobial activity of essential oils...... 89 4.3.1. Antibacterial activity of essential oils...... 90 4.3.2. Antifungal activity of essential oils...... 98 4.4. Evaluation of interactions between essential oils against bacterial strains...... 116 4.5. Determination of both minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)……... 151 4.6. Determination of Synergistic by Checker board method……... 169 4.7. Antioxidant activity of essential oils...... 177 4.7.1. Preliminary selection experiment...... 187 4.7.2. Rancimat test...... 194 4.7.3. DPPH radical scavenging method...... 197 4.8. Chemical composition of irradiated essential oil...... 220 4.9. Application of adding cumin, rosemary and thyme essential oils and their mixtures in food system...... 229 5. SUMMARY…………………………………………………... 243 6. REFERENCES……………………………………………… 249 ARABIC SUMMARY……………………………………….. LIST OF TABLES
No. Title page 1. Effect of gamma irradiation on physiochemical properties of different essential oils…………………………………………. 72 2. Chemical composition of cumin (Cuminum cyminum) essential oils…………………………………………………………….. 80 3. Chemical composition of rosemary (Rosmarinus officinalis) essential oils…………………………………………………… 83 4. Chemical composition of thyme (Thymus vulgaris) essential oils……………………………………………………………… 86 5. Effect of treatments of thyme essential oil by different irradiation doses on the bacterial inhibition zone (mm)………. 92 6. Effect of treatments of cumin essential oil at different irradiation doses on the bacterial inhibition zone (mm)……….. 95 7. Effect of treatments of rosemary essential oil at different irradiation doses on the bacterial inhibition zone (mm)……….. 99 8. Effect of different essential oils in different concentrations (µl) on the inhibition zone (mm) of different bacterial strains…… 105 9. Effect of mixture made from non-irradiated cumin and
rosemary essential oils (C0 X R0) in different concentrations on the inhibition zone of selected bacteria………………………... 117 10. The antibacterial effect of individual non-irradiated cumin and rosemary essential oil and 1:1 mixture of them……………….. 120 11. Effect of mixture made from non-irradiated cumin and thyme
essential oils (C0 X T0) in different concentrations on the inhibition zone of selected bacteria……………………………. 123 12. The antibacterial effect of individual non-irradiated cumin and thyme essential oil and 1:1 mixture of them…………………… 125 13. Effect of mixture made from non-irradiated rosemary and
thyme essential oils (R0 X T0) in different concentrations on the inhibition zone of selected bacteria………………………... 127 iv
14. The antibacterial effect of individual non-irradiated rosemary and thyme essential oils and 1:1 mixture of them……………... 129 15. Effect of mixture made from irradiated cumin essential oil at
doses 2 and 4 kGy (C2 X C4) in different concentrations on the inhibition zone of selected bacteria……………………………. 132 16. The antibacterial effect of individual irradiated cumin (2 kGy) and irradiated cumin (4 kGy) essential oils and 1:1 mixture of them…………………………………………………………….. 134 17. Effect of mixture made from irradiated cumin and rosemary
essential oils at doses 2 and 6 kGy, respectively (C2 X R6) in different concentrations on the inhibition zone of selected bacteria…………………………………………………………. 136 18. The antibacterial effect of individual irradiated cumin (2 kGy) and irradiated rosemary (6 kGy) essential oils and 1:1 mixture of them…………………………………………………………. 138 19. Effect of mixture made from irradiated cumin and thyme
essential oils at doses 2 and 4 kGy, respectively (C2 X T4) in different concentrations on the inhibition zone of selected bacteria…………………………………………………………. 140 20. The antibacterial effect of individual irradiated cumin (2 kGy) and irradiated thyme (4 kGy) essential oils and 1:1 mixture of them……………………………………………………………. 142 21. Effect of mixture made from irradiated cumin and rosemary
essential oils at doses 4 and 6 kGy, respectively (C4 X R6) in different concentrations on the inhibition zone of selected bacteria………………………………………………………… 144 22. The antibacterial effect of individual irradiated cumin (4 kGy) and irradiated rosemary (6 kGy) essential oils and 1:1 mixture of them…………………………………………………………. 145 23. Effect of mixture made from irradiated cumin and thyme
essential oils at doses 4 and 4 kGy, respectively (C4 X T4) in different concentrations on the inhibition zone of selected v
bacteria…………………………………………………………. 148 24. The antibacterial effect of individual irradiated cumin (4 kGy) and irradiated thyme (4 kGy) essential oils and 1:1 mixture of them…………………………………………………………….. 150 25. Effect of mixture made from irradiated rosemary and thyme
essential oils at doses 6 and 4 kGy, respectively (R6 X T4) in different concentrations on the inhibition zone of selected bacteria…………………………………………………………. 152 26. The antibacterial effect of individual irradiated rosemary (6 kGy) and irradiated thyme (4 kGy) essential oils and 1:1 mixture of them………………………………………………… 154 27. Minimum inhibitory concentration (MIC) and Minimum bactericidal concentrations (MBC) of both irradiated and non- irradiated different essential oils ……………………………… 157 28. Minimum inhibitory concentration (MIC) and Minimum bactericidal concentrations (MBC) of both irradiated and non- irradiated different essential oils mixtures ……………………. 162 29. Fraction inhibitory concentration of either irradiated or non- irradiated essential oils mixtures against selected bacterial strains. ………………………………………………………… 173 30. Fraction inhibitory concentrations (FICs) of both irradiated and non-irradiated different essential oils and their mixtures……… 174 31. Mean threshold values and standard errors for cumin, rosemary and thyme essential oils added to sunflower oil……………….. 188 32. Main components and olfactive notes of cumin, rosemary and thyme essential oil either irradiated or non-irradiated………… 192 33. Effect of different concentrations of rosemary, thyme and cumin essential oils either irradiated or non-irradiated on the oxidative stability of sunflower oil assessed by Rancimat test… 196 34. Effect of gamma irradiation on the DPPH scavenging activity of cumin, rosemary and thyme essential oil…………………… 199 35. Effect of gamma irradiation on the DPPH scavenging activity vi
of mixture made from cumin, rosemary and thyme essential oils……………………………………………………………… 207 36. Effect of gamma irradiation on the DPPH scavenging activity of mixture made from cumin, rosemary and thyme essential oils…………………………………………………………….. 213 37. Chemical components of cumin, rosemary and thyme essential oil before and after irradiation………………………………… 221 38. Chemical composition of irradiated cumin (Cuminum cyminum) essential oil (4kGy) ………………………………… 223 39. Chemical composition of irradiated rosemary (Rosmarinus officinalis) essential oil (6 kGy) ………………………………. 226 40. Chemical composition of irradiated thyme (Thymus vulgaris) essential oils (4 kGy) ………………………………………… 230 41. Mean threshold values and standard errors for the odor of adding cumin, rosemary and thyme essential oils to mayonnaise…………………………………………………….. 234 42. Total count of the handmade mayonnaise supplemented with essential oils mixture…………………………………………… 241 LIST OF FIGUERS
No. Title page 1. The structure of an isoprene molecule and an isoprene unit… 6 2. Locations and mechanisms in the bacterial cell thought to be sites of action for essential oil components…………………. 25 3. Isobologram to show areas of synergy, addition and antagonism…………………………………………………... 62 4. Effect of gamma irradiation on the refractive index of thyme, cumin and rosemary essential oil……………………. 73 5. Effect of gamma irradiation on the specific gravity of thyme, cumin and rosemary essential oil……………………………. 74 6. Effect of gamma irradiation on the acid value of thyme, cumin and rosemary essential oil……………………………. 75 7. Chromatographic profile of the essential oil cumin…………. 81 8. Chromatographic profile of the essential oil rosemary……… 84 9. Chromatographic profile of the essential oil thyme…………. 87 10. Effect of treatments of thyme essential oil at different irradiation doses on the bacterial inhibition zone (mm)…….. 93 11. Effect of treatments of cumin essential oil at different irradiation doses on the bacterial inhibition zone (mm)…….. 96 12. Effect of treatments of rosemary essential oil at different irradiation doses on the bacterial inhibition zone (mm)…….. 100 13. Effect of different irradiation doses on the inhibition zones (mm) of cumin, rosemary and thyme essential oil against Aspergillus flavus……………………………………………. 103 14. Effect of irradiated thyme essential oil (4 kGy) and non- irradiated thyme essential oil on growth inhibition of various bacterial strains……………………………………………… 107 15. Antimicrobial potential of both non-irradiated and irradiated thyme essential oils at different concentrations on the bacterial inhibition zone in mm…………………………….. 108 viii
16. Effect of irradiated cumin essential oil (2 and 4kGy) and non-irradiated cumin essential oil on growth inhibition of various bacterial strains……………………………………... 111 17. Antimicrobial potential of both non-irradiated and irradiated cumin essential oils at different concentrations on the bacterial inhibition zone in mm…………………………….. 112 18. Effect of irradiated rosemary essential oil (6 kGy) and non- irradiated rosemary essential oil on growth inhibition of various bacterial strains……………………………………... 114 19. Antimicrobial potential of both non-irradiated and irradiated rosemary essential oils at different concentrations on the bacterial inhibition zone in mm……………………………... 115 20. Antimicrobial potential of mixture made from non-irradiated
cumin and rosemary essential oils (C0 X R0) at different concentrations on the bacterial inhibition zone in mm……… 118 21. Antimicrobial potential of mixture made from non-irradiated
cumin and thyme essential oils (C0 X T0) at different concentrations on the bacterial inhibition zone in mm……… 124 22. Antimicrobial potential of mixture made from non-irradiated
rosemary and thyme essential oils (R0 X T0) at different concentrations on the bacterial inhibition zone in mm……… 128 23. Antimicrobial potential of mixture made from irradiated
cumin essential oil at doses 2 and 4 kGy (C2 X C4) at different concentrations on the bacterial inhibition zone in mm…………………………………………………………... 133 24. Antimicrobial potential of mixture made from irradiated
cumin and rosemary essential oils at doses 2 and 6 kGy (C2
X R6) at different concentrations on the bacterial inhibition zone in mm…………………………………………………... 137 25. Antimicrobial potential of mixture made from irradiated
cumin and thyme essential oils at doses 2 and 4 kGy (C2 X
T4) at different concentrations on the bacterial inhibition ix
zone in mm…………………………………………………... 141 26. Antimicrobial potential of mixture made from irradiated
cumin and rosemary essential oils at doses 4 and 6 kGy (C4
X R6) at different concentrations on the bacterial inhibition zone in mm………………………………………………….. 145 27. Antimicrobial potential of mixture made from irradiated
cumin and thyme essential oils at doses 4 and 4 kGy (C4 X
T4) at different concentrations on the bacterial inhibition zone in mm………………………………………………….. 149 28. Antimicrobial potential of mixture made from irradiated
rosemary and thyme essential oils at doses 6 and 4 kGy (R6
X T4) at different concentrations on the bacterial inhibition zone in mm………………………………………………….. 153 29. Minimum Inhibitory Concentration (MIC) of non-irradiated
cumin (C0), rosemary (R0) and thyme (T0) essential oil…….. 158 30. Minimum Bactericidal Concentration (MBC) of non-
irradiated cumin (C0), rosemary (R0) and thyme (T0) essential oil………………………………………………….. 159 31. Minimum Inhibitory concentration (MIC) of mixtures made
from non-irradiated cumin (C0), rosemary (R0) and thyme
(T0) essential oil…………………………………………….. 163 32. Minimum Bactericidal concentration (MBC) of mixtures
made from non-irradiated cumin (C0), rosemary (R0) and
thyme (T0) essential oil……………………………………… 164 33. Minimum Inhibitory Concentration (MIC) of irradiated cumin, rosemary and thyme essential oil……………………. 166 34. Minimum Bactericidal Concentration (MBC) of irradiated cumin, rosemary and thyme essential oil……………………. 167 35. Minimum Inhibitory concentration (MIC) of mixtures made
from irradiated cumin (C2), cumin (C4) rosemary (R6) and
thyme (T4) essential oil……………………………………… 170 36. Minimum Bactericidal concentration (MBC) of mixtures x
made from irradiated cumin (C2), cumin (C4) rosemary (R6)
and thyme (T4) essential oil………………………………… 171 37. Isoboles for zero-interaction, synergism and antagonism….. 175 38. The isobole method describing the effect of non-irradiated cumin and rosemary essential oil mixtures against selected bacterial strains……………………………………………… 178 39. The isobole method describing the effect of non-irradiated cumin and thyme essential oil mixtures against selected bacterial strains……………………………………………… 179 40. The isobole method describing the effect of non-irradiated rosemary and thyme essential oil mixtures against selected bacterial strains……………………………………………… 180 41. The isobole method describing the effect of irradiated cumin (2kGy) and cumin (4kGy) essential oil mixtures against selected bacterial strains…………………………………… 181 42. The isobole method describing the effect of irradiated cumin (2kGy) and rosemary (6kGy) essential oil mixtures against selected bacterial strains…………………………………….. 182 43. The isobole method describing the effect of irradiated cumin (2kGy) and thyme (4kGy) essential oil mixtures against selected bacterial strains…………………………………….. 183 44. The isobole method describing the effect of irradiated cumin (4kGy) and rosemary (6kGy) essential oil mixtures against selected bacterial strains…………………………………….. 184 45. The isobole method describing the effect of irradiated cumin (4kGy) and thyme (4kGy) essential oil mixtures against selected bacterial strains…………………………………….. 185 46. The isobole method describing the effect of irradiated rosemary (6kGy) and thyme (4kGy) essential oil mixtures against selected bacterial strains…………………………….. 186 47. Threshold level of adding cumin essential oil to sunflower oil……………………………………………………………. 189 xi
48. Threshold level of adding rosemary essential oil to sunflower oil………………………………………………… 190 49. Threshold level of adding thyme essential oil to sunflower oil…………………………………………………………… 191 50. Diphenyl-2-picrylhydrazyl (Free Radical)………………….. 197 51. Diphenyl-2-picrylhydrazyl (Non-Radical)………………….. 197 52. The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of non-irradiated and irradiated cumin essential oils and of synthetic antioxidant (BHT)……. 200 53. The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of non-irradiated and irradiated rosemary essential oils and of synthetic antioxidant (BHT)… 204 54. The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of non-irradiated and irradiated thyme essential oils and of synthetic antioxidant (BHT)……. 205 55. The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of non-irradiated cumin and rosemary essential oils combination and of synthetic antioxidant (BHT) ………………………………………….. 208 56. The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of non-irradiated cumin and thyme essential oils combination and of synthetic antioxidant (BHT) …………………………………………... 209 57. The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of non-irradiated rosemary and thyme essential oils combination and of synthetic antioxidant (BHT) ………………………………………….. 210 58. The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of irradiated cumin and cumin essential oils combination and of synthetic antioxidant (BHT) ……………………………………………………….. 214 59. The reduction of alcoholic DPPH solutions: the rate of xii
hydrogen donation ability of irradiated cumin and rosemary essential oils combination and of synthetic antioxidant (BHT) ………………………………………………………. 215 60. The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of irradiated cumin and thyme essential oils combination and of synthetic antioxidant (BHT) ……………………………………………………….. 216 61. The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of irradiated cumin and rosemary essential oils combination and of synthetic antioxidant (BHT) ……………………………………………………… 217 62. The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of irradiated cumin and thyme essential oils combination and of synthetic antioxidant (BHT) ……………………………………………………… 218 63. The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of irradiated rosemary and rosemary essential oils combination and of synthetic antioxidant (BHT) ………………………………………….. 219 64. Chemical components of irradiated cumin essential oil at dose 4 kGy…………………………………………………... 224 65. Chemical components of irradiated rosemary essential oil at dose 6 kGy………………………………………………… 227 66. Chemical components of irradiated thyme essential oil at dose 4 kGy…………………………………………………... 231 67. Threshold level of the odor of adding cumin essential oil to mayonnaise recipe…………………………………………… 235 68. Threshold level of the odor of adding rosemary essential oil to mayonnaise recipe……………………………………… 236 69. Threshold level of the odor of adding thyme essential oil to mayonnaise recipe…………………………………………… 237 70. Threshold level of the taste of adding cumin essential oil to xiii
mayonnaise recipe…………………………………………… 238 71. Threshold level of the taste of adding rosemary essential oil to mayonnaise recipe……………………………………… 239 72. Threshold level of the taste of adding thyme essential oil to mayonnaise recipe…………………………………………… 240
LIST OF ABBREVIATIONS
ANOVA Analysis of Variance ATP Adenosine Triphosphate BGA Brilliant Green Agar BHA Butylated hydroxyanisole BHT Butylated hydroxytoluene
C0 Non-irradiated cumin essential oil
C2 Irradiated cumin essential oil at dose 2 kGy
C4 Irradiated cumin essential oil at dose 4 kGy CFU Colony Forming Unite CH Hydrocarbon Compounds CHO Oxygenated Compounds DMSO Dimethyl Sulfuoxide DPPH 2, 2-diphenyl-1-picrylhydrazyl EB Electron-Beam EMCC Egyptian Microbial Culture Collection FAO Food Agriculture Organization FDA Food and Drug Administration FIC Fraction Inhibitory Concentration GC/MS Gas Chromatography and Mass Spectroscopy GRAS Generally Recognized As Safe IAEA International Atomic Energy Agency kGy Kilo Gray LPS Lipopolysaccharides LSD Least Significant Differences MBC Minimum Bactericidal Concentration MIC Minimum Inhibitory Concentration MRSA Methicillin resistant Staphylococcus aureus OD Optical Density OSI Oil Stability Index ppm Part Per Million xv
PROC Procedure PSFO Purified Sunflower Oil
R0 Non-irradiated rosemary essential oil
R6 Irradiated rosemary essential oil at dose 6 kGy ROS Reactive Oxygen Spices S. E. Standard Error SD Steam Distillation
T0 Non-irradiated thyme essential oil
T4 Irradiated thyme essential oil at dose 4 kGy WHO World Health Organization µl Micro Littre 60Co Cobalt-60 60Ni Nikal-60 1. INTRODUCTION Although desired antimicrobial activity of several essential oils against pathogenic and spoilage microorganisms are performed in vitro test, it has generally been found that a higher concentration is needed to achieve the same effect in foods (Shelef et al., 1984). This fact may lead to an organoleptic impact as the use of natural preservatives can alter the taste of food and exceed the flavor threshold acceptable to consumers (Hsieh et al., 2001 and Nazer et al., 2005). Gutierrez et al. (2008b) found that lettuce samples treated with 500 or 1000 ppm concentration of thyme and lemon balm essential oils were rejected because their bad effects on the organoleptic acceptability of foods. A number of studies have focused on the synergistic activity of the essential oil in combination with antibiotic to minimize the side effects of the antibiotic (Mahboubi and Bidgoli, 2010; Rosato et al., 2009 and Tohidpour et al., 2010), while the combinations of essential oil with other natural antibacterial compounds (e.g., nisin) was used in food to reduce the minimum effective dose of essential oil (Solomakos et al., 2008). However, a few studies regarding the synergistic effects of essential oil combinations to obtain effective antimicrobial activity at sufficiently low concentrations and consequently reduce negative sensory impact were performed (Gutierrez et al., 2009 and De Azeredo et al., 2011). Essential oils in plants generally are mixtures of abundant components (Burt, 2004), therefore, the synergistic effects are perhaps more likely to achieve in essential oil combinations. The concept of synergy is a central theme for many aromatherapy arguments and interventions. Its existence is often assumed whenever essential oils are combined with one another in blends and its manifestation within individual essential oils is often cited in arguments for obtaining and working with complete, pure and unrefined essential oils. The synergistic rationale for using combination products looks to producing a dynamic product that has multiple modes of action, respecting the principle that the 2
action of the combined product is greater than the sum total of known and unknown chemical components (Harris, 2002). Synergistic effects can be produced if the constituents of an extract affect different targets or interact with one another in order to improve the solubility and thereby enhance the bioavailability of one or several substances of an extract. A special synergy effect can occur when antibiotics are combined with an agent that antagonizes bacterial resistance mechanisms (Wagner and Ulrich- Merzenich, 2009). Early in vitro assays with essential oils showed they had promising antimicrobial and antioxidant properties, however when applied to food matrices amounts required to substantially inhibit the bacterial growth were often higher than would be organoleptically acceptable (Naveena et al., 2006). Regarding that these high concentrations is likely to impart a certain flavor to foods, the addition of sub-inhibitory amounts of essential oils in mixtures may be a way to provide the balance between sensory acceptability and antimicrobial and antioxidant efficacy. Although some studies have concluded that whole essential oils have a greater antibacterial activity than the major components mixed (Gill et al., 2002 and Mourey and Canillac, 2002), the combination of these major components with other components that have a weaker activity can achieve a synergistic effect (Ultee et al., 2000). In the Middle East, thyme (Thymus vulgaris L.) is found in spice mixtures such as zahtar from Jordan and dukkah from Egypt. It is used with roasted sesame seeds, black pepper, and other spices to flavor meat or is eaten with bread and olive oil (Raghavan, 2007). Cumin (Cuminum cyminum L., Apiaceae [Umbelliferae]) is a traditional plant, originally cultivated in Iran and the Mediterranean region (Grieve, 2005 and Tbaileh et al., 2007). Cumin has been in use since ancient times. Seeds excavated at the Syrian site Tell ed-Der have been dated to the second millennium BC. They have also been reported from several New Kingdom levels of ancient Egyptian 3
archaeological sites (Zohary and Hopf, 2000). The seed is widely used as a food flavoring agent in cheeses, pickles, sausages, soups and bean dishes, and has several important medicinal uses. Rosemary (Rosmarinus officinalis L.) is a perennial herb with fragrant evergreen needle-like leaves. It is native to the Mediterranean region and it has been cultivated for a long time. It belongs to the Lamaiaceae family, which comprises up to 200 genera and about 3500 species. The leaves are evergreen, with dense short woolly hairs. Rosemary has been a significant herb since antiquity, although rosemary is more familiar to contemporary Westerners as a kitchen herb used to add a spicy or slightly medicinal flavor to some foods, it was traditionally used as an antiseptic, astringent, and food preservative before the invention of refrigeration. Rosemary’s antioxidant properties are still used to extend the shelf life of prepared foods (Cuvelier et al., 1996). Here we report the investigation of an enhancing antibacterial effect when the essential oils from cumin, rosemary and thyme were used in combination against autochthonous microflora and some bacteria associated with the contamination of minimally processed foods and food matrices. Moreover, the influence of irradiation treatments on these essential oils on the antimicrobial activity was investigated. The treatment of essential oils with gamma irradiation did not apply since 1961 as mentioned by (Fazakerley et al., 1961). Where, irradiation of many essential oils, which are substantially compounds containing only carbon, hydrogen and oxygen, induces changes in flavor, odor and chemical composition which are beneficial. The aim of our investigation, despite essential oils are expected to be widely applied as antimicrobials as well as antioxidants, the organoleptical impact should be considered as the use of naturally derived preservatives can alter the taste of food or exceed acceptable flavour thresholds. Furthermore, 4
combinations of cumin, rosemary and thyme essential oil either irradiated or non-irradiated may help to minimize concentrations and consequently reduce sensory impact. Thus, the objectives of this study were to evaluate and quantify the effect of a range of cumin, rosemary and thyme essential oil either irradiated or non-irradiated oils in combinations, against four pathogens, B. cereus, E. coli, S. typhimurium and S. aureus, and to determine interactive effects of these essential oils and their combinations as antioxidant in order to optimize applications in food. 2. REVIEW OF LITERATURE 2.1. Essential oils and essential oils formation: Essential oils are volatile, natural, complex compounds characterized by a strong odor and are formed by aromatic plants as secondary metabolites. They are oily liquid, volatile, limpid and rarely colored, lipid soluble in organic solvents with a generally lower density than that of water. These oils can be synthesized by all plant parts including buds, flowers, leaves, stems, twigs, seeds, fruits, roots, wood or bark and are stored in secretory cells, cavities, canals, epidremic cells or glandular trichomes. They have been shown to possess antimicrobial, antifungal, antiviral, insecticidal and antioxidant properties (Kordali et al., 2005a; Bakkali et al., 2008 and Nanasombat and Wimuttigosol, 2011). Among vegetative volatiles, the most intensively studied substance is isoprene, a simple five-carbon terpene emitted from the foliage of many woody species (Sharkey and Yeh, 2001a). The function of isoprene is still controversial, and this compound may act to increase the tolerance of photosynthesis to high temperatures by stabilizing the thylakoid membranes (Sharkey et al., 2001b) or by quenching reactive oxygen species (Loreto and Velikova, 2001). Terpenoid molecules are made via the mevalonic acid biosynthetic pathway. The first step is to make mevalonic acid molecules (with six carbon atoms), hence the name of the pathway. Mevalonic acid molecules are then rearranged by a series of enzymatic transformations to form molecules known as isopentenyl pyrophosphate. Isopentenyl pyrophosphate consists of a branched 5-carbon molecule (known as an isoprene unit) joined to two phosphate groups. The isoprene unit as shown in Figure (1) is the starting point for manufacture of terpenoids compounds, the main type of molecule found in essential oils. The illustration of the unit demonstrates the branching structure of the 5-carbon atom chain, but does not specify double bonds. The double bonds rearrange during the formation of terpenoid molecules, but isoprene molecules usually contain at least one C=C bond. Isoprene 6
molecules are easily linked together to form long, branched chains of carbon atoms. When rubber was first analyzed in the late 1800s, it was found to contain hundreds of isoprene units all linked together. Rubber molecules are called polymer molecules (from the Greek poly, ‘many’ and mer, ‘unit’).
Fig. (1): The structure of an isoprene molecule and an isoprene unit
Terpenoid molecules in essential oils have either two or three isoprene units. The name ‘terpene’ derives from ‘turpentine’, a liquid solvent derived from the resin of Pinaceae species. When the structure of terpenoid molecules was first discovered, researchers thought the simplest molecule was one containing ten carbon atoms and started the naming system at this point, calling these molecules monoterpenes (from the Latin mono, ‘one’). The later discovery of isoprene units (an isoprene molecule has five carbon atoms) has not changed use of the earlier naming system, so ‘monoterpene’ still means two isoprene units joined together. Terpenoid molecules with three isoprene units are known as sesquiterpenes (from the Latin sesqui, ‘one and a half’). There are sometimes trace amounts of diterpenes in essential oils with four isoprene units, but the triterpenes with six isoprene units are waxes and are not found in steam-distilled essential oils because they are not volatile enough (Bowles, 2003 and Bakkali et al., 2008). 7
2.2. Spices essential oils: Spices are the tropical plants which have pungency or flavour of aromatic substances. They have long been used in foods and can be added as whole spices, ground spices or spice extracts. The most forms of spice extracts are oleoresins and essential oils (Nanasombat and Wimuttigosol, 2011). The spices essential oils have been a fertile area for research for many years due to their important antimicrobial, antioxidant, pharmacological and medicinal properties. More than 400 spices are used in the different countries in the world (Ceylan and Fung, 2004). Among them, cumin has been used as a spice since ancient times and is a very popular spice worldwide due to its unique aroma (Azeez, 2008). Whereas, thyme and rosemary are commonly used and their essential oils possess important antioxidant and antimicrobial properties (Baranauskiene et al., 2003; Yanishlieva et al., 2006 and Ojeda-sana et al., 2013). 2.2.1. Cumin essential oil: 2.2.1.1. Essential oil content of cumin (Cuminum cyminum L.): Walsh (2000) mentioned that the essential oil content in cumin seeds was about 2% to 5%. While, Amin (2001) reported that the yield of cumin oil production varies from 2.5 to 4.5%, depending on whether the entire seed or the coarsely ground seed is distilled. Li and Jiang (2004) observed that cumin seeds yielded 3.8% essential oil. While, Behera et al. (2004) showed that cumin seeds contained 5.6 ± 0.05%essential oil. Raghavan (2007) mentioned that cumin has 2.5% to 4.5% essential oil (depending on age and regional variations), which is pale to colorless, mainly containing monoterpene aldehydes. Azeez (2008) found that essential oil content of cumin seeds ranges from 2.3% to 5%. Li et al. (2009) studied the effect of different extraction methods on the essential oil content on cumin seeds. The obtained data 8
showed that content of essential oil in the super critical fluid extraction was 5.24% while, the combination of organic solvent and steam distillation was 4.87% and the content of essential oil extracted by steam distillation was 3.16%. On the other hand, cumin seeds generally contain up to 5% of the volatile oil (Burdock, 2010). Good quality cumin seeds, on crushing and steam distillation, yield 4 - 5% essential oil (Attokaran, 2011). On the other hand, Bettaieb et al. (2011) indicated that the contents of essential oil in Tunisian and Indian cumin seeds were 1.21 and 1.62%, respectively. Sowbhagya (2013) found that the essential oil content of cumin (Cuminum cyminum) seeds was ranged from 3 to 4%. 2.2.1.2. Physiochemical properties of cumin essential oils: The physiochemical properties of essential oil distilled from Mediterranean Sea cumin were as follow: specific gravity at 15 oC was 0.9170 – 0.9240, while the optical rotation at 15 oC was +4° 22́ ̶ +5° 6́. Refractive index at 20 oC was 1.5011 ̶ 1.5039 and the oil was soluble in 5 ml or more of 80% ethanol as reported by (Guenther, 1961). Moreover, EL- Baroty (1988) determined the physiochemical properties of cumin seeds essential oil. Where, specific gravity at 20 oC was 0.9254, refractive index at 20 oC was 1.5013 and optical rotation at 20 oC was + 64°. Amin (2001) reported that the specific gravity of cumin essential oil at 25oC ranged from 0.905 - 0.925, whereas the optical rotation of the oil at 20oC ranged from + 3 to + 8 and its refractive index ranged from 1.501 - 1.506. In addition, the cumin essential oil was soluble in 8 volumes of 80% ethanol and contained 40 - 52% aldehydes (as cuminic aldehyde). Behera et al. (2004) showed that the cumin essential oil had an optical rotation of +2.860 and refractive index (30 oC) of 1.4958. Azeez (2008) mentioned that the specific gravity (at 25°C) of cumin essential oil ranged from 0.900 - 0.935 and its optical rotation (at 20°C) 9
ranged from +4° to +8°, while its refractive index (at 20°C) ranged from 1.495 to 1.509. Moreover, the oil was almost insoluble in water, but soluble in ten volumes of each of 80% alcohol, ether and chloroform. Viuda-Martos et al. (2008) found that the physiochemical properties of cumin essential oil which obtained by steam distillation were: the refractive index at 20°C was 1.503 and the density at 20°C was 0.915 g/ml.
In other study, the refractive index of cumin essential oil at 20°C was found to be between 1.500 and 1.506, while its specific gravity at 25°C ranged from 0.905 - 0.925 (Burdock, 2010). Sowbhagya et al. (2011) studied the effect of various enzymes on the extraction of the volatile oil of cumin. The refractive index of the control oil sample was 1.4912 as against 1.4922 – 1.4970 in different enzyme-treated samples. Specific gravity of the control sample was 0.8967 as against 0.8912 – 0.8939 for enzyme-treated samples. Optical rotation values were higher (+4.3o) for all enzyme-treated cumin oil samples than for the control (+3.6o). 2.2.1.3. Chemical composition of cumin essential oil: Hirasa and Takemasa (1998) illustrated that cuminaldehyde (4- isopropylbenzaldehyde) was the major constituent of the cumin essential oil and represented 40–65% of the oil. They showed that it is an important aroma compound and responsible for the reported bitterness in cumin. Meanwhile, Walsh (2000) mentioned that the chief components in cumin essential oil were cuminaldehyde, gamma-terpenes, beta-pinenes, ρ-cymene, 1, 3-ρ-menthandial. Amin (2001) studies that the chemical composition of cumin essential oil showed the presence of the following components: α-pinene (0.5%), myrcene (0.3%), limonene (0.5%), 1-8-cineole (0.2%), ρ-menth-3-en-7-ol (0.7%), p-mentha-1, 3-dien-7-ol (5.6%), caryophyllene (0.8%), β-bisabolene (0.9%), β-pinene (13.0), ρ-cymene (8.5%), β- phellandrene (0.3%), D- terpinene (29.5%), cuminic aldehyde (32.4%), cuminyl alcohol (2.8%), β- 10
farnesene (1.1%) together with much smaller quantities of α phellandrene, α- terpinene, cis and trans sabinene, myrtenol, α-terpineol and phellandral. Weiss (2002) indicated that the characteristic flavour of cumin (which is best described as penetrating, irritating, fatty, overpowering, curry-like, heavy, spicy, warm and persistent) is probably due to dihydrocuminaldehyde and monoterpenes. Li and Jiang (2004) identified 37 components, representing 97.97% of the cumin essential oil and cuminal (36.31%), cuminic alcohol (16.92%), γ-terpinene (11.14%), safranal(10.87%), ρ-cymene (9.85%) and β-pinene (7.75%) were the major components. Whereas, Raghavan (2007) found that the chemical composition of cumin essential oil chiefly contained cuminic aldehyde (33%), β-pinene (13%), terpinene (29.5%), ρ-cymene (8.5%), ρ-mentha-1,3- dien-7-al (5.6%), cuminyl alcohol (2.8%), and β-farnesene (1.1%). Hajlaoui et al. (2010) identified twenty-one components in the cumin essential oil and found that cuminlaldehyde (39.48%), γ-terpinene (15.21%), O-cymene (11.82%), α-pinene (11.13%), 2-caren-10-al (7.93%), trans- carveol (4.49%) and myrtenal (3.5%) were the major components. They added that the cumin’s distinctive flavor and strong and warm aroma is due to its essential oil content and the main important aroma compound is cuminaldehyde (4-isopropylbenzaldehyde). Meanwhile, the important aroma compounds of the toasted cumin are the substituted pyrazines, 2-ethoxy-3- isopropylpyrazine, 2-methoxy-3-s-butylpyrazine, and 2-methoxy-3- methylpyrazine.
Pajohi et al. (2011) showed that cuminaldehyde (29.02%) and α- terpinen-7-al (20.70%) constituted the highest amount of the cumin essential oil. While, Vazin (2013) indicated that ρ-Menta-1, 3-diene-7-al, cuminaldehyde, γ-Terpinene, ρ-cymene and β-Pinene were the main constituents in the cumin essential oil. 11
2.2.2. Rosemary essential oil: 2.2.2.1. Essential oil content of rosemary (Rosmarinus officinalis L.): Prakasa Rao et al. (1999) reported that most of the oil (90%) comes out within the first 60 minutes of distillation, though distillation can be continued for 120 minutes for full recovery of rosemary oil under field distillation conditions. Thus, the essential oil yield was varied from 0.5% to 0.9%.
Walsh (2000) observed that rosemary herbs yielded about 1% to 2.5% essential oil from herbs. While, Atti-Santos et al. (2005) extracted Rosmarinus officinalis L. by steam distillation in a pilot plant, they found that the essential oil yields ranged from 0.37% to 0.49%. Raghavan (2007) mentioned that rosemary has 0.5% to 2.5% essential oil. Traditional hydro-distillation and innovative microwave hydrodiffusion and gravity methods have been compared and evaluated for their effectiveness in the isolation of essential oil from fresh Rosmarinus officinalis leaves (Bousbia et al., 2009); hydrodistillation method yielded about 0.33% ± 0.09 essential oil. While, microwave hydrodiffusion and gravity methods yielded about 0.35% ± 0.07 essential oil. However, the great difference between these methods was the shorter time that obtained in microwave hydrodiffusion and gravity method (15min against 3 hours in Hydro-distillation). Burdock (2010) illustrated that the leaves of rosemary contain 0.5% to 2.5% essential oil. Zermane et al. (2010) presented experimental results concerning the supercritical CO2 extraction of essential oil from Algerian rosemary leaves. The obtained yield was 1.35%, at a pressure of 10MPa and temperature of 40 °C. Meanwhile, Yosr et al. (2013) found that essential oils were present in high concentration (1.23 and 1.43%) in rosemary leaves (Rosmarinus officinalis var. typicus Batt.) being collected during the vegetative and flowering stages. 12
2.2.2.2. Physiochemical properties of rosemary essential oils: El-Laban (1998) studied the physiochemical properties of rosemary essential oil and found that its specific gravity was 0.9008, while its refractive index was 1.4709. Atti-Santos et al. (2005) mentioned that the specific gravity of rosemary essential oil averaged 0.8887, while its refractive index and optical rotation averaged 1.4689 and +11.82°, respectively.
Clarke (2008) illustrated that the physical properties of rosemary essential oil were; specific gravity (20 °C), 0.890 – 0.915; optical rotation (20°C), -5° to +10°; refractive index (20 °C), 1.460 – 1.480. Bousbia et al. (2009) found that the physical properties of rosemary essential oil were as follow: specific gravity 0.9, refractive index + 1.470 and optical rotation in degree +3, respectively. Burdock (2010) found that the refractive index and specific gravity of rosemary essential oil were between (1.464 and 1.476) at 20 °C and between (0.894 and 0.925) at 25 °C. 2.2.2.3. Chemical composition of rosemary essential oil: Prakasa Rao et al. (1999) showed that rosemary essential oil contained 1,8-cineol, camphor, borneol, bornyl acetate, α-pinene , β-pinene, linalool, camphene, subinene, myrcene, α-phellandrene, α-terpinene, limonene, ρ- cymene, terpinolene, thujene, copalene, terpinen-4-ol, α-terpineol, caryophyllene, methyl chavicol and thymol. Among them, 1,8-cineol, camphor, borneol, bornyl acetate and α-pinene constituted the major components and their percentages reached 30 - 40%, 15 - 25%, 16 - 20%, up to 7% and 25%, respectively. Walsh (2000) identified the main constituents of rosemary herb essential oil as; 1,8-cineole ( 20 to 50%), alpha-pinene (15 to 25%), camphor (10 to 25%), including as well camphene, borneol, bornyl acetate, betacaryophyllene, ρ-cymene, limonene, linalool, myrcene, alpha-terpineol and verbenone 13
Atti-Santos et al. (2005) identified twenty compounds in rosemary essential oil and showed that the major components were found to be α- pinene (40.55 - 45.10%), 1,8-cineole (17.40 - 19.35%), camphene (4.73 - 6.06%) and verbenone (2.32 - 3.86%), respectively. Raghavan (2007) mentioned that the rosemary essential oil mainly contained 1,8-cineol (30%), borneol (16% - 20%), camphor (15% - 25%), bornyl acetate (2% - 7%) and α-pinene (25%). He added that the varieties of rosemary differ in their flavor depending on their essential oil constituents and the compound 1,8-cineol gives the rosemary its cool eucalyptus aroma.
Clarke (2008) indicated that 1,8-cineole, camphor and α-pinene constituted the main components of the rosemary essential oil. Burdock (2010) illustrated that the monoterpene hydrocarbons constituted the major components of the rosemary essential oil and included α-pinene (~22%), β- pinene (~2%), camphene (~11%), camphor (10-20%), limonene, cineole, borneol (~2%), linalool and verbinol. Szumny et al., (2010) evaluated the influence of drying methods on volatile compounds of rosemary. Then, volatile compounds of rosemary samples were extracted by steam-hydrodistillation and analyzed by GC/MS. Thirty-four compounds were tentatively identified, with α-pinene, bornyl acetate, camphene and 1,8-cineole being the major components. Zermane et al. (2010) showed that the major compound detected in the rosemary essential oil was camphor, at 48.89%. Meanwhile, Jordán et al. (2013) mentioned that three chemotype of rosemary essential oils have eucalyptol, camphor and pinene as major compounds. In addition, Ojeda-Sana et al. (2013) found that the major components in rosemary essential oil were 1,8- cineol, camphor and α-pinene. 14
2.2.3.Thyme essential oil: 2.2.3.1. Essential oil content of thyme (Thymus vulgaris L.): Rey (1993a and 1994a) found that the essential oil content of thyme of thymol chemotype was more than 3.5%. While, the essential oil content in thyme was 2.5% or below as described by (Walsh, 2000). It should be noted that the Swiss Pharmacopoeia VII had fixed the minimum essential oil content at 1.5 per cent, in the dry herbs of thyme. Today the European Pharmacopoeia demands a minimum of 1.2 per cent oil content for thyme herb (Rey and Sáez, 2002). Moreover, in study to determine the content and the chemical composition of Italian thyme (Thymus vulgaris L.) essential oil at different dates of harvest, Venskutonis, (2002) found that the essential oil content was 1.75%, 1.40%, 2.90%, 2.86% 2.14%, 3.24 and 4.29% in 25May, 6June, 16June, 28June, 7July, 19July and 7August, respectively. Stahl-Biskup (2004) stated that the dried plant material of thyme herbs contains 1 - 2.5% of an essential oil. Meanwhile, the essential oil content of thyme ranges from 1.5% to 5% and has mainly phenols (Raghavan, 2007). On the other hand, thyme (Thymus vulgaris L.) yielded approximately 0.6% of essential oil as mentioned by (Burdock, 2010). Jia et al. (2010) indicated that the yields of essential oils from different thyme varieties ranged between 1.22% ± 0.01 and 0.16% ± 0.01 (weight/dry weight). Sárosi et al. (2013) studied the effect of different drying techniques (natural way, convective drying at 30 °C, 40 °C and 50 °C and lyophilization) on the quantity and quality parameters of the thyme essential oil and found that the measured essential oil amounts were between 0.69 ml/100 g and 1.84 ml/100 g (calculated on the dry weight basis). The highest drying temperature (50 °C) and lyophilization caused the most considerable essential oil loss. 15
In other study, the effect of six different drying treatments (sun, shade, oven 50 °C, oven 70 °C, microwave and freeze–drying) on the essential oil yield of thyme leaves was investigated. It was found that the highest essential oil yields were obtained by freeze–drying (1.7%) followed by oven 50 °C (1.46%), sun drying (1.42%), oven 70 °C (1.01%), shade drying (0.91%) and microwave (0.89%). In addition, air drying at ambient temperature significantly increased the essential oil yield (Rahimmalek and Goli, 2013). 2.2.3.2. Physiochemical properties of thyme essential oils: Lawrence and Tuker (2002) mentioned that the physiochemical properties of Thymus zygis were: specific gravity at 20oC was 0.912 to 0.935, refractive index at 20oC was 1.495 to 1.505. On the other hand, the physiochemical properties of Spanish thyme (Thymus capitatus) were: specific gravity at 20oC was 0.9380 – 0.9630, refractive index at 20oC was 1.5000 – 1.5130, optical rotation at 20oC was - 2o to + 3o and the solubility in 70% v/v (1:4 volume). Meanwhile, another kind of Spanish thyme (Thymus masticbina) have physiochemical properties as follow: refractive index 20oC was 1.4620 – 1.4680, optical rotation at 20oC was - 6o to + 10o. Stahl-Biskup (2004) and Lawrence and Tuker (2002) reported that the physiochemical properties of thymus vulgaris essential oil were: specific gravity at 25oC 0.915 to 0.935, optical rotation at 20 oC Laevorotary; but not more than - 3o, refractive index at 20oC 1.495 to 1.505 and solubility in 80% v/v aqueous ethanol 1:2 volumes. (Burdock, 2010) reported that the refractive index of the thyme essential oil was between 1.495 and 1.505 at 20 oC. while, the specific gravity was between 0.915 and 0.935 at 20 oC. 2.2.3.3. Chemical composition of thyme essential oil: Walsh (2000) found that the chief components in thyme essential oil were thymol (20-55%), ρ-cymene (14-45%), carvacrol (1-10%), gamma terpinene (5-10%), borneol (up to 8%) and linalool (up to 8%). 16
Stahl-Biskup (2004) reported that most of the volatiles detected in thyme oil belong to the monoterpene group with thymol, a phenolic monoterpene, as the main representative (30–55%). Thymol is always accompanied by some monoterpenes, which are closely connected by biogenetical processes, namely carvacrol (1–5%), an isomer terpene phenol, as well as ρ-cymene (15–20%) and γ-terpinene (5–10%). The latter two are precursors in the biogenetic pathway of thymol and carvacrol. Often the methyl ethers of thymol and carvacrol are present. Further monoterpenes are linalool (1-5%) and, in smaller percentages (0.5-1.5%), borneol, camphor, limonene, myrcene, β-pinene, trans-sabinene hydrate, α-terpineol and terpinen-4-ol. Sesquiterpenes are not very important in thyme oils. Only β- caryophyllene (1–3%) is worth mentioning. Raghavan (2007) mentioned that thyme contains predominantly thymol (45%), and carvacrol (18%). Spanish thyme (which provides 90% of the world’s thyme oil) has 12% to 61% thymol and 0.4% to 20.6% carvacrol, 1,8-cineole (0.2% to 14.2%), ρ-cymene (9.1% to 22.2%), linalool (2.2% to 4.8%), borneol (0.6% to 7.5%), α-pinene (0.9% to 6.6%), and camphor (0% to 7.3%). Clarke (2008) showed that the main components in thyme essential oil were Thymol, Carvacrol, ρ-cymene and 1,8-cineole; where the relative percentage of that compounds in the essential oil were 48%, 5.5%, 21.4 and 15.3, respectively. Bașaran (2009) mentioned that the main constituents of thyme essential oil were thymol, carvacrol, borneol, cineol and pinene. Jia et al. (2010) indicated that the main components in the essential oils of Thymus marschallianus and Thymus proximus were thymol (28.0- 32.9%), ρ-cymene (7.7- 25.4%), and γ-Terpinene (18.0- 22.4%). Viuda-Martos et al. (2011) found that the Egyptian thyme (Thymus vulgaris) had a high content of total phenols reaching 913.17 mg Gallic acid equivalents /L). Ait-Ouazzou et al. (2011) reported that a high content of borneol (23.48%) has been described for the first time in the thyme (Thymus 17
algeriensis) essential oil. El Bouzidi et al. (2013) analyzed the hydrodistilled oils obtained from wild and cultivated thyme species by GC–MS. They found that, in total, 41 components were identified representing more than 98% of the oils, with carvacrol (26.0 – 71.6%), borneol (5.0 – 20.1%), γ- terpinene (4.0 – 8.9%) and ρ-cymene (5.2 – 10.3%) as the main constituents. They also found that similar oil profiles were obtained from wild and cultivated Thymus maroccanus, whereas some quantitative differences were noted between oils obtained from wild and cultivated Thymus broussonetii and Thymus satureioides. 2.3. Irradiation of spices and herbs: There have been efforts, since 1986, to harmonize the law of the member states with regard to food irradiation; but only in 1999, a Directive was adopted. It includes, at present, only a single item, spices; and it enforces strict labelling. The European Commission was charged to develop a final ‘positive list’ of permitted items until the end of 2000; and the contents of this list are still under dispute between member states today. The Member States had to implement the provisions of the directive in 2000; however, only a few states completed their legal procedures. As long as the ‘positive list’ is not adopted, member states can maintain their existing regulations except for spices. This whole picture must be put in the context of the existing general standard for Irradiated Foods of the Codex Alimentarius, which does not allow for the exclusion of specific food items (Ehlermann, 2002). Doellstaedt and Huenber (1985) reported that in October 1980 the acceptance of food irradiation up to an overall average dose of l0 kGy was recommended by an Expert Committee of the Food and Agriculture Organization, the International Atomic Energy Agency (IAEA) and the World Health Organization (WHO). The conclusion of this Committee was that food irradiation up to the above dose does not imply any toxicological hazards. This is of great significance for any further work in this field and for 18
the use of radiation preservation of foods. On the basis of this recommendation extensive research work has been carried out in the recent years. Examples of the application of food and feed irradiation are; sprouting inhibition in onions, potatoes and garlic, reduction or control of microbiological contamination in spices and enzyme preparations, control of certain pathogenic microorganisms, such as salmonellae in eviscerated chicken, in meat or farm animal feed and irradiation of feed for specific pathogen-free laboratory animals. Nowadays the use of ionizing radiation is one of the most effective means of microbial decontamination of dried herbs and phytopreparations (Migdal et al., 1998). In this respect, the final microbial status of herbs and spices is determined by the natural content of microorganisms in plants and by the harvesting, drying, transporting, and packaging processes (Gould, 1996). To inactivate microorganisms of spices and aromatic herbs they usually subjected to disinfectant treatments. The most employed treatments are essentially three (Leistritz, 1997), fumigation with ethylene oxide, thermal treatment with steam, and irradiation with γ-rays or high-energy electrons. The use of irradiation instead of ethylene oxide to ensure hygienic quality of spices and herbs has increased in the past 20 years, because of the phasing out of ethylene oxide in many countries due to possible toxic residues and occupational health hazard for workers in the fumigation plants. Moreover, irradiation offers a broader spectrum for application for sanitizing dry ingredients than thermal treatments, often at a more competitive cost (Farkas, 1998). WHO (1981, 1999) reported that the application of ionizing radiation treatment of foods on an industrial scale was started at the beginning of the 1980s after the joint Food and Drug Administration / International Atomic Energy Authority/World Health Organization (FAO/IAEA/WHO) expert committee accepted the application of a 10 kGy overall average dose for foods. Moreover, based on the findings of WHO study group, the large number of toxicological studies including carcinogenicity bioassays and 19
multigeneration reproductive toxicology evaluations did not demonstrate any short-term toxicity related to irradiation process.
Farkas (1998) proved that radiation treatment at doses of 2–7 kGy, depending on condition of irradiation and the food, can effectively eliminate potentially pathogenic non-sporeforming bacteria including both long-time recognized pathogens such as Salmonella and Staphylococcus aureus as well as emerging or ‘‘new’’ pathogens such as Campylobacter, Listeria monocytogenes or Escherichia coli O157:H7 from suspected food products without affecting sensory, nutritional and technical qualities. Radioisotopes, chemical elements that have the inherent property to emit radiation, emit gamma radiation. The most usual radioisotope in food industrial applications is cobalt (60Co), which is obtained from 59Co (natural isotope) bombardment with neutrons in a nuclear reactor. Each 60Co disintegration causes an emission of one beta particle and two photons with energy of 1.17 MeV, originating 60Ni, a stable element. As all radioisotopes, the activity of 60Co is reduced in time according to an exponential law, its half-life being 5.3 years (Camargo et al., 2008) Food irradiation is approved for use in over 55 countries worldwide for various applications and purposes in a wide variety of foodstuffs; however, its use as a post-harvest phytosanitary treatment is still limited. Examples of countries with legislation allowing phytosanitary uses of irradiation include Argentina, Australia, Bangladesh, Brazil, China, India, Mexico, Philippines, Russian Federation, Thailand, Turkey, Ukraine, the United States of America, and Vietnam (IAEA, 2009). In many countries all over the world such as Iran, various products especially spices and herbs are conventionally irradiated up to 10 kGy to increase the hygienic quality and also prolong the shelf life of spices by reducing the number of pathogenic and spoilage microorganisms (Fatemi et al., 2011). 20
2.4. Effect of gamma irradiation on the chemical composition of essential oils: The development of unpleasant flavours and odours in irradiated natural products is not uncommon, and often occurs when foodstuffs are treated with large doses of radiation. Fazakerley et al. (1961) found that the irradiation of many essential oils and isolates, which are substantially compounds containing only carbon, hydrogen and oxygen, induces changes in flavour and odour which are beneficial. The reports on the effect of irradiation on the essential oil content of spices and chemical composition are controversial and often contradictory. Klaus and Wilhelm (1990) reported a 20% increase in ρ-cymene content of nutmeg irradiated to a dose of 10 kGy while in black pepper irradiation even at doses <10 kGy brought about major quantitative changes in the volatile oil constituents (Ljubica, 1983). A reduction in cinnamaldehyde content by 33% and an increase in cinnamylacetate and eugenol by 27% were reported by Farkas (1988) in cinnamon bark samples after irradiation at 10 kGy. In contrast, Maija et al. (1990) were unable to detect any significant differences in the volatile oil constituent of control and irradiated spices even up to doses of 50 kGy. (IAEA, 1992) proved that irradiation had no effect on the composition of the volatile oil of turmeric as compared to the equivalent non-irradiated ones. The irradiation also did not affect the percent yield of the major compounds identified by GC/MS such as; α-phellandrene, p-cymene, 1:8 cineol. Whereas, the percent yield of them were 1.89%, 1.3%, and 1.3%, respectively for control and 1.44%, 0.89%, and 1.26%, respectively for irradiated turmeric. In addition, the overall yield of the volatile oil remained unaffected after irradiation.
The effect of γ-irradiation on the volatile oil constituents of several spices has been extensively studied and reported in literature (Anon., 1992). However, the few reports available on the effect of γ-irradiation on turmeric 21
aroma are not definitive and are contradictory. Chosdu et al. (1985) were unable to detect any changes in essential oil constituent of turmeric subjected to γ-irradiation up to a dose of 10 kGy. On the other hand, Zaied et al. (1996) reported a reduction in cineol content of γ-irradiated spices including turmeric. Ito and Islam (1994) mentioned that the utilization of high dose rate of Electron-Beam (EB) irradiation suppressed the increase of peroxide values in spices at high dose irradiation up to 80 kGy. However, components of essential oils in spices were not changed even irradiated up to 50 kGy with EB and γ-rays. Wu and yang (1994) studied the effect of irradiated fresh ginger at dose 50 Gy on the flavour compounds; it was observed that after three months of storage the major volatile compounds (zingibrein, β-sesquiphellandrene and ar-curcumene) were significantly lower in irradiated than in non-irradiated samples. No differences were however, found in these constituents immediately after irradiation. Antonelli et al. (1998) irradiated dried basil leaves with two different doses (5 kGy and 10kGy) of gamma rays. They found that linalool and estragol showed the greatest increases with gamma irradiation (28.23% and 13.06% for 5kGy and 27.16% and 13.69% for 10kGy, respectively) while eugenol decreased with the same doses (9.63% for 5kGy and 10.52% for 10kGy, respectively); in comparing with control basil leaves (14.92% for linalool, 4.54% for estragol, and 12.13% for eugenol. Variyar et al. (1998) studied the effect of irradiation at dose 10 kGy used for decontamination of spices on the chemical composition of clove, cardamom and nutmeg using gas liquid chromatography (GLC). The results found that there were no qualitative and major quantitative changes in the essential oil constituents of irradiated clove and cardamom. However in case of irradiated nutmeg a 6-fold increase in the content of myristicin accompanied by a decrease of similar magnitude in elimicin content. 22
Meanwhile, Chatterjee et al. (2000) studied the effect of γ-irradiation at dose 10 kGy on the volatile constituents of irradiated turmeric essential oil. The results found that the percentage content of main component (ar- Turmerone + turmerone) was increased from 68 ± 1.4 (non-irradiated) to 70 ± 1.2 (irradiated). While, some compounds were decreased related to irradiation dose i.e. ρ-Cymene from 1.3 ± 0.3 to 0.89 ± 0.2, Curlone from 15 ± 2.8 to 12 ± 2. By using ionizing radiation at dose of 10 kGy, a satisfactory result of microbiological decontamination of medical herbs could be observed. The content of essential biologically active substances such as essential oils, flavonoids, glycosides, anthocyans, antra-compounds, poliphenoloacids, triterpene saponins, oleanosides and plants mucus did not significantly change after irradiation. Pharmacological activity of medicinal herbs has been found satisfactory after microbiological decontamination by irradiation (Owczarczyk et al., 2000). Fan and Sokorai (2002) investigated the effect of irradiation on volatile compounds of fresh cilantro leaves. Fresh cilantro leaves (Coriandrum sativum L.) were irradiated with 0, 1, 2, or 3 kGy γ-radiation and then stored at 3 °C up to 14 days. The obtained results conducted that the amounts of linalool, dodecanal, and (E)-2-dodecenal in irradiated samples were significantly lower than those in non-irradiated samples at day 14. Koseki et al. (2002) reported that whether high doses of gamma radiation (10, 20 and 30 kGy) affects the characteristics or amount of phenolics, flavonoids, essential oils, tannins and β-carotene of rosemary (Rosmarinus officinalis L.), watercress (Nasturtium officinale), artichoke (Cynara scolymus L.) and sweet basil (Ocimum basilicum L.). The results showed that; there were no changes in the chemical composition of essential oil except for the radiation dose 10 kGy, phenolic content had a small change also after 10 kGy irradiation of rosemary. Finally there was no evidence that radiation caused a significant degradation of total β-carotene. 23
Moussaid et al. (2004) studied the irradiation of Moroccan orange peel (Maroc Late) with doses up to 1 kGy, which did not result in significant (p > 0.05) quantitative changes in the essential oils components including D- limonene, linalool, citral and methyl anthranilate. Irradiation with 2 kGy stimulated synthesis of D-limonene, while enhancing the degradation of linalool, methyl anthranilate and citral. Topuz and Ozdemir (2004) irradiated sun-dried and dehydrated paprika samples at doses from 2.5 to 10 kGy and analyzed capsaicinoid contents. They found that the contents of capsaicin, dihydrocapsaicin and homodihydrocapsaicin increased significantly by about 10% in samples irradiated at a dose of 10 kGy. Gyawali et al. (2006) studied the effect of gamma irradiation at different doses 0, 1, 3, 5, 10 and 20 kGy on the volatile compounds of dried Welsh onion after extraction by steam distillation method. A total of 35 volatile compounds were identified by GC/MS in non-irradiated and 1 kGy irradiated samples and 36 volatile compounds were identified in 3, 5, 10 and 20 kGy irradiated samples so far belong to chemical classes of acid, alcohol, aldehyde, ester, furan, ketone and S-containing compound. S-containing compounds were detected as major volatile compounds of all experimental samples. The results revealed that the content of majority of compounds was increased after different doses of γ-irradiation, the content of major S- compounds in control such as 1-propanethiol, 5-methyl thiazole, propenyl propyl disulfide, dipropyl disulfide and 3,5-diethyl-1,2,4-trithiolane was decreased after the process. Seo et al. (2007) determined the effects of gamma irradiation on the volatile flavor components including essential oils, of Angelica gigas Nakai. The volatile organic compounds from non- and irradiated A. gigas Nakai at doses of 0, 1, 3, 5, 10 and 20 kGy were extracted by a simultaneous steam distillation and extraction (SDE) method and identified by GC/MS analysis. The major volatile compounds were identified 2,4,6-trimethyl heptane, α- 24
pinene, camphene, α-limonene, β-eudesmol, α-murrolene and sphatulenol. Among these compounds, the amount of essential oils in non-irradiated sample were 77.13%, and the irradiated samples at doses of 1, 3, 5, 10 and 20 kGy were 84.98%, 83.70%, 83.94%, 82.84% and 82.58%, respectively. Oxygenated terpenes such as β-eudesmol, α-eudesmol, and verbenone were increased after irradiation but did not correlate with the irradiation dose. The yields of active substances such as essential oil were increased after irradiation; however, the yields of essential oils and the irradiation dose were not correlated. Thus, the profile of composition volatiles of A. gigas Nakai did not change with irradiation.
Shim et al. (2009) studied the effect of γ-irradiation at doses of 0, 1, 3, 5 and 10 kGy on the contents of volatile compounds from Paeoniae Radix such as paeonol, carveol, eugenol acetate. They found that the concentration of this compound increased with the increase of irradiation dose level. Khattak and Simpson (2010) irradiated Glycyrrhiaz glabra roots at doses 0, 5, 15, 20 and 25 kGy to represent the effect of irradiation doses on the chemical composition and the phenolic content. The results revealed that the phenolic content increased in samples irradiated with 20 and 25 kGy. Irradiation of cumin essential oil at doses 10 and 25 kGy has been investigated by Fatemi et al. (2012). The chemical composition of essential oil after irradiation was as follow; the content of γ-terpinine in control, 10kGy and 25kGy were 20.09%, 21.91% and 15.46, respectively. While, cuminaldehyde in control, 10kGy and 25kGy samples were 19.03%, 17.98% and 20.51, respectively. On the other hand, antioxidant activity of irradiated cumin essential oil was determining by free-radical scavenging activities. The obtained results were 32.99, 34.17 and 31.47% measured as DPPH% in control, 10kGy and 25kGy, respectively. 25
2.5. Antimicrobial activity of essential oils: Although the antimicrobial properties of essential oils and their components have been reviewed in the past, the mechanism of action has not been studied in great detail (Burt, 2004). Considering the large number of different groups of chemical compounds present in essential oils, it is most likely that their antibacterial activity is not attributable to one specific mechanism but that there are several targets in the cell The locations or mechanisms in the bacterial cell thought to be sites of action for essential oil components are indicated in Figure (2). Not all of these mechanisms are separate targets; some are affected as a consequence of another mechanism being targeted. Thus, Degradation of the cell wall Described by Helander et al. (1998). Sikkema et al. (1994) and Ultee et al., (2000 and 2002) mentioned that the mechanism of essential oil activity is related to damage to cytoplasmic membrane. While, Ultee et al. (1999) illustrated the mechanism is due to damage of membrane proteins.
Fig. (2): Locations and mechanisms in the bacterial cell thought to be sites of action for essential oil components
Gustafson et al. (1998); Helander et al. (1998); Cox et al. (2000) and Lambert et al. (2001) found that leakage of cell contents was happened as a 26
reason of essential oils treatment, Gustafson et al. (1998) reported that coagulation of cytoplasm could be done related to essential oils treatment and depletion of the proton motive force might be happened as described by (Ultee and Smid, 2001 and Ultee et al., 1999). 2.5.1. Antimicrobial activity of cumin essential oil: Farag et al. (1989) examined the antimicrobial activity of the cumin essential and found that the oil had no or very little effect against Pseudomonas fluorescens, Escherichia coli, and Serratia marcescens. Also Davidson and Naidu (2000) showed that the cumin essential oil had a limited antimicrobial activity. Iacobellis et al. (2005) examined the antimicrobial activity of the cumin essential oil through the determination of minimal inhibitory and minimal bactericidal concentrations (MIC and MBC).They found that Escherichia coli was the most sensitive microorganism with the lowest MBC value (1 µl/ml), while Staphylococcus aureus and Listeria monocytogenes had MIC values of 1 and 2 µl/ml, respectively. Similarly, Gachkar et al. (2007) showed that Escherichia coli was the most sensitive microorganism to the cumin's essential oil with the lowest minimum bactericidal concentration (MBC) value of 1 µl/ml, whereas Listeria monocytogenes required higher oil concentration (2 µl/ml) for complete elimination (with a greater zone of inhibition reaching 17.67 mm). Viuda-Martos et al. (2008) reported that cumin essential oil was effective as antibacterial showing inhibition zones between 31.23 mm on Lactobacillus sakei and 38.17 mm on Enterobacter gergoviae. Hajlaoui et al. (2010) determined the antimicrobial activity of the essential oil of cumin against different microbial species included Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis CIP 106510, Micrococcus luteus NCIMB 8166, Bacillus cereus ATCC 11778, B. cereus ATCC 14579, Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium LT2 DT104, Listeria 27
monocytogenes ATCC 19115, Enterococcus feacalis ATCC 29212, 14 strains belonging to Vibrio genus, four Candida species (albicans, parapsilosis, glabrata and krusei) and one Saccharomyces cerivisae strain. They showed that the essential oil of cumin cyminum had substantial antimicrobial activity against the tested bacterial and yeast species. From zones of growth inhibition (mm), the data demonstrated that Gram-positive bacteria exhibited the highest diameters of growth inhibition (10 - 35 mm) and the oil was particularly effective against M. luteus NCIMB 8166 with a diameter of inhibition about 35 mm. Meanwhile, Gram-negative bacteria were less sensitive to cumin essential oil with a diameter of growth inhibition ranging from 10 (E. feacalis ATCC 29212) to 12 mm (E. coli ATCC 35218). Furthermore, the diameters of growth inhibition for Vibrio spp. strains ranged from 11 mm (Vibrio alginolyticus ATCC 33787) to 23 mm (Vibrio cholerae ATCC 9459) and the oil was effective against all the tested yeasts with a diameter of growth inhibition ranging from 17 to 22.67 mm. Allahghadri et al. (2010) studied the antimicrobial activity of cumin essential oil against several bacterial strains i.e., E. coli, S. aureus and S. faecalis. The results revealed that the inhibition zones of cumin essential oils were 13, 10 and 10.33mm, respectively. 2.5.2. Antimicrobial activity of rosemary essential oil: Benjilali et al. (1984) tested the antifungal activities of essential oils of three chemotypes of mugwort, thyme, rosemary and eucalyptus against 13 strains of Penicillium, nine of Aspergillus. Overall, thyme was the most effective followed by mugwort, rosemary and eucalyptus. Phenol containing essential oils (thyme) were more effective than ketone containing essential oils (eucalyptus). Each essential oil differed in its antifungal activity against different fungi and strains of the same fungus. In a study of antilisterial effect of some spices in laboratory medium and meat system, rosemary (≥ 0.5% w/v) was antilisterial to Listeria monocytogenes Scott A in laboratory medium (Pandit and Shelef, 1994). 28
Rosemary essential oil at a concentration of 10 µL/100mL exhibited bactericidal effect against Listeria monocytogenes in broth. Among four major components of rosemary essential oil (cineole, bornole, α-pinene, and camphor), α-pinene at 1 µL/100mL produced listeriostatic effect in laboratory medium and rosemary oleoresins had no antilisterial effect up to 100 mg/100mL concentration. Addition of 0.5% (w/w) ground rosemary and 1% (v/w) rosemary essential oil delayed the growth of Listeria monocytogenes in pork liver sausage emulsion stored at 5 oC for 50 days. Smith-Palmer et al. (1998) found that the bacteriostatic concentration of rosemary essential oil against Salmonella enteritidis, Listeria monocytogenes, Escherichia coli and Staphylococcus aureus was > 1%, 0.02%, > 1% and 0.04%, respectively. Meanwhile, essential oil extracted from rosemary (Rosmarinus officinalis) presented an inhibitory concentration of 10 – 250 ppm against Brotytis cinerea as described by (Bouchra et al., 2003). Holly and Patel (2005) showed that the essential oil of rosemary - which had chemical composition; borneol (26.5%), α-terpinene (15.6%) and α- pinene (12.7%) - had an antimicrobial activity against some bacterial strains and represented highly sensitive effect against Listeria monocytogenus, Esherchia coli, Staphylococcus aureus and Sarcina spp. Especially in sausage, cheese and bakery products. On the other hand, Sacchetti et al. (2005) studied the antimicrobial activity of rosemary (Rosmarinus officinalis) essential oil against various microorganisms i.e. Candida albicans, Rhodotorula glutinis, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Yarrowia lypolitica. The results illustrated that the minimum inhibitory concentrations of rosemary essential oil were 0.09, 0.12, 0.06, 0.18 and 0.12mg/ml. The antilisterial activity of the rosemary essential oils and the sub-lethal concentration of lactic acid were established by the agar well diffusion method. The bactericidal kinetics of the diluted oils (50 ppm, 100 ppm, 200 29
ppm and 300 ppm) and their mixtures with 50 ppm of lactic acid were determined by optical density (OD600) measurements. The results suggest that a sub-lethal dose of lactic acid noticeably increased the antilisterial activity, especially of rosemary but that the synergistic effects were reduced with higher concentrations of oils (Dimitrijević et al., 2007). Meanwhile, Fu et al. (2007) showed that rosemary essential oil possessed significant antimicrobial effects against Staphylococcus epidermidis, Escherichia coli and Candida albicans showing MICs of 0.125-1.000% (v/v). Viuda-Martos et al. (2008) indicated that rosemary (Rosmarinus officinalis) essential oil had an inhibitory effect on different bacteria commonly used in the food industry including Lactobacillus curvatus, Lactobacillus sakei, Staphylococcus carnosus and Staphylococcus xylosus or related to food spoilage including Enterobacter gergoviae and Enterobacter amnigenus. Bousbia et al. (2009) studied the antimicrobial activity of rosemary essential oil; the obtained inhibition zones in mm. were 12.5 ± 0.04 in Staphylococcus aureus and 15.5 ± 0.05 in Escherichia coli. While, Romano et al. (2009) studied the antioxidant and antibacterial activity of rosemary extract containing 30% carnosic acid, 16% carnosol and 5% rosmarinic acid in vitro alone and in combination with the antioxidant food additives butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). The study showed that synergistic interaction of methanol rosemary extract with BHA to inhibit Escherichia coli and Staphylococcus aureus growth was observed.
Ait-Ouazzou et al. (2011) found that rosemary (Rosmarinus officinalis) essential oil had no significant antimicrobial effect against Salmonella enteritidis (CECT 4155), Escherichia coli O157:H7 (CECT 4267), Pseudomonas aeruginosa (CECT 110), Staphylococcus aureus (CECT 239), Enterococcus faecium (CECT 410), Listeria monocytogenes 4b (CECT 935) and Listeria monocytogenes EGD-e.16. 30
Ojeda-Sana et al. (2013) assessed the relationship between the chemical composition of rosemary essential oils and their antibacterial activity against human pathogenic and food decay bacteria. Their results suggested a relationship between the antibacterial activity of rosemary essential oil, against Gram-positive and Gram-negative bacteria and the content of α- pinene. They also showed that 1,8-cineole, the other main compound present in rosemary essential oils, was found to disrupt the cell membrane of E. coli at ½ X MIC (4 µL/mL). Teixeira et al. (2013) found that the mean radius of inhibition zones (mm) of 20µL undiluted rosemary essential oil tested against Brochothrix thermosphacta, Escherichia coli, Listeria innocua, Listeria monocytogenes, Pseudomonas putida, Salmonella typhimurium and Shewanella putrefaciens were 37, 6, 4, 8, 4, 3, and 27 mm, respectively. They also determined the effect of minimum inhibitory concentration (MIC) obtained with the disc diffusion method in solid medium and found that the mean bacterial CFU (colony forming units) logarithmic reductions per mL were 4.5, 0.0, 8.0, 7.3, 1.1 and 1.7 cfu/ml, respectively. 2.5.3. Antimicrobial activity of thyme essential oil: Several studies have focused on the antimicrobial activity of the essential oils of thyme in order to identify the responsible compounds. Thymol and carvacrol seem to play an outstanding role. These terpene phenols join to the amine and hydroxylamine groups of the proteins of the bacterial membrane altering their permeability and resulting in the death of the bacteria (Juven et al., 1994). In addition, thymol and carvacrol were shown to induce a decrease of the intracellular adenosine triphosphate (ATP) pool of Escherichia coli and an increase of the extracellular ATP (Helander et al., 1998). Antibacterial activity was also observed for the aliphatic alcohols, especially geraniol, and ester components. A variety of activities was presented by the esters; in some cases they were more active than their 31
corresponding free alcohols, but sometimes less active (Megalla et al., 1980). According to Agarwal and Mathela (1979) and Agarwal et al. (1979) the antifungal activity of the essential oil of Thymus serpyllunz is attributable to thymol and carvacrol. They cause the degeneration of the fungal hyphae which seems to empty their cytoplasmic content (Zambonelli et al., 1996). The terpenic alcohols as well as the aldehydes, ketones and some esters, also presented considerable activities, whereas the hydrocarbons showed only low activities Purified compounds derived from essential oils such as carvacrol (form thyme essential oil), eugenol, linalool, and thymol (form thyme essential oil) inhibit a variety of micro-organisms (Didry et al., 1993a and Hulin et al., 1998). In general, levels of essential oils and their compounds necessary to inhibit microbial growth are higher in foods than in culture media. This is due to interactions between phenolic compounds and the food matrix as figured out by (Nychas and Tassou, 2000). Partitioning of the hydrophobic antibacterial essential oil components into the fat content of the food may prevent them from coming into contact with bacterial cells growing in the hydrophilic regions in the food (Gill et al., 2002). Essential oils with high concentrations of thymol and carvacrol e.g. oregano, savory and thyme, usually inhibit Gram-positive more than Gram-negative pathogenic bacteria (Nevas et al., 2004). Rota et al., (2004) found that the essential oil extracted from thymus vulgaris has cytotoxic effect against various pathogenic bacterial strains; Salmonella enteritidis, Salmonella typhimurium, Escherichia coli, Yersinia enterocolitica, Shigella flexneri, Listeria monocytogenes and Staphylococcus aureus. Minimum inhibitory concentration (MIC) was used in determining the inhibition concentration. MIC ranged from 0.1 to 5 µl/ml. Meanwhile, Sacchetti et al. (2005) studied the antimicrobial activity of thyme (Thymus 32
vulgaris) essential oil against various microorganisms i.e. Candida albicans, Rhodotorula glutinis, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Yarrowia lypolitica. The results illustrated that the minimum inhibitory concentrations of thyme essential oil were 0.06, 0.06, 0.06, 0.03 and 0.03 mg/ml, respectively. Thyme and oregano essential oils can inhibit some pathogenic bacterial strains such as E. coli, Salmonella enteritidis, Salmonella choleraesuis and Salmonella typhimurium (Penalver et al., 2005), with the inhibition directly correlated to the phenolic components carvacrol and thymol. Meanwhile, Viuda-Martos et al. (2007) determined the antifungal potential of essential oil of thyme (Thymus vulgaris) against two molds related to food spoilage, Aspergillus niger and Aspergillus flavus. In the case, it can be seen that thyme essential oil reduced fungal growth for A. niger when used at 2 mL/18 mL culture medium, but the total inhibition was only attached when 8 mL was used. Meanwhile, the oil reduced mycelial growth of A. flavus at 2,4 and 6 mL and inhibition was total at 8 mL meaning that A. flavus was more sensitive to thyme essential oil than A. niger. Essential oils and methanol extracts obtained from aerial parts of Thymus vulgaris and Pimpinella anisum seeds were evaluated for their single and combined antibacterial activities against nine Gram-positive and Gram- negative pathogenic bacteria: Staphylococcus aureus, Bacillus cereus, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Salmonella typhi, Salmonella typhimurium, Klebsiella pneumoniae and Pseudomonas aeruginosa. Thyme essential oil inhibits the 90% of growth of the previous bacterial strains at concentrations; 31.2, 15.6, 62.5, 31.2, 62.5, 250.0, 125.0, 500.0 and > 500 µg/ml, respectively (Al-Bayati, 2008). Solomakos et al. (2008) showed that the antibacterial activity of thyme essential oil in different concentrations alone and in combination with nisin on E. coli O157:H7 in minced beef meat under refrigeration condition was an additive effect against the pathogen. They added that the effect of thyme 33
essential oil individually at different concentrations (0.3, 0.6 and 0.9%, v/v) and at different time of incubation (0, 4, 8, 12, 16, 24 and 32 hrs) showed no microbial growth at the concentrations 0.6 and 0.9% after all incubation time while, concentration 0.3% showed 4.1, 2.8, 3.61, 4.69, 5.98 7.1 and 8.3 log cfu/g at the previous incubation time, respectively. On the other hand, Rota et al. (2008) determined the antimicrobial activity of the essential oils of different thyme varieties including Thymus vulgaris (thymol chemotype), Thymus zygis subsp. gracilis (thymol and two linalool chemotypes) and Thymus hyemalis Lange (thymol, thymol/linalool and carvacrol chemotypes). They found that essential oils from T. hyemalis (thymol) followed by T. hyemalis (carvacrol), T. zygis (thymol) and T. vulgaris possessed antimicrobial activities against different pathogenic bacterial strains including Salmonella enteritidis, Salmonella typhimurium, Escherichia coli, Yersinia enterocoltica, Shigella flexneri , Shigella sonnei, Listeria monocytogenes and Staphylococcus aureus. While, Goze et al. (2009) studied the antimicrobial effect of Tyhmus Fallax essential oil - using disc diffusion methods - on several pathogenic strains; Staphylococcus aureus, Escherichia coli, Bacillus subtilis, pseudomonas aeruginosa and Salmonella typhi. The obtained inhibition zones in millimeter were 90, 90, 70, 11 and 38 mm., respectively. In study to evaluate the antibacterial effect of two traditional plants essential oils, Thymus vulgaris and Eucalyptus globulus against clinical isolates of Methicillin resistant Staphylococcus aureus (MRSA) and other standard bacterial strains through disk diffusion and agar dilution methods, Results revealed both of oils to possess degrees of antibacterial activity against Gram (+) and Gram (–) bacteria. Thymus vulgaris essential oil showed better inhibitory effects than Eucalyptus globulus essential oil (Tohidpour et al., 2010). Jia et al. (2010) studied the antimicrobial activities of the essential oils from Thymus marschallianus and Thymus proximus against Escherichia coli, 34
Staphylococcus aureus, Bacillus subtilis, yeast, Rhizopus and Penicillium. Results from the antimicrobial disc-diffusion assay showed that the tested organisms were affected by the 2 thyme essential oils tested and the formed inhibition zones depended on the strain, the kind and the concentration of extract. On the whole, S. aureus was the most sensitive strain for the thyme oils, showing the largest inhibition zone diameter in the presence of the oils, while yeast and Rhizopus were the most resistant strains for essential oils of T. proximus T. marschallianus, respectively. The values of inhibition zones for the tested organisms were in the range of 5.0 - 35.7 mm in diameter, while their MIC values ranged from 1.81 to 4.52 μL/mL. Viuda-Martos et al. (2011) found that the essential oil of thyme showed inhibitory effects on Listeria innocua, Serratia marcescens and Pseudomonas fluorescens with inhibition zones of 41.00, 20.25 and 23.50 mm for these bacteria, respectively. Desai et al. (2012) observed that the thyme oil at concentration of 0.5% resulted in 4 log CFU/mL reduction of Listeria monocytogenes within 30 min in broth, whereas 0.1% concentrations required more than 24 h for the same level of reduction. Gorran et al. (2013) found that the thyme (Thymus daenensis) essential oil could completely inhibit the growth of Aspergillus flavus in the liquid culture at concentration of 375 mg/l. 2.6. Antioxidant activity of essential oils: The free radicals, chemical species or molecules are generated in vivo in form of reactive oxygen spices (ROS) such as superoxide anion, hydroxyl radical, and hydrogen peroxide which are highly reactive. They contain one or more unpaired electrons causing damage to other molecules. Over- production of ROS or called ‘oxidative stress’ causes damage of cellular proteins, lipids, carbohydrates, and DNA which involved in many diseases such as Alzheimer’s disease, mild cognitive impairment, Parkinson’s disease, multiple sclerosis, cardiovascular disease, cardiac failure, and cancer Ali et al., (2008). 35
Therefore, the consumption of foods with natural antioxidants such as fruits, vegetables, nuts, grains, spices, and herbs may provide health benefit. Natural antioxidants have been shown to have ability to protect cells from damage induced by oxidative stress which was considered as a cause of aging, degenerative diseases, and cancer (Yanishlieva et al., 1999). A number of synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), have been developed, but their use has begun to be restricted due to their toxicity (Ito et al., 1983 and Namiki, 1990). Vitamin E (α-tocopherol) is an effective natural antioxidant but has limited usage (Fang and Wada, 1993). As a result, there is considerable interest in the food industry and in preventive medicine in the development of natural antioxidants from botanical sources (Schuler, 1990; Okuda et al., 1993 and Andersson et al., 1996). The oxidation of lipids has long been classified as the major deterioration process affecting both the sensory and the nutritional quality of foods. The principle route of deterioration and possible economic loss of vegetable oils is through rancidity resulting from oxidation which takes place at the double bond sites in the triacylglycerol (lipid) molecules (LH). This free radical chain process proceeds via initiation [reaction (1)], propagation [reactions (2) and (3)] and termination steps [reactions (4) to (6)] as shown by (Yanishlieva and Marinova, 2001):
Initiator
(1) LH (Triacylglycerol molecules) → L• Initiation (2) L• + O • (Peroxyl radical) 2 → LOO Propagation (3) LOO• + LH → LOOH + L• (4) L• + L• → L – L Termination (5) LOO• + L• → LOOL 36
(6) LOO• + LOO• → Non-radical products Hydroperoxides are the primary oxidation products. They are unstable compounds which produce a number of secondary products such as alkanes, alcohols, aldehydes and acids, some of which smell badly at low threshold values. The chain breaking antioxidants AH are able to compete with the substrate for the chain-propagating species, the peroxyl radicals LOO•, which are normally present in high concentration in the system [reaction (7)]:
(7) LOO• +AH (Antioxidant) → LOOH + A• Usually A• is a radical of low reactivity which does not propagate the oxidation chain according to: A• + LH → AH + L•. The efficient inhibitors are well known to terminate free radical chain oxidation by trapping two peroxyl radicals according to reactions (7) and (8).
(8) LOO• + A•→ A – OOL (Non-radical recombination products) The synthetic antioxidants are less expensive than natural antioxidants. It is generally accepted that natural antioxidants are more potent, more efficient and safer than synthetic antioxidants. 2.6.1. Antioxidant activity of cumin essential oil: Singh et al. (2005) observed that the cumin essential oil exerted strong and comparable antioxidative activity in mustard oil system as determined by peroxide and thiobarbituric acid methods as well as the absorbance of the DPPH radical. Allahghadri et al. (2010) studied the antioxidant activity of cumin essential oil. The study revealed that the oil showed higher antioxidant activity compared with that of BHT and BHA. The cumin essential oil exhibited a dose dependent scavenging of DPPH radicals and 5.4 μg of the oil was sufficient to scavenge 50% of DPPH radicals/mL. The antioxidant activity of cumin essential oil might contribute to its cytotoxic activity. 37
Hajlaoui et al. (2010) investigated the antioxidant activity of cumin essential oil using four different tests then compared with BHT. Results showed that cumin essential oil exhibit a higher activity in each antioxidant system with a special attention for β-carotene bleaching test (IC50: 20 µg/ml) and reducing power (EC50: 11 µg/ml). Meanwhile, El-Ghorab et al. (2010) mentioned that the major components in cumin volatile oil are cuminal, γ - terpinene, and pinocarveol which represented antioxidant activity. They added that cumin essential oil is better at reducing Fe3+ ions than dried or fresh cumin. However, antioxidant activities of the methanolic extract and essential oil of Cuminum cyminum in the bulk of purified sunflower oil (PSFO) and emulsion (emulsified PSFO) systems were investigated. On the basis of the different methods of antioxidant activity evaluation, the methanolic extract at levels 800 ppm exhibited higher antioxidative activities than the essential oil at levels (200 – 2000 ppm) (Einafshar et al., 2012). 2.6.2. Antioxidant activity of rosemary essential oil: Rosemary oleoresin was comparable to a commercial mixture of BHA, BHT and citric acid in freeze-dried sausages for inhibition of lipid oxidation. Although commercial antioxidants were more effective inhibiting of lipid oxidation in frozen sausages, rosemary oleoresins significantly reduced lipid oxidation compared to control samples with no antioxidants (Barbut et al., 1988). The effect of increasing levels (150, 300 and 600 ppm) of rosemary essential oil on lipid and protein oxidation and the increase of non-heme iron (NHI) content during refrigeration (+4 °C /60 days) of frankfurters produced with tissues from Iberian pigs or white pigs, was studied. The effect of the addition of rosemary essential oil on the oxidative stability of frankfurters depended on the level of added essential oil and the characteristic of the frankfurter. The rosemary essential oil successfully inhibited the development of lipid and protein oxidation in Iberian pigs and the 38
antioxidant effect was more intense at higher concentrations of essential oil. In white pigs, 150 ppm rosemary essential oil showed an antioxidant effect and significantly reduced the generation of lipid and protein oxidation products. At higher levels (300 and 600 ppm) the essential oil had, in general, no effect on lipid oxidation while significantly enhanced the oxidation of proteins and the release of iron from myoglobin (Estévez and Cava, 2006a).
Estévez et al. (2006b) evaluated the antioxidant effect of essential rosemary oil on protein oxidation, modification of heme (HI) and non-heme iron (NHI) concentrations in refrigerated stored porcine liver pâté (4 °C/90 days) as compared with the synthetic antioxidant (BHT). They showed that rosemary essential oil exhibited similar antioxidant properties to BHT denoting their suitability as alternatives to synthetic antioxidants. The oil significantly lowered the amount of carbonyls from protein oxidation during refrigerated storage, successfully protected heme molecules from degradation and significantly inhibited the increase of NHI in refrigerated stored liver pâtés. Estévez et al. (2007) also studied the antioxidant effect of rosemary essential oil in refrigerated stored liver pâté (4 °C/90 days) in comparison with synthetic antioxidant (BHT). They illustrated that rosemary essential oil significantly lowered the decrease of polyunsaturated fatty acids during refrigerated storage of porcine liver pâté than in samples of control and BHT pâté. Moreover, TBARS numbers in treated pates were significantly smaller than in the control counterparts and the addition of oil significantly reduced the total amount of oxidized lipid-derived volatiles isolated from liver pâté. They added that the essential oil inhibited the oxidative deterioration of liver pates to a higher extent than BHT did. Olmedo et al. (2008) evaluated the antioxidant effect of rosemary essential oil on the oxidative stability of fried–salted peanuts and found that fried–salted peanuts with essential oils showed protection against the lipid 39
oxidation process during storage when compared with the control samples. In other study, the inhibition of linoleic acid peroxidation and bleaching of β- carotene in linoleic acid system were used to determine the antioxidant activity of rosemary essential oil. It was found that rosemary essential oil presented moderate antioxidant activity with 50.7% - 59.1% inhibition of peroxidation and slight decrease in the absorbance of β-carotene (Hussain, 2009).
(Bousbia et al., 2009) mentioned that the microwave hydrodistillation gravity extraction method of rosemary essential oil increased the antioxidant activity of rosemary essential oil and provides a more valuable essential oil (with high amount of oxygenated compounds). Meanwhile, Romano et al. (2009) studied the antioxidant and antibacterial activity of rosemary extract containing 30% carnosic acid, 16% carnosol and 5% rosmarinic acid in vitro alone and in combination with the antioxidant food additives butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). Rosemary enhanced the antioxidant efficiency of BHA and BHT. In study to determine the antioxidant activity of rosemary essential oil, inhibition of linoleic acid peroxidation and bleaching of β-carotene in linoleic acid system were used. The results showed that rosemary essential oil presented moderate antioxidant activity with 50.7% - 59.1% inhibition of peroxidation and slight decrease in the absorbance of β-carotene (Hussain, 2009). Moreover, rosemary (Rosmarinus officinalis L.) plants grow worldwide and have been cultivated since long ago for its strong antioxidant activities. Where, both of rosemary essential oil and their main components act as donors of hydrogen atoms or electrons for the transformation of DPPH into its reduced form DPPH● as described by (Ojeda-Sana et al., 2013). The obtained results showed a significant antioxidant activity of myrcene with an
IC50 4.5 µl/ml followed by α-pinene with an IC50 18 µl/ml. 40
Olmedo et al. (2013) evaluated the effect of rosemary essential oil on the oxidative and fermentative stabilities of flavored cheese prepared with cream cheese base. They found that rosemary essential oil demonstrated a protective effect against lipid oxidation and fermentation in flavored cheese prepared with cream cheese base. Cream cheese with the addition of rosemary essential oil exhibited lower peroxide value than the control samples which had much higher rancid flavor intensities during storage.
2.6.3. Antioxidant activity of thyme essential oil: Lee and Shibamoto (2002) extracted volatile oils from thyme and basil. Antioxidant activities against hexanal oxidation were noticed, especially noteworthy; where, thyme and basil volatile extracts inhibited the oxidation of hexanal for 40 days at the levels of 10μg/mL and 50μg/mL, respectively. Sokmen et al. (2004) evaluated the antioxidant properties of essential oil from Thymus spathulifolius (Hausskn. and Velen.) using the inhibition of free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and β-carotene–linoleic acid systems. They observed that the essential oil was able to reduce the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) with an IC50 of 243.2± 7.20 µg/ml compared to 19.80± 0.50 µg/ml for the synthetic BHT. While the inhibition value of linoleic acid oxidation was calculated as 92% for oil compared to 96% for BHT. Tepe et al. (2005) compared the antioxidant potential of thyme essential oils from two Thymus species (Thymus sipyleus subsp. sipyleus var. sipyleus and Thymus sipyleus subsp. sipyleus var. rosulans) by using 2,2-diphenyl-1- pic-rylhydrazyl (DPPH) and b-carotene/linoleic acid assays. In the first case, the free radical scavenging activity of the essential oil of T. sipyleus subsp. sipyleus var. rosulans was superior to var. sipyleus oil (IC50 ¼ 220 ± 0.5 and 2670±0.5 µg/ml, respectively). In the case of linoleic acid system, oxidation of linoleic acid was effectively inhibited by T. sipyleus subsp. sipyleus var. rosulans (92.0%) and the inhibition rate was close to the synthetic antioxidant BHT (96.0%), while the var. sipyleus oil had no activity. 41
Tomaino et al. (2005) demonstrated that the essential oils of basil, cinnamon, clove, nutmeg, oregano and thyme, traditionally used for their aromatic properties in the preparation of Mediterranean food, exhibit good properties as free radical-scavengers/antioxidants. This fact can support their use to control lipid oxidation during food processing. Lee et al. (2005) identified aroma compounds in the extracts of basil leaves (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L.) by gas chromatography/mass spectrometry (GC/MS). The chromatogram (aroma constituents) observed that linalool, eugenol, thymol, carvacrol, 1,8-cineol, α -terpineol and 4-allylphenol were the main components of basil and thyme leaves. Furthermore, Eugenol, thymol, carvacrol, and 4-allylphenol showed stronger antioxidant activities than did the other components tested in the assay. They all inhibited the oxidation of hexanal by almost 100% for a period of 30 days at a concentration of 5μg/ml compare to those of the known antioxidants, α-tocopherol and butylated hydroxy toluene (BHT). Bounatirou et al. (2007) evaluated the antioxidant activities of thyme essential oils from the aerial parts of Tunisian Thymus capitatus during the different phases of plant development and from different locations. The antioxidant activity of the oils (100–1000 mg / L), as compared with that of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), was assessed by measurement of metal chelating activity, the reductive potential, the free radical scavenging (DPPH) and by the TBARS assay. They found that both the essential oils and BHA and BHT showed no metal chelating activity. Although with the other methodologies, there was a general increase in the antioxidant activity, with increasing oil concentration, maxima being obtained in the range of 500 and 1000 mg/ L for flowering and post- flowering phase oils. Safaei-Ghomi et al. (2009) determined the antioxidant activity of thyme essential oil from Thymus caramanicus as well as the oil main component (carvacrol) by means of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and β- 42
carotene/linoleic acid bleaching assays. In DPPH test, the concentrations that led to 50% inhibition (DPPH IC50) were found to be 263.09 ± 0.62 and 448.05 ± 3.62 µg/ml for oil and carvacrol, respectively, while was 19.72 ± 0.80 µg/ml for the standard commercial synthetic antioxidant (BHT). Meanwhile, the percentages of β-carotene/linoleic acid inhibition were 79.03 ± 0.54 %, 50.18 ± 0.34 % and 93.3% for oil, carvacrol and BHT, respectively.
Jia et al. (2010) concluded that the thyme essential oils (from T. marschallianus and T. proximus) had antioxidant properties and were effective scavengers of free radicals as they contained higher concentration of phenolic components. On the other hand, Sarikurkcu et al. (2010) extracted Thymus longicaulis subsp. longicaulis var. longicaulis using solvent extraction (hexane, ethyl acetate, methanol and water). The extracts attained the remarkable activity to prevent lipid oxidation. In general, methanol and water extracts exhibited the strongest activity profiles. Viuda-Martos et al. (2011) determined the antioxidant activity of the Egyptian essential oils from thyme (Thymus vulgaris) and other spices by means of three different antioxidant tests. They showed that the thyme oil presented the best antioxidant profile giving the highest percentaage of inhibition of TBARS (80.76 %). Anthony et al. (2012) evaluated the antioxidant and scavenging activities of different thyme essential oils from different countries using series of dilutions to estimate their EC50 (DPPH
EC50 μg/mL) and found that the EC50 of these oils ranged from 260 to 480 μg/mL. While, Teixeira et al. (2013) determined the antioxidant activity of thyme essential oil as measured by the DPPH method and found that its EC50 was 0.25 ± 0.01 mg/mL, while its antioxidant activity index was 0.3±0.0. El Bouzidi et al. (2013) screened the wild and cultivated thyme essential oils for their possible antioxidant activity by two test systems, namely DPPH free radical scavenging and the ferric ion reduction assay. They observed that all essential oils obtained from wild and 43
cultivated Thymus species showed antioxidant activity in both assays. For the DPPH assay, the most potent oil was obtained from wild and cultivated T. maroccanus (IC50 = 82.87 ± 2.41 µg/mL and 88.42 ± 3.88 µg/mL, respectively), but they were less potent than the pure compounds used as positive controls, namely the synthetic antioxidant
BHT (IC50 = 4.21 ± 0.08 µg/mL) and the flavonol quercetin (IC50 = 1.07 ± 0.01 µg/mL). Similarly, the highest ferric reductive capacity resided with the oils obtained from wild and cultivated T. maroccanus
(EC50 = 139.31 ± 1.08 and 149.41 ± 1.13 µg/mL, respectively) compared with quercetin (EC50 = 2.29 ± 0.1 µg/mL) and BHT (EC50 = 7.09 ± 0.1 µg/mL). 2.7. Effects of irradiation on the antimicrobial and antioxidant activity of essential oils: The germicidal activity of irradiated orange essential oil was studied by Harris et al. (1930), who found that irradiated orange essential oil has no germicidal effect on selected pathogenic bacterial strains. Several studies on spices treated with doses up to 10-15 kGy have shown that no substantial changes occurred in volatile oils, flavour profiles, spicing power as mentioned by IAEA (1992) and in the antioxidants properties as mentioned by Kuruppu et al. (1985). EL-Khawas (1995) studied the antioxidant activity of gamma irradiated cumin essential oil at dose 10 kGy. He found that the antioxidant activity of irradiated cumin essential oil was increased related to irradiation dose. Antioxidant properties of caraway, cumin, anise and fennel essential oils extracted from untreated, γ-irradiated and microwaved seeds were evaluated by Farag and EL-Khawas (1998). As they found, neither γ-irradiation at 10 kGy nor microwave treatments affects the antioxidant status of the essential oils under study in any way. 44
By using ionizing radiation at dose of 10 kGy, a satisfactory result of microbiological decontamination of medical herbs could be observed. The content of essential biologically active substances such as essential oils, flavonoids, glycosides, anthocyans, antra-compounds, poliphenoloacids, triterpene saponins, oleanosides and plants mucus did not significantly change after irradiation. Pharmacological activity of medicinal herbs has been found satisfactory after microbiological decontamination by irradiation (Owczarczyk et al., 2000). Fan and Sokorai (2002) investigated the effect of irradiation on volatile compounds of fresh cilantro leaves. Fresh cilantro leaves (Coriandrum sativum L.) were irradiated with 0, 1, 2, or 3 kGy γ-radiation and then stored at 3 °C up to 14 days. The obtained results conducted that the amounts of linalool, dodecanal, and (E)-2-dodecenal in irradiated samples were significantly lower than those in non-irradiated samples at day 14. Khattak and Ali (2004) studied the antifungal activity of irradiation doses on the antimicrobial activity of Nigella sativa. The obtained results showed that the antifungal activity did not affect after irradiation doses. On the other hand, Kitazuru et al. (2004) studied the effect of ionizing radiation on natural cinnamon antioxidants. Samples were irradiated with 60Co using doses 0, 5, 10, 15, 20, 25 kGy at room temperature. The study illustrated that irradiation did not abolish the antioxidant activities of cinnamon to a great extent; although a small decrease was observed at 20–25 kGy. Topuz and Ozdemir (2004) irradiated sun-dried and dehydrated paprika samples at doses from 2.5 to 10 kGy and analyzed capsaicinoid contents. They found that the contents of capsaicin, dihydrocapsaicin and homodihydrocapsaicin increased significantly by about 10% in samples irradiated at a dose of 10 kGy. There are not many reports of the influence of irradiation procedures on antioxidant activity of herbs and spices. The effects of this processing technique on antioxidant properties of seven dessert spices (anise, cinnamon, 45
ginger, licorice, mint, nutmeg, and vanilla) were evaluated by Murcia et al. (2004). With respect to the non-irradiated samples, water extracts of irradiated spices at 1, 3, 5, and 10 kGy did not show significant differences of antioxidant activity in the radical scavenging assays used. Khattak et al. (2008) studied the effect of irradiation doses on the antioxidant activity of irradiated Nigella sativa at doses 0, 2, 4, 6, 8, 10, 12 and 16 kGy using DPPH scavenging radical activity. The results found that, in the control samples, the DPPH radical-scavenging activity was 79.4%, 79.1% and 92.0% for water, acetone and methanol extracts, respectively, at 5 mg/ml concentration. Thus, gamma irradiation enhanced the scavenging activity in acetone and methanol extracts by 10.6% and 5.4%, respectively, at 16 kGy. In addition, the same trend of the preceding study for the same authors Khattak et al. (2009) but on the antioxidant activity of Nelumbo nucifera rhizome irradiated at doses 0, 1, 2, 4 and 6 KGy was investigated. The results showed that free radical scavenging activity was enhanced after gamma irradiation. Alothman et al. (2009) summarized that the effects of ionizing (gamma and electron beam) radiation on the compositional changes induced in health-promoting phytochemicals and antioxidants of plant origin. The main advantage was, the short processing time; in addition to the minor effect irradiation had on the antioxidants in plant produce. Meanwhile, Kim et al., (2009) studied the effect of gamma irradiation on the natural antioxidants of cumin essential oil by evaluating the radical-scavenging effect on the 1,1- diphenyl-2-picryl hydrazyl (DPPH) radicals, determination of ferric reducing antioxidant power (FRAP), total polyphenol content (TPC) and the antioxidant index by β-carotene/linoleic acid co-oxidation. Cumin seeds were irradiated in a cobalt-60 irradiator to 0, 1, 3, 5 and 10 kGy at ambient temperature. The results showed that there was a good maintenance and slight increase in the DPPH scavenging activity. A significant increase in the antioxidant index was found in the 5 and 10 kGy irradiated cumin. Reducing power of all the irradiated cumin was maintained to that of the non- 46
irradiated. A slight increase and maintenance in the total polyphenolic content was found in all irradiated cumin. Lacroix et al. (2009) studied the effect of oregano essential oil alone and in combination with gamma irradiation on the radiosensitivity of Listeria monocytogenes and E. coli O157: H7. The results indicated that, the use of essential oils in combination with irradiation has permitted an increase of the bacterial radiosensitization by more than 3.1 times and that combination effect was synergistic. Shim et al. (2009) studied the effect of γ-irradiation at doses of 1, 3, 5 and 10 kGy on the contents of volatile compounds from Paeoniae Radix such as paeonol, carveol, eugenol acetate. They found that the concentration of this compound increased with the increase of irradiation dose level. Khattak and Simpson (2010) irradiated Glycyrrhiaz glabra roots at doses 0, 5, 15, 20 and 25 kGy to represent the effect of irradiation doses on the chemical composition, the phenolic content, antimicrobial activity and DPPH scavenging properties. The results revealed that the phenolic content increased in samples irradiated with 20 and 25 kGy, these results may be beneficial for plant antioxidant activity as well as, the DPPH scavenging activity increased in all irradiated samples. While, the antibacterial and antifungal activities did not affected up to 20 kGy. Fatemi et al. (2011) suggested that the conventional method of gamma irradiation used for preservation of medicinal plants sustains the composition and biochemical properties of the caraway essential oils even at sterilization dose of 25 kGy. Gamma-irradiation maintained the antioxidant properties of caraway oils as judged by DPPH and β-carotene bleaching tests. These evidences together with the results of GC/MS analysis clearly show that there are no major changes in active components and properties of caraway seeds following irradiation. 47
2.8. Synergistic effect of essential oils as antimicrobials and antioxidants: Synergy needs to be distinguished from simple dose addition or additive responses. Synergy represents a form of interaction / dynamic interplay as opposed to a simple addition response. It is also important to acknowledge that synergy is not necessarily always positive – antagonism can occur as well as potentiation and that both situations can exist within a given essential oil and within a blend of essential oils depending on the dose, application and properties (Harris, 2002). Despite the high efficiency of the essential oils and their constituents against food-borne pathogens and spoilage microorganisms when in vitro tests are conducted (Chorianopoulos et al., 2004 and Fisher and Phillips, 2006), the same effect in food is only achieved with higher concentration of essential oils (Burt, 2004 and Hulin et al., 1998). This fact may imply an organoleptic impact, caused by altering the natural taste of the food by exceeding the acceptable flavor thresholds (Hsieh et al., 2001 and Nazer et al., 2005). Few approaches have been proposed to minimise essential oil concentrations and reduce the sensory effect. One solution would consist of combining plant extracts. Although essential oils were concluded to have greater activity than mixtures of their major components (Gill et al., 2002 and Mourey and Canillac, 2002), the combination of these major components with other constituents with a weaker activity might result in a synergistic, additive or antagonist effect (Ultee et al., 2000). Fu et al. (2007) studied the combination of clove and rosemary essential oils on the antimicrobial activity against Staphylococcus epidermidis, Escherichia coli and Candida albicans. The results showed that both essential oils possessed significant antimicrobial effects against all microorganisms tested. The MICs of clove oil ranged from 0.062% to 0.500% (v/v), while the MICs of rosemary oil ranged from 0.125% to 48
1.000% (v/v). The antimicrobial activity of combinations of the two essential oils indicated their additive, synergistic or antagonistic effects against individual microorganism tests. The time-kill curves of clove and rosemary essential oils towards three strains showed clearly bactericidal and fungicidal processes of 1/2 × MIC, MIC, MBC and 2 × MIC. In a study to evaluate the efficacy of plant essential oils in combination as antimicrobial agent and to investigate the effect of food ingredients on their efficacy; the essential oils of lemon balm, marjoram, oregano, rosemary, sage and thyme were used. Fractional inhibitory concentrations (FIC) were calculated and interpreted as synergy, addition, indifference or antagonism. The results showed that all the oregano combinations showed additive efficacy against B. cereus, and oregano combined with marjoram, thyme or basil also had an additive effect against E. coli and P. aeruginosa. The mixtures of marjoram or thyme also displayed additive effects in combination with basil, rosemary or sage against L. monocytogenes (Gutierrez et al., 2008a). Gutierrez et al. (2009) evaluated some essential oils (lemon balm, marjoram, oregano and thyme) and their combinations for their antimicrobial activies; where, the minimum inhibitory concentrations were determined against Enterobacter spp., Listeria spp., Lactobacillus spp., and Pseudomonas spp. The results showed that the essential oil of oregano combined with thyme had an additive effect against spoilage organisms. Combining lemon balm with thyme yielded additive activity against Listeria strains. Rosato et al. (2009) investigated the synergistic effects between essential oils (O. vulgare, P. graveolens and M. alternifolia) and the antifungal compound Nystatin against Candida species. The results showed that an experimental occurrence of a synergistic interaction between O. vulgare and M. alternifolia essential oils and Nystatin. Some combinations of Nystatin and P. graveolens essential oils did not have any synergistic interactions for 49
some of the strains considered and associations of Nystatin with M. alternifolia essential oil had only an additive effect. Saad et al. (2010) studied a possible synergistic effect of the essential oils of two Moroccan endemic thymes (Thymus broussonetii and Thymus maroccanus) with amphotericin B and fluconazol against Candida albicans. The fractional inhibitory concentration indices of Thymus maroccanus and Thymus broussonetii essential oils combined with amphotericin B and fluconazol, calculated from the checkerboard titer assay. Results indicate that the synergistic effect of essential oils with fluconazol was stronger than the combination with amphotericin B. All these data highlight that the essential oils tested potentiate the antifungal action of amphotericin B and fluconazol, suggesting a possible utilization of these essential oils in addition to antifungal drugs for the treatment of some candidiasis due to Candida albicans. 2.9. Utilization of essential oils in food industry: The increased demand for safe and natural food, without chemical preservatives, provokes many researchers to investigate the antimicrobial effects of natural compounds. Numerous investigations have confirmed the antimicrobial action of essential oils in model food systems and in real food (Koutsoumanis et al., 1998 and Tsigarida et al., 2000). In addition, many natural compounds found in dietary plants, such as extracts of herbs and fruit extracts, possess antimicrobial activities against L. monocytogenes (Cowan, 1999; Hao et al., 1998 and Kim et al., 1995). Several constituents of essential oil exhibit significant antimicrobial properties when tested separately (Kim et al., 1995 and Lambert et al., 2001). However, there is evidence that essential oils are more strongly antimicrobial than is accounted for by the additive effect of their major antimicrobial components; minor components appear, therefore, to play a significant role. Since essential oils are considered as generally recognized as safe (GRAS) (Kabara, 1991), the possibility of reinforcing their natural 50
antimicrobial effects by the addition of small amounts of other natural preservatives may be a way to attain a balance between sensory acceptability and antimicrobial efficacy. Billing and Sherman (1998) had two hypotheses about how people started using spices. First, people who used spices, especially in hot countries, suffered less from foodborne illnesses and stored their food for longer periods of time. Second, adding spices changed the taste and flavor of food, and made it more palatable and safe for consumption. In the food industry, rosemary is a very frequently used herb and its extracts are added to some products to improve their oxidative stability and to ameliorate the organoleptic profiles (Sui et al., 2012). 2.10. Application of synergistic effect of essential oils on some food products: Spices and herbs can be used as an alternative preservative and pathogen- control method in food materials. Generally, effective essential oils in decreasing order of antimicrobial activities are: oregano > clove > coriander > cinnamon > thyme > mint > rosemary > mustard > cilantro/sage (Burt, 2004). There are differences between in vitro and in-food trials of plant origin antimicrobials, mainly because only small percentages of essential oils are tolerable in food materials. Finding the most inhibitory spices and herbs depends on a number of factors such as type, effects on organoleptic properties, composition and concentration and biological properties of the antimicrobial and the target micro-organism and processing and storage conditions of the targeted food product (Naidu, 2000, Gutierrez et al., 2008a and Romeo et al., 2008). Despite some positive reports in regard to application of plant-origin natural antimicrobials, two major issues are faced regarding application of plant- origin antimicrobials in food: odors created mostly by the high concentrations, and the costs of these materials (Silva et al., 2007 and Proestos et al., 2008). 51
Sadeghi et al. (2012) evaluated the effect of Cuminum cyminum L. essential oil and Lactobacillus acidophilus (a probiotic) on growth of Staphylococcus aureus in white brined cheese. The experiment included different levels of essential oils (0, 7.5, 15 and 30 mL/ 100 mL milk) and L. acidophilus (0 and 0.5%) to assess their effects on S. aureus count during the manufacture, ripening and storage of Iranian white brined cheese for up to 75 days. The obtained results revealed that the best inhibitory effect was obtained at combination of 15 mL/100 mL cumin essential oil and 0.5% probiotic.
3. MATERIALS AND METHODS 3.1. Materials: 3.1.1. Spices: Cumin (Cuminum cyminum L.), rosemary (Rosmarinus officinalis L.) and thyme (Thymus vulgaris L.) were obtained at 2011 from AMD verde company, Cairo, Egypt. 3.1.2. Ingredients: Whole Egg, Lemon juice, Vinger, Mustard, Sugar, White pepper were purchased from local market. While, Sunflower oil (Refined and free from artificial antioxidants) was purchased from ARMA for food industry Co., 10th of Ramadan 3.1.3. Chemicals: Butylated hydroxytoluene (BHT) as a synthetic antioxidant (of purity 99.9 %) was purchased from Naarden International U.K. Limited, England, [Polyoxyethylene (20) sorbitan monooleate (Tween 80), Dimethyl sulfoxide (DMSO) and Barium chloride (BaCl2) were purchased from Alpha Chemika™, Mumbi, India], Sodium chloride was obtained from Cook΄s Company, United food Industries, 6th October City, Egypt and 2,2- diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich, Germany.
3.1.4. Microbial strains: Escherichia coli, Salmonella typhimurium, Bacillus cereus and Staphylococcus aureus strains were obtained from the Egyptian Microbial Culture Collection (EMCC) Faculty of Agriculture, Ain Shams University, Cairo, Egypt. While Aspergillus flavus strain was obtained from the Regional Center for Mycology and Biotechnology, Faculty of Science, Elazhar University, Cairo, Egypt. 53
3.1.5. Microbiological media: All of the following microbiological media (Oxoid, 2006) were purchased from the most famous companies of chemicals in Cairo, Egypt. 3.1.5.1. Nutrient agar: The medium used for bacterial growth was consisted of: Lab Lemco powder 1.0 gm. Yeast extract 2.0 gm. Peptone 5.0 gm. Sodium chloride 5.0 gm. Agar 15.0 gm. Distilled water 1.0 L pH 7.4 ± 0.2 3.1.5.2. Mueller Hinton Agar: This medium was used for bacterial growth consisted of: Beef, dehydrated infusion form 300 gm. Casein hydrolysate 17.5 gm. Starch 1.5 gm.
Agar 17 gm. Distilled water 1.0 L pH 7.3 ± 0.2 3.1.5.3. Mueller Hinton Broth: This medium was used for bacterial growth consisted of: Beef, dehydrated infusion form 300 gm. Casein hydrolysate 17.5 gm. 54
Starch 1.5 gm. Distilled water 1.0 L pH 7.3 ± 0.1 3.1.5.4. Sabouraud`s Dextrose Agar: This medium was used for fungal growth consisted of: Glucose 40 gm. Mycological peptone 10 gm. Agar 15 gm. Distilled water 1.0 L pH 5.6 ± 0.2 3.1.5.5. Potato Dextrose Agar: This medium was used for fungal growth consisted of: Glucose 20 gm. Potato extract 4 gm. Agar 15 gm. Distilled water 1.0 L pH 5.6 ± 0.2
3.1.5.6. Peptone Water: A basal medium was used for inoculation the bacterial strains Peptone 10 gm. Sodium chloride 5 gm. Distilled water 1.0 L pH 5.6 ± 0.2 55
3.1.5.7. Brilliant green agar: A selective medium used for the isolation of Salmonellae Protease peptone 10 gm. Yeast Extract 3 gm. Lactose 10 gm. Sucrose 10 gm. Sodium chloride 5 gm. Phenol red 0.08 gm. Brilliant green 0.0125 gm. Agar 12 gm. Distilled water 1.0 L pH 6.9 ± 0.2 3.1.5.8. Selenite broth base (Lacotse): Peptone 5 gm. Lactose 4 gm. Sodium phosphate 10 gm. Distilled water 1.0 L pH 7.1 ± 0.2 3.2. Methods: 3.2.1. Extraction of essential oils: The essential oils of cumin (C), rosemary (R) and thyme (T) were individually extracted by steam distillation using a stainless steel apparatus then the essential oils yield was measured according to Guenther (1961). The extracted volatile oils from each spice were collected, dried over anhydrous sodium sulphate, divided into 11 dark glass bottles and kept at – 20 oC until needed. 56
3.2.2. Irradiation of essential oils: Samples of the extracted essential oils were subjected to gamma irradiation at doses of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 kGy. Irradiation treatments were carried out using an experimental 60Co Russian Gamma chamber (providing a dose rate of 3.7 kGy/hr), Cyclotron Project, Nuclear Research Center, Atomic Energy Authority, Egypt. 3.2.3. Analysis of the extracted essential oils: 3.2.3.1. Percentages of essential oils: The percentages of essential oil contents in cumin, rosemary and thyme spices were determined according to AOAC (1996). 3.2.3.2. Physiochemical properties: 3.2.3.2.1. Specific gravity: The specific gravity of essential oil was determined at 20 oC using a pycnometer 10 cm3, Guenther (1961). 3.2.3.2.2. Refractive index: The refractive index value of the essential oil was determined at room temperature using Abbe refractometer. A correction factor of 0.00044 was used for every degree above or below 25 oC, Guenther (1961). 3.2.3.2.3. Acid value:
The acid value was determined according to the procedure stated by Guenther (1961). 3.2.3.3. Identification of chemical components of essential oils: The Gas Chromatography / Mass Spectrometry (GC/MS) technique was used for separation and identification of chemical components of non- irradiated cumin, rosemary and thyme essential oils as well as those irradiated at the chosen doses, based on the results of preliminary experiments for antimicrobial and antioxidant activity. Analyses were performed using a TRACE 2000 GC-MS (THERMO) equipped with a flame 57 ionization detector. A capillary column (30 M length and 0.25 mm ID) packed with DB-5 (5% phenyle) methylpolysiloxane was used for separation, while the instrument was operated under electron-impact (EI) mode at 70 ev. The auxzone and injector temperatures were 250 oC and 220 oC, respectively. Helium was used as a carrier Gas at flow rate of 20 cm/sec. The oven Temperature was kept at 40 oC for 2 min and increased up to 250 oC at a rate of 5 oC /min, then kept constant at this temperature for 3 min. The injected sample was 1 μl (Kose et al., 2010). 3.2.4. Experiments for antimicrobial activity of the essential oils: 3.2.4.1. Evaluation of antimicrobial activity using agar well-diffusion method: Agar well-diffusion method was performed according to the method designed by Bennett et al. (1966) 3.2.4.1.1. Microbial inoculation: A suspension from each of the studied microbial strains was prepared in a sterilized peptone water to give a count of 107 -108 cfu/ml for the organism. The density of organism suspension was adjusted using the 0.5 McFarland
Standard which made as follow; Add 0.5 ml of 0.048 M BaCl2 (1.17% w/v
BaCl2. H2O) to 99.5 ml of 0.18 M H2SO4 (1% v/v) with constant stirring. Distribute the standard into screw cap tubes of the same size and with the same volume as those used in growing the broth cultures. Seal the tubes tightly to prevent loss by evaporation. Store protected from light at room temperature. Vigorously agitate the turbidity standard on a vortex mixer before use. Standards may be stored for up to 6 months, after which time they should be discarded (Andrews, 2001). 3.2.4.1.2. Preparation of plates: Seven ml of the appropriate sterilized media (Mueller Hinton Agar for bacteria and Potato Dextrose Agar for fungi) were poured into a sterile 90 mm Petri dish and allowed to harden. Further 7.0 ml. portion of the same media but inoculated with a suspension of the tested organism was poured 58 over the surface of the harden media in the dish and allowed to harden (Andrews, 2001). 3.2.4.1.3. Antimicrobial activity assay: The antibacterial activity of irradiated and non-irradiated control essential oils under investigation was assayed against the examined microorganisms using the prepared plates as described by Pérez et al. (1999). Three wells were symmetrically formed around the center in the plate containing the seeded media using a sterile cork borer. Portions of 30, 50 and 70 μl of the studied essential oil were transferred individually to the three formed wells in the plate and diffusion was allowed in the refrigerator over-night. Plates which inoculated with bacteria were incubated at 37 oC / 48 hr, while those inoculated with the fungus were incubated at 25 oC / 72 hr, then the inhibition zone was measured in mm after the incubation period. 3.2.4.1.4. Choosing of the optimum irradiation dose: Based on the observed results, samples of the examined essential oils which showed the highest antimicrobial activity were recorded. Subsequently, the optimum irradiation doses for each of the studied essential oils were chosen to be 2 and 4 kGy for cumin essential oil, 6 kGy for rosemary essential oil and 4 kGy for thyme essential oil. These irradiation doses are also used in the making binary mixtures (1:1 v/v) from both non-irradiated and irradiated essential oils.
3.2.4.2. Measurement of Synergy: The synergistic antibacterial effect of both non-irradiated and the chosen irradiated cumin, thyme and rosemary essential oils were calculated for each of Escherichia coli, Staphylococcus aureus, Bacillus cereus and Salmonella typhimurium separately as follows: 59
3.2.4.2.1. Evaluation of Minimum Inhibitory Concentration (MIC): 3.2.4.2.1.1. Preparation of essential oil dilutions in Dimethy sulfoxide (DMSO):
Prepare 3 bottles; the first one contains 10000 ppm, the second one contains 1000 ppm and the last one contains 100 ppm. All the previous bottles contain essential oil diluted in DMSO. Then, label 11 universal containers as follows: 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0 ppm.
From the 10000 ppm stock, dispense the following amounts with a micropipette:
256 µl into the container labeled 128
128 µl into the container labeled 64
64 µl into the container labeled 32
32 µl into the container labeled 16
From the 1000 ppm stock, dispense the following amounts with a micropipette:
160 µl into the container labeled 8
80 µl into the container labeled 4
40 µl into the container labeled 2
From the 100 ppm stock, dispense the following amounts with a micropipette:
200 µl into the container labeled 1
100 µl into the container labeled
50 µl into the container labeled 0.25 60
The previous methods according to Andrews, 2001.
3.2.6.1.1. Evaluation of Minimum Inhibitory Concentration (MIC):
Dilutions of cumin, rosemary and thyme essential oil were made for evaluation of the interactions. The type of interaction was studied on the Escherichia coli, Staphylococcus aureus, Bacillus cereus and Salmonella typhimurium bacterial strain. Dilutions from the 0.5 McFarland bacterial cultures were prepared and distributed into varying concentrations of the different essential oils (each from cumin, rosemary and thyme). The inoculated tubes were incubated at 37 °C for 24 h and then evaluated for bacterial growth. Whereas, the minimum inhibitory concentration was defined as the lowest essential oil concentration that results in no visible growth (turbidity) (Vigil et al., 2005).
3.2.4.2.2. Determination of Fraction Inhibitory Concentration (FIC): To assess the activities of oil combinations, fraction inhibitory concentration was determined based on the determined MIC and using the checker board methods (Houghton, 2009) as follows:
Where: FICI = the fractional inhibitory concentration indices were calculated
as FICA + FICB, where, FICA and FICB are the minimum concentrations that inhibited the bacterial growth for oil samples A and B, respectively.
MICA combination = MIC of oil A in the presence of oil B
MICB combination = MIC of oil B in the presence of oil A
MICA alone = MIC of one single oil sample 61
MICB alone = MIC of another single oil sample From the observed FIC for each of oils, the mean fractional inhibitory concentration (FIC) indices were calculated as follows:
FICI = FICA + FICB Then the results of FICI were categorized as synergism, addition, indifference or antagonism as follows: synergistic (< 0.5), additive (0.5 –1.0), indifferent (>1– 4) or antagonistic (> 4.0). 3.2.4.2.3. Use of Isoboles and Isobolograms: Isobolograph analysis was performed according to Tallarida (2001), to evaluate the presence of synergism or antagonism. It requires experimental data for agents used alone and in different dose combinations at equieffective levels. The data is plotted on graphs as shown in Figure (3) with the axes representing the doses of each agent. If two agents do not interact, the line that forms with the points corresponding to the different combination of doses representing the sum of the effects will be a straight line. When agents in combination are more effective than what might be expected from their dose-response curves (synergy), smaller amounts will be needed to produce the effect under consideration, and a concave-up isobole results. On the other hand, when agents in combination are less effective than expected (antagonism), greater doses than expected will be needed to produce the same effect, and a concave-down isobole is generated. Different doses of the compounds were selected using the checkerboard method (Davidson and Parish, 1989). 62
2.0
Antagonism 1.5
Addition 1.0
MIC Effect (A) Effect MIC Synergism 0.5
0.0 0.0 0.5 1.0 1.5 2.0 MIC Effect (B) Fig. 3. Isobologram to show areas of synergy, addition and antagonism 63
3.2.4.3. Evaluation of the minimum bactericidal concentration (MBC) of essential oils: The Minimum bactericidal concentration (defined as the essential oil concentration which kills 99.9% or more of the initial inoculum) was determined for the examined essential oils and their mixtures against the same bacterial strains (Escherichia coli, Staphylococcus aureus, Bacillus cereus and Salmonella typhimurium) separately according to Vigil et al. (2005). Tubes of each bacterial suspension containing the different concentrations of essential oils were prepared as mentioned above (in the determination of the minimum inhibitory concentration). However, tubes were prepared with individual essential oils as well as with a mixture of two oils (1:1v/v) interchangeably but representing the same concentrations of the individual oils. For each of individual oils and essential oils mixtures, tubes of 4 concentrations were chosen (representing the minimum inhibitory concentration in addition to the first lower concentration and the first two higher concentrations). For each bacterial strain, inoculums from each of the prepared tubes were transferred into Mueller Hinton Agar plates. Then plates were incubated at 37 oC for 48 hr and checked for viable colonies. Plates with no viable colonies presented the MBC of the examined oil or oils mixture. 3.2.4.4. Evaluation of the antimicrobial activity of essential oils mixtures in mayonnaise (as a food system): 3.2.4.4.1. Preparation of control mayonnaise samples: Mayonnaise was prepared as described by (Kishk, 1997) except that whole egg was substituted with egg yolk only according to (Xiong et al., 2002). Therefore, the main formula of mayonnaise was consisted of sunflower oil (70.28 %), egg Yolk (22.17 %), lemon juice (2.20 %), vinegar (0.63 %), mustard flour (2.51 %), Sugar (0.64%), salt (1.26 %) and white pepper (0.31 %). 64
Ingredients of salt, sugar, mustard and white pepper were first mixed with the egg yolk, vinegar and lemon juice using electronic mixer on puree velocity (low speed) for about 5 seconds. The oil was then slowly (drop by drop) added to the system during the first 30 seconds and more rapidly after the mass begins to thicken with continued mixing for 30 sec at the same velocity followed by mixing on blend velocity (medium speed) for 20 seconds. Then all ingredients were mixed on liquefy velocity (maximum speed) for 15 seconds. 3.2.4.4.2. Determination of the maximum acceptable concentration of essential oils in mayonnaise: The maximum acceptable concentration of the studied essential oils in mayonnaise was determined through sensory evaluation tests. For each of the examined oils (cumin, thyme and rosemary essential oils), different mayonnaise samples were prepared by adding different concentrations of the individual non-irradiated essential oil or oils mixture (1:1 v/v) to the sunflower oil. The volume of the added essential oil was deducted from the main volume of the sunflower oil, while the final concentrations of the added essential oil in mayonnaise (w/w) were 0.0%, 0.5%, 0.7% and 0.9% for cumin essential oil, 0.0%, 0.2%, 0.4%, and 0.6% for thyme essential oil and 0.0%, 0.4%, 0.6% and 0.8% for rosemary essential oil. The different samples of the prepared mayonnaise were subjected to sensory evaluation for their odor and taste. Sensory evaluation was performed by nine non-expert members of our laboratory (Food irradiation unit) using a 9-point scale, where 9 = like extremely, 8 = like very much, 7 = like moderately, 6 = like slightly, 5 = neither like nor dislike, 4 = dislike slightly, 3 = dislike moderately, 2 = dislike very much and1 = dislike extremely according to (Pajohi et al., 2011). Based on the results of sensory evaluation, the maximum acceptable concentrations of the individual essential oils in mayonnaise (w/w) were determined and the possible concentrations of the mixtures of essential oils were selected to be 0.4% for 65 cumin essential oil, 0.1% for thyme essential oil, 0.2% for rosemary essential oil, 0.2% for essential oils mixtures. 3.2.4.4.3. Preparation of mayonnaise samples : 3.2.4.4.3.1. Un-inoculated mayonnaise samples: Based on the results of sensory evaluation of mayonnaise samples and the highest determined antimicrobial properties of selecting irradiation doses for essential oils (well-diffusion method), seven groups of mayonnaise samples were prepared using sunflower oil only (control) as well as sunflower oil containing the different mixtures of essential oil mixtures at concentration 0.2% (w/w) as follow: 1- Sunflower oil (0% essential oils).
2- Non-irradiated cumin oil + Non-irradiated thyme oil (C0 X T0).
3- Non-irradiated cumin oil + Non-irradiated rosemary oil (C0 X R0).
4- Non-irradiated rosemary oil + Non-irradiated thyme oil (R0 X T0).
5- 4kGy irradiated cumin oil + 4kGy irradiated thyme oil (C4 X T4).
6- 4kGy irradiated cumin oil + 6kGy irradiated rosemary oil (C4 X R6).
7- 6kGy irradiated rosemary oil + 4kGy irradiated thyme oil (R6 X T4). Note: concentration of each of the added essential oils mixture was based on the maximum acceptable concentration of oils having the lower acceptable value. Samples of the prepared mayonnaise were left at room temperature for 24 hr before analysis. 3.2.4.4.3.1.1. Microbiological analysis of un-inoculated mayonnaise: For samples of the un-inoculated mayonnaise, 25 g samples were aseptically removed to prepare the initial 1/10 dilution which was used for the preparation of other serial dilutions in 0.1% peptone water. Then total 66 bacterial count was determined in by plating on plate count agar medium and incubation at 30˚C for 3 days according to APHA (1992). 3.2.4.4.3.2. Inoculated mayonnaise samples: As with the un-inoculated mayonnaise samples, other similar groups of samples were prepared under controlled conditions using decontaminated ingredients, in which, salt, sugar, white pepper mustard and egg yolk were gamma irradiated at dose 5 kGy for decontamination (Muhamad et al., 1986) and keeping the sensory evaluation and aroma (Farkas, 1988), while lemon juice and vinegar were exposed to UV irradiation for 20 min., and distributed in appropriate vials of 100 ml capacity. Then samples were inoculated with a suspension of Salmonella typhimurium (as a possible pathogen of concern in mayonnaise) in peptone water to give approximately 105 cfu of the organism per gram sample. The inoculated samples were well mixed for better distribution of the inoculum in samples and stored at 37 oC for 72 hr (Xiong et al., 2002). 3.2.4.4.3.2.1. Microbiological analysis of inoculated mayonnaise: For the inoculated mayonnaise samples, 10 g contaminated mayonnaise was then added to 90 ml of 1% Buffered Peptone Water and the mixture was homogenized well for 1 min. Serial tenfold dilutions in 0.1% peptone diluent were plated onto Briliant green (BG) agar using the surface spread method. The BG agar plates were incubated at 37 oC for at least 24 hr. and then the viable colonies were enumerated (Xiong et al., 2002). 3.2.5. Experiments for antioxidant activity of the essential oils: 3.2.5.1. Determination of the maximum acceptable concentration of essential oils in sunflower oil: The essential oil of cumin, rosemary and thyme were studied in this trail to detect the proper concentration of each essential oil which can be used as antioxidant for sunflower oil. Sensory evaluation test was performed to illustrate the effect of adding cumin, rosemary and thyme essential oils 67
(individually) to sunflower oil according to the method reported by Ranganna (1978). To sets of beakers 20ml (6 each) containing 10ml sunflower oil were mixed separately with different concentration (0.0%, 0.1%, 0.2%, 0.3%, 0.4% and 0.5%, v/v) of each cumin, rosemary and thyme essential oil. The odour was checked by smell in an ascending order according to essential oil concentration using an appropriate blotter. Sensory evaluation was carried out by eleven non-expert members of food irradiation unit and the intensity of the odor was described according to El-Baroty (1988) using the following scale: 0 = no odour, 1 = very weak odour, 2 = higher acceptable odour, 3 = non acceptable odour. 3.2.5.2. Determination of the antioxidant activity of essential oils: 3.2.5.2.1. Measurement of scavenging Activity: The hydrogen atoms or electrons donation ability of the corresponding extracts and some pure compounds were measured from the bleaching of purple coloured methanol solution of 2, 2-diphenyl-1-picrylhydrazyl (DPPH). The effect of oils on DPPH radical was estimated according to Kose et al. (2010). 1 ml of various concentrations (200, 300, 400 and 500 µg/ml) of the essential oils and Butylated Hydroxy Toluene (BHT) in methanol was added to a 4 ml of DPPH radical solution in methanol (0.004%). The mixture was shaken vigorously and allowed standing for 30 min; the absorbance of the resulting solution was measured at 517 nm with a spectrophotometer (Shimadzu UV-1601, Kyoto, Japan). Inhibition of free radical DPPH in percent (I%) was calculated in following way:
I% = 100 x (AControl – ASample) /Acontrol 68
Where, AControl is the absorbance of the control reaction (containing all reagents except the test compound), and ASample is the absorbance of the test compound. Butylated Hydroxy Toluene (BHT) was used as a control. 3.2.5.2.2. Measurement of sunflower oil stability by Rancimat apparatus: The oxidative stability of sunflower oil as affected by the addition of the different studied essential oil mixtures or BHT was determined using A Metrohm Rancimat model 679 (Herisau, Switzerland) was used. The Rancimat test is based on automatically determining the time (known as the oxidative/oil stability index (OSI)) before the maximum rate change of oxidation. A stream of air was bubbled into oil samples (3.0 g) contained in a reaction vessel placed in an electric heating block. The effluent air that contained volatile organic acids from the oil sample was collected in a measuring vessel containing distilled water (60 mL). The conductivity of the water as oxidation proceeded was measured automatically. Filtered, cleaned, dried air was allowed to bubble through the hot oil at a rate of 15 L/h. The OSIs of the oil samples were automatically recorded at 120 oC. In each time, eight oil samples were accommodated in the equipment and analyzed simultaneously. The oil samples for all determinations were randomized to determine their position in the heating block (Farhoosh and Moosavi, 2007). 3.2.5.3. Evaluation of interaction between essential oils as antioxidants: The selective doses of irradiation were selected according to the highest antioxidant properties (Rancimat apparatus test and Scavenging activity test) of all individually treated essential oils. Thus, the selected doses used in making essential oils mixtures were; 2 and 4 kGy for cumin essential oil, 6 kGy for rosemary essential oil and 4 kGy for thyme essential oil. Thus, eight groups of essential oils mixtures (1:1, v/v) were prepared from non-irradiated essential oils (control) as well as irradiated essential oils as follow:
1- Non-irradiated cumin oil + Non-irradiated thyme oil (C0 X T0).
2- Non-irradiated cumin oil + Non-irradiated rosemary oil (C0 X R0). 69
3- Non-irradiated rosemary oil + Non-irradiated thyme oil (R0 X T0).
4- 2kGy irradiated cumin oil + 4kGy irradiated thyme oil (C2 X T4).
5- 2kGy irradiated cumin oil + 6kGy irradiated rosemary oil (C2 X R6).
6- 4kGy irradiated cumin oil + 4kGy irradiated thyme oil (C4 X T4).
7- 4kGy irradiated cumin oil + 6kGy irradiated rosemary oil (C4 X R6).
8- 6kGy irradiated rosemary oil + 4kGy irradiated thyme oil (R6 X T4). These essential oils mixtures were subjected to scavenging activity test and the Rancimat apparatus test to evaluate all probability interactions. 3.2.6. Statistical analysis: All the experiments were carried out in triplicate and mean and standard error (S.E.) were calculated. Then, the results were subjected to PROC ANOVA followed by Duncan`s significant differences using SAS program (version 9.1.3) software (Cary, NC). Significant levels were defined as P < 0.05 (SAS, 2004). 4. RESULTS AND DISCUSSION 4.1. Effect of gamma irradiation on the physiochemical properties of essential oil: Determining the quality and purity of essential oils faces similar difficulties to establishing the quality of raw spices, given the accepted variations in and mixing of varieties within a particular spice. There are, however, a number of properties that can be used to set quality standards. These include physical characteristics such as; specific gravity and refractive index. It is also possible to use chemical properties to benchmark quality. These include determination of acids value (Guenther, 1974). It’s known that during irradiation of foods, chemical and physical properties of the food constituents are sometimes modified (Olson, 1998). 4.1.1. Effect of gamma irradiation on the physiochemical properties of cumin essential oil: Physiochemical properties of cumin essential oil were determined in both irradiated essential oil and non-irradiated essential oil. 4.1.1.1. Refractive Index: As shown in Table (1) and Figure (4) the refractive index of irradiated cumin essential oil was 1.5000, 1.5050 and 1.5045 at doses 2, 4, and 6 kGy, respectively. The refractive index was 1.502 in non-irradiated cumin essential oil. Similar value was obtained by Guenther (1974); Azeez (2008) and (Burdock, 2010). It was obvious from Table (1) and Figure (4) that various gamma irradiation doses undertaken caused only changes on the right two decimal numbers of the refractive index of the essential oil. Same results obtained by Abdelaleem (2008) who mentioned that the refractive index of irradiated coriander essential oil at doses 2, 4 and 8 kGy changed only in the fourth decimal numbers. 71
4.1.1.2. Specific Gravity: From Table (1) and Figure (5) it is clear that the specific gravity of non- irradiated cumin essential oil was 0.908. Whereas, all irradiated samples had the same specific gravity. The results in the line with Abdelaleem (2008) and Fazakerley et al. (1961), who reported that the specific gravity of irradiated coriander and peppermint essential oil was not changed after 71 irradiation with 10 kGy.
4.1.1.3. Acid Value: Table (1) and Figure (6) showed that the acid value of cumin essential oil in irradiated samples at doses 2, 4 and 6 kGy were slightly higher (2.5, 2.5 and 2.7, respectively) than the control one which had acid value 2.4. The data in accordance with those obtained by Amin (2001) and Azeez (2008). 4.1.2. Effect of gamma irradiation on the physiochemical properties of rosemary essential oil: 4.1.2.1. Refractive Index: Data in Table (1) and Figure (4) represented that the refractive index of all irradiated rosemary essential oil at doses 2, 4 and 6 kGy were (1.471) the same and there were no differences. While non-irradiated essential oil had a refractive index 1.468. The obtained datum in control sample was in agreement with that obtained by El-Laban (1998) and Clarke (2008). Fazakerley et al. (1961) mentioned that the refractive index of irradiated peppermint essential oil was not changed after irradiation with 10 kGy. 4.1.2.2. Specific Gravity: The specific gravity of irradiated rosemary essential oil was slightly increased compared to non-irradiated rosemary essential oil as shown in Table (1) and Figure (5). Whereas, specific gravity was 0.892 in non- irradiated sample and 0.906, 0.899 and 0.895 in 2, 4 and 6 kGy samples. 72
Table (1): Effect of gamma irradiation on physiochemical properties of different essential oils Thyme essential oil Cumin essential oil Rosemary essential oil Irradiation Treatments (kGy) 0 2 4 6 0 2 4 6 0 2 4 6
1.4930A 1.4921A 1.4950A 1.4950A 1.5020B 1.5000B 1.5050B 1.5045B 1.4680C 1.4710C 1.4710C 1.4710C ± 0.005 ± 0.006 ± 0.005 ± 0.011 ± 0.005 ± 0.005 ±0.005 ± 0.011 ± 0.005 ± 0.011 ± 0.005
index ± 0.005 Refractive
0.9230D 0.9250D 0.9270D 0.9200D 0.9080E 0.9060E 0.9180E 0.9078E 0.8920F 0.9060F 0.8990F 0.8955F
cific ± 0.005 ± 0.006 ± 0.005 ± 0.011 ± 0.005 ± 0.011 ± 0.006 ± 0.011 ± 0.049 ± 0.003 ± 0.007 ± 0.000 gravity Spe 7 2
1.2G ± 1.3G ± 1.3G ± 1.24G ± 2.4H ± 2.5H ± 2.5H ± 2.7H ± 0.91I ± 0.9I ± 1.1I ± 1.2I ± 0.057 0.057 0.115 0.005 0.057 0.057 0.057 0.057 0.014 0.057 0.115 0.057 Acid value
Each value represents the mean S.E.; The mean value with different superscript alphabets in the same row (under the same spice essential oil in each determination) indicate significant differences (P0.05) 73
1.51 Cumin Rosemary Thyme
1.5
1.49
1.48
1.47 Refractive Index Refractive 3 7 1.46
1.45
1.44 0 kGy 2 kGy 4 kGy 6 kGy Irradiation Treatment
Fig. (4): Effect of gamma irradiation on the refractive index of thyme, cumin and rosemary essential oil 74
Cumin Rosemary Thyme 0.93
0.92 C) °
25 0.91 C / C ° 25
0.9 7 4
0.89 Specifig gravity ( Specifig gravity 0.88
0.87 0 kGy 2 kGy 4 kGy 6 kGy
Irradiation Treatment Fig. (5): Effect of gamma irradiation on the specific gravity of thyme, cumin and rosemary essential oil 75
3 Cumin Rosemary Thyme
2.5
2
1.5 5 AcidValue 7 1
0.5
0 0 kGy 2 kGy 4 kGy 6 kGy
Irradiation Treatment Fig. (6): Effect of gamma irradiation on the acid value of thyme, cumin and rosemary essential oil 76
The previous data are in similar to Bousbia et al. (2009). Thus, Abdelaleem (2008) mentioned that the specific gravity of irradiated coriander essential oil increased non-significantly when irradiated with 2, 4, and 8 kGy doses. 4.1.2.3. Acid Value: Data in Table (1) and Figure (6) showed that the acid value increased non-significantly in non-irradiated samples (0.91) compared to irradiated samples at doses 2, 4 and 6 kGy (0.9, 1.1 and 1.2, respectively). Same results were obtained by El-Hady (1982) who mentioned that the acid value of irradiated geranium essential oil was increased gradually by increasing of gamma irradiation. 4.1.3. Effect of gamma irradiation on some physiochemical properties of thyme essential oil: The physiochemical properties of thyme essential oil were determined in both irradiated essential oil and non-irradiated essential oil. 4.1.3.1. Refractive Index: Table (1) and Figure (4) revealed that the refractive index of irradiated thyme essential oil and non-irradiated thyme essential oil was as follows; 1.4930, 1.4921, 1.4950 and 1.4950 in 0, 2, 4 and 6 kGy ,respectively. The previous data was in the similar with those obtained from (Burdock, 2010). There were no significant changes in refractive index in the irradiated samples compared with non-irradiated samples. The obtained data are in agreement with Fazakerley et al. (1961). 4.1.3.2. Specific Gravity: The specific gravity of essential oil from irradiated thyme essential oil was 0.923 in the control sample, at zero time, as shown in Table (1) and Figure (5) this value was in accordance with the results reported by Stahl- Biskup (2004) and Burdock (2010). However, the data in Table (1) and 77
Figure (5) indicated that the treatment of thyme essential oil with gamma irradiation at 2, 4 and 6 kGy caused little changes in specific gravity. 4.1.3.3. Acid Value: As shown in Table (1) and Figure (6) acid value was slightly increased in all samples due to irradiation treatment. The data in Table (1) indicated that acid value in control samples at zero time was 1.2. Acid values of irradiated samples at doses 2, 4 and 6 kGy were as follow; 1.3, 1.3 and 1.24, respectively. 4.2. Chemical composition of essential oil:
The variations in the composition of the essential oil could be due to factors such as plant age, plant part, development stage, growing place, harvesting period and principally by chemotype since they influence the plant biosynthetic pathways and consequently the relative proportion of the main characteristic compounds (Viuda-Martos et al., 2011). Thus, the chemical constituents of cumin, rosemary and thyme essential oils were fractionated and identified by gas chromatography and mass spectroscopy technique (GC/MS). 4.2.1. Chemical composition of cumin essential oil: Essential oil of cumin was extracted by steam distillation (SD), fractionated and identified by Gas Chromatography and Mass Spectroscopy (GC/MS) technique. The obtained data are tabulated in Table (2) and illustrated by Figure (7). From these results, it could be indicated that there are 19 compounds were isolated from cumin essential oil. Sixteen components were identified and classified into tenth chemical categories namely; aliphatic terpenes (0.68 %), monocyclic terpenes (20.6 %), bicyclic terpenes (18.42 %), aliphatic terpene alcohols (1.6 %), cyclic terpene alcohols (0.17 %), monoterpen phenol (0.15%), monoterpene cyclic aldehydes (24.93 %), monoterpene aldehydes (25.43 %), aromatic hydrocarbons (6.81%) and sequiterpene hydrocarbon (0.41%). These 78
identified compounds accounted for 99.2 % of the composition of cumin essential oil. The remaining portion 0.82% representing three unknown constituents. These compounds are the similar with Vazin (2013). The first chemical group in cumin essential oil was aliphatic terpenes which consisted of one compound namely; β-myrcene (0.68%). This compound was reported as a constituent of cumin essential oil by Pajohi et al. (2011) who found that the amount of that compound was 1.1 % and also Lucchesi et al. (2004b); Jirovetz et al. (2005); Allahghadri et al. (2010); Bettaieb et al. (2011) and Sadeghi et al. (2012) reported β-myrcene as a component of cumin essential oil. The second recorded chemical group was monocyclic terpenes which consisted of four compounds namely; α-thujene (0.26 %), sabinene (0.43 %), α-terpinene (0.42 %) and γ-terpinene (19.49 %). These compounds were reported as constituents of cumin essential oil according to Bettaieb et al. (2011), Sadeghi et al. (2012) and Vazin (2013). The third identified chemical group was bicyclic terpenes which consisted of two compounds namely; α-pinene (0.98%) and β-pinene (17.44%). The fourth chemical group was aliphatic alcohols consists of one compound namely citranellol (1.6%). These results are agreed with Bettaieb et al. (2011); Pajohi et al. (2011) and Sadeghi et al. (2012). The fifth chemical group recorded and identified in cumin essential oil was cyclic terpene alcohols which consists of one compound namely α- terpineol (0.17%) the found result in this group are in agreement with Allahghadri et al. (2010). The seventh chemical group was aromatic hydrocarbons consists of two constituents namely; o-Cymene (0.09%) and ρ-Cymene (6.72%). The eighth chemical group was monoterpene aldehyde mainly has cuminaldehyde (24.93%) classified as the predominant compound in cumin essential oil. This compound was classified in Tunisian and Indian cumin essential oil where, the percentage content of cuminaldehyde was 15.31% 79
and 22.29%, respectively (Bettaieb et al., 2011). Also, Vazin (2013) found that cuminaldehyde content was 26.49% in Iranian cumin. The ninth identified chemical group was monoterpene aldehyde consists of two compounds namely; α-Terpinene-7-al (10.55%) and γ-Terpinene-7-al (14.88%). these compounds were also recorded by Pajohi et al. (2011) who found the percentage of two constituents were 20.70% and 8.90%, respectively.
Finally, the tenth chemical group was sesquiterpene hydrocarbon consists of only one compound namely; β-Bisabolene (0.41%). All the above mentioned data are in agreement with Masada (1980) and Vazin (2013) who identified and classified β-pinene, ρ-cymene, γ-terpinene and cuminaldehyde as main components of cumin essential oil. 4.2.2. Chemical composition of rosemary essential oil: Essential oil of rosemary was extracted by steam distillation, fractionated and identified by Gas Chromatography and Mass Spectroscopy technique. The obtained data are tabulated in Table (3) and illustrated by Figure (8). From these results, it could be indicated that there are 32 compounds were isolated from rosemary essential oil. Twenty three components were identified and classified into eleventh chemical categories namely; aliphatic terpenes (0.97 %), monocyclic terpenes (8.81 %), bicyclic terpenes (24.59 %), cyclic ether monoterpene (33.2%), aliphatic alcohols (4.17 %), cyclic terpene alcohols (3.39 %), cyclic terpene alcohol (14.5 %), bicyclic terpene ketone (2.25%), aromatic hydrocarbons (0.63%), sequiterpene hydrocarbons (4.49%) and oxygenated sesquiterpenes (0.09%). These identified compounds accounted for 97.09 % of the composition of rosemary essential oil. The remaining portion 2.91% is representing eight unknown constituents. 80
Table (2): Chemical composition of cumin (Cuminum cyminum) essential oils Compounds Peak Number by Figure (7) % Aliphatic terpenes -Myrcene 5 0.68 Total 0.68 Monocyclic terpenes -Thujene 1 0.26 Sabinene 3 0.43 -Terpinene 6 0.42 -Terpinene 9 19.49 Total 20.6 Bicyclic terpenes -Pinene 2 0.98 -Pinene 4 17.44 Total 18.42 Aliphatic alcohols Citranellol 11 1.6 Total 1.6 Cyclic terpene alcohols α-Terpineol 10 0.17 Total 0.17 Monoterpene phenol Carvacrol 13 0.15 Total 0.15 Aromatic hydrocarbons o-Cymene 7 0.09 ρ-Cymene 8 6.72 Total 6.81 Monocyclic terpene aldehyde Cuminaldehyde 12 24.93 Total 24.93 Monoterpenes aldehyde α-Terpinene-7-al 14 10.55 γ-Terpinene-7-al 15 14.88 Total 25.43 Sesquiterpene hydrocarbone β-Bisabolene 16 0.41 Total 0.41 Total known 99.2 Total unknown 17, 18, 19 0.82 Total oxygenated compounds 52.82 Total non-oxygenated compounds 46.92 81 1 8
6 5 6 5 7
Fig. (7): Chromatographic profile of the essential oil cumin 82
The first chemical group in rosemary essential oil was aliphatic terpenes which consisted of one compound namely; β-myrcene (0.97%). This compound was reported as a constituent of rosemary essential oil by Giordani et al. (2004) who found that the amount of that compound was 1.01 % and also Kubeczka (2002) reported β-myrcene as a component of rosemary essential oil. The second recorded chemical group was monocyclic terpenes which consisted of six compounds namely; sabinene (0.92 %), α-terpinene (0.53 %), limonene (5.93%), γ-terpinene (0.27%), α-terpinolene (0.12%) and pinocarvone (1.04%). These compounds were reported as constituents of rosemary essential oil according to Szumny et al. (2010), Sui et al. (2012), Jordán et al. (2013), Teixeira et al. (2013) and Yosr et al. (2013). The third identified chemical group was bicyclic terpenes which consisted of four compounds namely; α-pinene (17.1%), camphene (6.35%), β-pinene (0.92%) and Δ-3-Carene (0.22%). The fourth identified chemical group was cyclic ether monoterpene consists of only one compound 1,8-cineol (33.2%) classified as the predominant component of rosemary essential oil. Giordani et al. (2004) mentioned that the content of 1,8-cineol was 24.36%. The fifth chemical group was aliphatic alcohols consists of two compound namely linalool (1.38%) and geraniol (2.79%). These results are agreed with Bousiba et al. (2009) and Usai et al. (2011). The sixth chemical group recorded and identified in rosemary essential oil was cyclic terpene alcohols which consists of two compounds namely borneol (0.48%) and α-terpineol (2.91%) the found result in this group are in agreement with Dimitrijević et al. (2007). The seventh chemical group was consists of one constituents namely; camphor (14.5%). Dimitrijević et al. (2007) mentioned that the camphor content was (10.08%) while, the content was 7.82%, 14.5% and 21.8% in Bousbia et al. (2009), Yosr et al. (2013) and Machado et al. (2013). 83
Table (3): Chemical composition of rosemary (Rosmarinus officinalis) essential oils Compounds Peak Number by Figure (8) % Aliphatic terpenes -Myrcene 5 0.97 Total 0.97 Monocyclic terpenes Sabinene 3 0.92 -Terpinene 8 0.53 Limonene 9 5.93 -Terpinene 11 0.27 -Terpinolene 12 0.12 Pinocarvone 15 1.04 Total 8.81 Bicyclic terpenes -Pinene 1 17.1 Camphene 2 6.35 -Pinene 4 0.92 Δ-3-Carene 6 0.22 Total 24.59 Cyclic ether monoterpene 1,8-Cineole 10 33.2 Total 33.2 Aliphatic alcohols Linalool 13 1.38 Geraniol 19 2.79 Total 4.17 Cyclic terpene alcohols Borneol 16 0.48 α-Terpineol 17 2.91 Total 3.39 Cyclic terpene ketone Camphor 14 14.5 Total 14.5 Bicycylic ketone terpene Verbenone 18 2.25 Total 2.25 Aromatic hydrocarbons o-Cymene 7 0.63 Total 0.63 Sesquiterpene hydrocarbons Caryophyllene 23 0.07 α-Humulene 24 3.76 γ-Curcumene 26 0.49 β-Sesquiphellandrene 29 0.17 Total 4.49 Oxygenated sesquiterpene Caryophyllene oxide 32 0.09 Total 0.09 Total known 97.09 Total unknown 2.91 Total oxygenated compounds 20, 21, 22, 25, 27, 28, 30, 31 58.63 Total non-oxygenated compounds 38.50 84
1 10
14
2 4 8 9 24 Relative Abundance Relative
3 4 20 5 7 17 19 32 8 13 15 26 30 6 16 18 22 31 11 12 21 23 25 27 2829
Time (min)
Fig. (8): Chromatographic profile of the essential oil rosemary 85
The eighth and the ninth chemical groups were consists of verbenone (2.25%) and o-cymene (0.63%), respectively. The obtained results are in agreement with Dimitrijević et al. (2007) who found that verbenone was 1.86% and o-cymene was 0.59%. The tenth identified chemical group was sesquiterpene hydrocarbons consists of four compounds namely; caryophyllene (0.07%) α-Humulene (3.76%), γ-Curcumene (0.49%) and β-Sesquiphellandrene (0.17%). these compounds were also recorded by Bousbia et al. (2009). Finally, the eleventh chemical group was oxygenated sesquiterpene consists of only one compound namely; Caryophyllene oxide (0.09%). All the above mentioned data are in agreement with Kubeczka (2002) and Yosr et al. (2013) who identified and classified α-pinene, champor, camphene and 1,8-cineol as main components of rosemary essential oil. 4.2.3. Chemical composition of thyme essential oil: Thyme essential oil was extracted and then identified by GC/MS technique. The obtained data are tabulated in Table (4) and illustrated by Figure (9). The results indicated that there are 23 compounds were isolated from thyme essential oil. All isolated components were identified and classified into ninth chemical categories namely; aliphatic terpenes (5.18 %), monocyclic terpenes (18.61 %), bicyclic terpenes (3.51 %), aliphatic alcohols (1.22 %), cyclic terpene alcohols (1.53 %), monoterpene phenol ester (0.56%), monoterpene phenol (34.86%), aromatic hydrocarbons (33.6%) and sequiterpene hydrocarbons (0.93%). The first chemical group in rosemary essential oil was aliphatic terpenes which consisted of two compounds namely; β-myrcene (2%) and β-ocimene (3.18%). These compounds were reported as constituents of thyme essential oil by Usai et al. (2011) and Hussain et al. (2013). 86
Table (4): Chemical composition of thyme (Thymus vulgaris) essential oils Compounds Peak Number by Figure (9) % Aliphatic terpenes -Myrcene 5 2 -Ocimene 11 3.18 Total 5.18 Monocyclic terpenes α-Thujene 1 2.52 -Terpinene 8 0.17 Limonene 9 2.06 -Terpinene 12 13.1 α-Phellandrene 6 0.76 Total 18.61 Bicyclic terpenes -Pinene 2 1.92 Camphene 3 0.76 -Pinene 4 0.5 Δ-3-Carene 7 0.33 Total 3.51 Aliphatic alcohols Linalool 13 0.87 Geraniol 17 0.35 Total 1.22 Cyclic terpene alcohols Borneol 14 0.29 α-Terpineol 15 1.24 Total 1.53 Monoterpene phenol ester Carvcrol Methyl Ester 16 0.56 Total 0.56 Monoterpene phenol Thymol 18 31.7 Carvacrol 19 3.16 Total 34.86 Aromatic hydrocarbons ρ-Cymene 10 33.6 Total 33.6 Sesquiterpene hydrocarbons γ-Muurolene 20 0.13 α-Muurolene 21 0.19 γ-Cadinene 22 0.28 δ-Cadinene 23 0.33 Total 0.93 Total known 100 Total unknown 0 Total oxygenated compounds 38.5 Total non-oxygenated compounds 61.5 87
10
18
12 7 8
Relative Abundance Relative 1 19 2 9 11 5 3 4 8 6 7 13 23 14 15 16 17 20 21 22
Time (min) Fig. (9): Chromatographic profile of the essential oil thyme 88
The second recorded chemical group was monocyclic terpenes which consisted of five compounds namely; α-thujene (2.52%), α-terpinene (0.17%), limonene (2.06%), γ-terpinene (13.1%) and α-phellandrene (0.76%). These compounds were reported as constituents of thyme essential oil according to Giordani et al. (2004), Dimitrijević et al. (2007) and Hussain et al. (2013). The third identified chemical group was bicyclic terpenes which consisted of four compounds namely; α-pinene (1.92%), camphene (0.76%), β-pinene (0.5%) and Δ-3-carene (0.33%). The fourth chemical group was aliphatic alcohols consists of two compound namely linalool (0.87%) and geraniol (0.35%). The fifth chemical group identified in thyme essential oil was cyclic terpene alcohols which consists of two compounds namely borneol (0.29%) and α-terpineol (1.24%). These results are agreed with Dimitrijević et al. (2007). The sixth chemical group was monoterpene phenol ester consists of only one compound namely; carvacrol methyl ester (0.56%). The seventh chemical group was monoterpene phenol mainly consists of two constituents namely; thymol (31.7%) and carvacrol (3.16%). These compounds are the main constituents in thyme essential oil as mentioned by Giordani et al. (2004) and Usai et al. (2011).
The eighth chemical group were aromatic hydrocarbon consists of ρ- cymene (33.6%). The obtained results are in agreement with Usai et al. (2011) who found that ρ-cymene was 21.63%. Finally, the ninth identified chemical group was sesquiterpene hydrocarbons consists of four compounds namely; γ-Muurolene (0.13%) α- Muurolene (0.19%), γ-Cadinene (0.28%) and δ-Cadinene (0.33%). these compounds were also recorded by Giordani et al. (2004). 89
All the above mentioned data are agreed with Kubeczka (2002), Hussain et al. (2013) and Teixeira et al. (2013) who identified and classified thymol, carvacrol and ρ-Cymene as main components of thyme essential oil. 4.3. Antimicrobial activity of essential oils (in vitro): Secondary metabolites produced by plants constitute a source of bioactive substances. Nowadays the scientific interest on these metabolites has increased due to the search for new drugs from plant origin. In addition, the antimicrobial activity of plant volatile oils and extracts has been recognized for many years. However, few investigations have compared large numbers of volatile oils and extracts using methods that are directly comparable. In the present study, the technique used to determine the inhibitory properties of the thyme, cumin and rosemary essential oils were Well- Diffusion System that system was different from the paper disc method; which involved soaking discs with herb extracts and placing them on the surface of agar seeded with microorganisms. This technique (paper disk) does not always allow an accurate determination of the volume of oil added and although the disc was applied to the agar surface, the extract would have diffused in three dimensions, giving a hemispherical zone difficult to measure accurately (Deans and Ritchie, 1987). In this study, these problems were largely overcome by punching holes through the agar, creating wells into which measured volumes (30, 50 and 70 μl) of the plant volatile oils were placed. This ensured that radial diffusion from the well gave a clean and easily measured zone of inhibition (Bennett et al., 1966). 90
4.3.1. Antibacterial activity of essential oils: 4.3.1.1. Preliminary experiment to assess recommended doses for antibacterial: A preliminary experiment was set to evaluate the approximate irradiation doses that make the highest antimicrobial activity of irradiated essential oils against selected bacterial strains. Ascending gamma irradiation doses were chosen ranking from 1 to 10 kGy. As a point of view, regarding to the effect of gamma irradiation on the chemical functional group as mentioned by Fazakerley et al., 1961 and Seo et al., 2007, the effect of irradiation at different doses represented irregular changes in the inhibition zones which refers to the antibacterial activity of irradiated essential oil. However, Seo et al., 2007 mentioned that the effect of gamma irradiation on the chemical functional group of Angelica gigas essential oil possess to be irregular due ascending order doses from 1 kGy to 10 kGy . 4.3.1.1.1. Preliminary experiment for thyme essential oil: The effect of irradiation doses on the antibacterial activity of irradiated thyme essential oil was studied. The obtained data shown in Table (5) represented that irradiation doses make irregular significant differences between values. Where, the inhibition zone in the control sample in Escherichia coli was 30.33 mm, the largest inhibition zone was 42 mm at irradiation dose 4 kGy. On the other hand, the largest inhibition zone illustrated in S. typhimurium, B. cereus and S. aureus were 26.5 mm, 29 mm and 39 mm at doses 0, 1 and 4 kGy, respectively. As a point of view, irregular differences in inhibition zones as shown by Figure (10) appeared due to gamma irradiation, happened because each essential oil has many complicated chemical components. The essential oils 91
are composed of hydrocarbons (terpene derivatives) or terpenes (e.g., α- terpinene, α-pinene, camphene, limonene, myrcene, and sabinene), oxygenated derivatives of hydrocarbons (e.g., linalool, citronellol, geraniol, carveol , thymol, borneol, fenchone, tumerone, and nerol), benzene compounds (alcohols, acids, phenols, esters, and lactones) and nitrogen- or sulfur-containing compounds (indole, hydrogen sulfide, methyl propyl disulfide, and sinapine hydrogen sulfate) (Raghavan, 2007).
However, the effect of irradiation process could be result from chemical modification of terpenes including the oxidative effect; this effect of irradiation process was degreed according to the content of terpenes and other hydrocarbons in the essential oil. On the olfactory evidence, the order of resistance to irradiation was aldehydes > alcohols > ester > hydrocarbon > phenol (Fazakerley et al., 1961). On the other hand, Juven et al. (1994) examined the working of thymol against Salmonella typhimurium and Staphylococcus aureus and hypothesised that thymol binds to membrane proteins hydrophobically and by means of hydrogen bonding, thereby changing the permeability characteristics of the membrane. Thymol was found to be more inhibitive at pH 5.5 than 6.5. At low pH the thymol molecule would be undissociated and therefore more hydrophobic, and so may bind better to the hydrophobic areas of proteins and dissolve better in the lipid phase (Juven et al., 1994). 92
Table (5): Effect of treatments of thyme essential oil with different irradiation doses on the bacterial inhibition zone (mm) Inhibition Zone (mm) Irradiation dose Escherichia Salmonella Bacillus Staphylococcus (kGy) coli typhimurium cereus aureus
0 30.33E ± 0.882 26.50AB ± 0.288 24.50C ± 0.388 32.5CD ± 1.443
1 39.50AB ± 1.041 26.00AB ± 0.577 29.00A ± 0.577 29.5D ± 0.288
2 39.00AB ± 0.577 20.50B ± 0.288 29.00A ± 0.577 36.00BC ± 1.527
3 37.00BCD ± 0.577 22.00 B ± 0.577 27.00A ± 0.577 41.5A ± 0.288
4 42.00A ± 1.154 25.00AB ± 0.882 23.00BC ± 0.577 39.00AB ± 0.577
5 42.00A ± 1.154 23.50AB ± 0.288 20.00C ± 1.154 37.67ABC ± 1.856
6 40.00AB ± 1.154 25.50AB ± 0.288 21.00BC ± 0.577 39.00AB ± 0.577
7 36.00CD ± 1.154 22.50AB ± 1.041 19.50C ± 0.288 42.00A ± 0.577
8 35.00D ± 0.577 26.00AB ± 5.033 20.00C ± 0.577 38.00AB ± 4.509
9 35.00D ± 0.577 23.00AB ± 0.577 19.00C ± 0.577 38.00AB ± 1.154
10 38.00BC ± 1.154 28.33A ± 0.882 23.00B ± 1.527 36.5ABC ± 0.288
Each value represents the mean S.E.; The mean value with different superscript alphabets in the same column indicate significant differences (P0.05) using LSD test. 93 3 9
Fig. (10): Effect of treatments of thyme essential oil at different irradiation doses on the bacterial inhibition zone (mm) 94
4.3.1.1.2. Preliminary experiment for cumin essential oil: Cumin essential oil was irradiated at doses from 1 to 10 kGy. Then, the inhibition zone of irradiated essential oils was measured and data tabulated in Table (6). The highest inhibition zone observed at dose 2 kGy in E. coli and B. cereus. While, the highest inhibition zone appeared at dose 4 kGy in S. typhimiurium and S. aureus. From the obtained data it could be noticed that irradiation doses that has the better antimicrobial properties against selected bacterial strains were 2 and 4 kGy. The same reason of irregular changes in inhibition zones observed due to various chemical components in the structure of essential oil (Raghavan, 2007). Thus, the changes might be related to gamma irradiation (Fazakerley et al., 1961). As shown by Figure (11) it could be noticed that irradiation doses decreased the antibacterial activity of cumin essential oil against S. typhimurium and B. cereus. In case of S. typhimurium as gram-negative bacteria; the essential oils had been ascribed to their hydrophilic outer membrane which can block the penetration of hydrophobic compounds into target cell membrane (Bouhdid et al. 2008). On the other hand, S. aureus - one of the gram negative bacteria - had the highest inhibition zone in irradiated essential oil at dose 4 kGy compared to non-irradiated. Where, the inhibition zone was 29 mm at dose 4 kGy and 20 mm in non-irradiated essential oil. The reason might be due to the essential oil of cumin contain cumin aldehyde, gamma-terpinene and beta-pinene (as main components) have severe antibacterial activity against Gram-positive and Gram-negative bacteria (Iacobellis et al. 2005). 95
Table (6): Effect of treatments of cumin essential oil at different irradiation doses on the bacterial inhibition zone (mm)
Inhibition Zone (mm) Irradiation dose Escherichia Salmonella Bacillus Staphylococcus (kGy) coli typhimurium cereus aureus
0 18.50D ± 0.288 13.00A ± 0.577 14.00A ± 0.288 20.00B ± 6.245
1 29.00A ± 0.577 9.00DEF ± 0.577 9.50A ± 1.041 21.50AB ± 0.288
2 30.00A ± 0.577 8.50EF ± 0.288 14.50A ± 0.577 26.50AB ± 0.288
3 25.00B ± 0.577 10.50CD ± 0.288 11.33A ± 1.855 29.00A ± 0.577
4 23.00BC ± 0.577 12.50AB ± 0.866 10.50A ± 0.907 29.00A ± 0.577
5 24.00BC ± 0.577 11.00 BC ± 0.577 11.00A ± 1.527 25.00AB ± 4.041
6 22.50C ± 0.288 8.50EF ± 0.288 11.00A ± 0.577 27.00AB ± 1.527
7 23.50BC ± 0.763 10.50CD ± 0.288 11.50A ± 0.288 25.50AB ± 0.288
8 20.00D ± 0.577 10.00 CDE ± 0.577 11.50A ± 0.288 28.00AB ± 0.577
9 24.00BC ± 1.154 7.50F ± 0.288 11.00A ± 0.577 23.50AB ± 0.288
10 23.00BC ± 0.577 9.00DEF ± 0.577 9.80A ± 0.416 26.00AB ± 0.577
Each value represents the mean S.E.; The mean value with different superscript alphabets in the same column indicate significant differences (P0.05) using LSD test. 96 96
Fig. (11): Effect of treatments of cumin essential oil at different irradiation doses on the bacterial inhibition zone (mm) 97
The mechanisms by which essential oils can inhibit microorganisms have different reasons, and in some part may be due to their hydrophobicity. In this connection, the penetration of the oils into the lipid bilayer of the cell membrane makes the cells more permeable, leading to leakage of vital cell contents (Kim et al., 1995 and Burt, 2004). Moreover, the antimicrobial activity of essential oils may be due to the presence of synergy between the major components and other constituents of the oils leading to various degrees of antimicrobial activity (Bouhdid et al. 2008). 4.3.1.1.3. Preliminary experiment for rosemary essential oil: From Table (7) and Figure (12). it could be found that the inhibition zones in the four bacterial strains were smaller than that in cumin and thyme essential oil, especially in B. cereus and S. typhimurium. The same results achieved by Bousbia et al. (2009) who studied the antimicrobial activity of rosemary essential oil. Where, the obtained inhibition zones were 12.5 mm in Staphylococcus aureus and 15.5 mm in Escherichia coli. Irradiation doses made some changes in the inhibition zone compared to control one. But that changes represented slight decrease in the inhibition zone in irradiated samples compared to non-irradiated samples. Rosemary essential oil possesses to be the lowest antibacterial activity in essential oils under study.
Regarding to the data tabulated in Table (37) the content of α-pinene is decreased from 17.1% in non-irradiated rosemary essential oil to 13.33% in irradiated rosemary essential oil at 6 kGy, that finding might be the responsible for the decrease of inhibition zone in irradiated essential oil specially in case of S. typhimiurium. The obtained finding is in agreement with Jordán et al. (2013) who, found that the high proportion of α-pinene increases the effectiveness of the oil against S. typhimiurium, while the presence of eucalyptol (1,8-cineol), as the most abundant compound, considerably decreases the efficiency of rosemary oil. Furthermore, Ojeda- 98
Sana et al. (2013) explained that the α-pinene and 1,8-cineol exhibited board antibacterial spectrum against gram negative bacteria by inducing cell membrane disruption.
That data in the similar with Fatemi et al. (2011) who found that γ- irradiation of caraway seeds could not affect the antibacterial activity of its essential oils but it was indicated that the inhibition zones of the oil extracted from irradiated caraway were slightly different from the control group, but these changes were not significant (P > 0.05). Therefore, the antibacterial activity of essential oils of caraway seeds could not be significantly changed even after exposing to the gamma irradiation up to 25 kGy. 4.3.2. Antifungal activity of essential oils: 4.3.2.1. Preliminary experiment to assess recommended doses for antifungal: As shown by Figure (13) both non-irradiated and irradiated cumin and thyme essential oil showed no inhibition zones at concentrations 30, 50 and 70 µl/well in all plats from 0 kGy up to 10 kGy. The inhibition zones were 90 mm in all plates (plate diameter). Thus, both cumin and thyme essential oil either irradiated or non-irradiated represent a great antifungal activity especially on the growth of Aspergillus flavus at all used concentrations (30, 50 and 70 µl). This finding might be related to the phenolic content of thyme essential oil and the aldehyde content of cumin essential oil, these compounds classified as oxygenated compounds which represent the great antimicrobial activity. The obtained data are in the line with (Zuzarte et al., 2013) who mentioned that the antifungal activity of the oil was probably due to the activity of minor compounds or related with a synergistic effect between different compounds present in the oil since the main compounds tested alone showed low antifungal activity. Thymus oil was more active against all the tested strains, being the activity related to the phenolic nature of its main 99
Table (7): Effect of treatments of rosemary essential oil at different irradiation doses on the bacterial inhibition zone (mm)
Irradiation Inhibition Zone (mm) dose Escherichia Salmonella Bacillus Staphylococcus (kGy) coli typhimurium cereus aureus
0 15.33ABC ± 0.577 6.50BC ± 0.577 7.50C ± 0.288 17.57ABC ± 0.288
1 10.50CD ± 0.288 6.00BC ± 0.577 7.50C ± 0.288 15.00BCD ± 0.577
2 14.50BCD ± 1.041 7.50A ± 0.288 7.50C ± 0.288 17.00ABC ± 1.732
3 16.00ABC ± 0.577 5.50C ± 0.288 10.50A ± 0.288 14.00CD ± 1.732
4 17.00AB ± 1.527 4.66CD ± 0.882 8.33BC ± 0.882 16.50ABCD ± 0.288
5 14.50BCD ± 0.288 5.50C ± 0.288 5.50D ± 0.288 18.50AB ± 0.866
6 21.50A ± 0.288 7.00AB ± 0.577 8.50BC ± 0.866 19.00AB ± 0.577
7 20.00AB ± 1.154 2.50E ± 0.288 8.50BC ± 0.288 12.00 D ± 0.577
8 20.10AB ± 1.595 3.50DE ± 0.288 8.00BC ± 0.577 17.50ABC ± 0.288
9 19.50AB ± 1.041 6.00BC ± 0.577 9.50AB ± 0.288 18.50AB ± 0.288
10 19.50AB ± 6.007 6.00BC ± 0.577 8.50BC ± 0.866 19.50AB ± 0.288
Each value represents the mean S.E.; The mean value with different superscript alphabets in the same column indicate significant differences (P0.05) using LSD test. 100 100
Fig. (12): Effect of treatments of rosemary essential oil at different irradiation doses on the bacterial inhibition zone (mm) 101
compounds (carvacrol and thymol). Meanwhile, Tyagi and Anushree (2011) found that the growth of A. flavus was completely inhibited at 60 µl essential oil vapour indicating a higher fungicidal action by the Eucalyptus globulus oil used. Furthermore, cumin essential oil showed high antifungal activity against various pathogenic fungi (Rahman et al., 2000). The essential oil of T. vulgaris inhibits both mycelial growth and aflatoxin synthesis by Aspergilhs parasiticus (Tantaoui-Elaraki and Beraoud, 1994) at only 0.1 per cent in the medium. Therefore it is used as a preservative in agriculture, completely inhibiting aflatoxin production on lentil seeds up to eight weeks of incubation (El-Maraghy, 1995). In addition, the essential oil of T. vulgaris exerts a protective effect in corn against Aspergillus flavus, without producing phytotoxic effects on germination or corn growth (Montes and Carvajal, 1998). On the same trend, the results are in accordance with (Prakash et al., 2013) who found that the Cinnamomum glaucescens essential oil also showed a broad fungitoxic spectrum inhibiting almost all the tested food borne fungi such as (A. flavus, A. niger, A. fumigatus and A. terreus) strengthening complete protection of the treated food commodities from fungal contamination. The antifungal action of plant essential oils has been supposed to be because of their lipophilic nature; causing disruption of plasma membrane and negatively affecting the cellular constituents responsible for vital functioning of cell as has been supported by the transmission electron microscopy studies on essential oil treated Aspergillus flavus culture by Nogueira et al. (2010) and Tian et al. (2012). Several studies have shown that thyme oils possess antibacterial and antifungal activities, those of the phenol type being the most active (Rasooli and Abyanech, 2004; Giordani et al., 2004; Pinto et al., 2006 and Marzouk et al., 2009). 102
On the other hand, the agar plates contain Aspergillus flavus treated with non-irradiated rosemary essential oil showed delaying in the spore forming, and the mycelia growth was appeared in the range of 40 mm (inhibition zone). These obtained results might be related to the chemical composition of rosemary essential oil which has 1,8-cineol as a predominant compound as shown in Table (39). The previous data are in agreement with Vilela et al. (2009) and Tyagi and Anushree (2011) who reported that although 1,8-cineole was the major component (86.0%) in Eucalyptus globulus essential oil, it demonstrated very poor fungicidal activity against A. flavus and A. parasiticus in both contact and headspace volatile exposure assays. Data in Figure (13) showed that the inhibition zones were increased related to irradiation doses. Where, the inhibition zone of rosemary essential oil was 40, 35, 35 and 30mm at doses 0, 1, 2 and 3kGy and it was 90 mm (full inhibition) in all plates from 4 kGy up to 10 kGy. As a point of view, rosemary essential oil showed various changes after irradiation treatment might be the reason of the complete inhibition in all plates after 3 kGy. For example, irradiation dose 6 kGy represent an increase in the content of limonene and camphor from 5.93% to 15.37% in limonene and from 14.5% to 29.74% Table (39). Velázquez-Nuñez et al. (2013) mentioned that limonene represented 96.62% in orange peel, the growth of Aspergillus flavus decreased when the essential oil, which contains limonene, is increased. As far as non-phenolic components of essential oils are concerned, the type of alkyl group has been found to influence activity (alkenyl > alkyl). For example, limonene (1-methyl-4-(1-methylethenyl) cyclohexene) is more active in the antimicrobial activity than ρ-cymene (Dorman and Deans, 2000). 103
Cumin Thyme Rosemary
90 90 90 90 90 90 90
90 90 90 90 90 90 90 90 90 90 90
100 90 90 90 90 90 90 90 90 90 90 90
80 40 35 35 30 60
40 3 10 20
Inhibiton Zone in mm in Zone Inhibiton 0 ١٠10 9٩ 8٨ 7٧ 6٦ 5٥ ٤4 ٣3 2٢ 1١ 0٠
Irradiation Treatments (kGy) Fig. (13): Effect of different irradiation doses on the inhibition zones (mm) of cumin, rosemary and thyme essential oil against Aspergillus flavus 104
4.3.3. Effect of antibacterial recommended doses on bacterial strains growth: The preliminary experimental of all irradiated essential oils were recommended that the final dose of irradiated cumin essential oil was 2 and 4 kGy while, the selected dose of irradiated rosemary and thyme was 6 kGy and 4 kGy.
Antimicrobial potential of the thyme, cumin and rosemary essential oils were also observed in terms of the zone of inhibition generated by the well- diffusion of the essential oil components into the agar plate. The zone of inhibition increased with the increasing concentration (30, 50 and 70 µl) of all essential oils under investigation in each well Table (8). The obtained finding is in the line with (Viuda-Martos et al., 2008 and Tyagi and Anushree, 2011) who found that the inhibition zones were increased related to the increase of the essential oil concentrations. 4.3.3.1. Effect of thyme essential oil on growth inhibition of bacterial strains: As shown in Table (8) and Figures (14 and 15) the inhibition zones of non-irradiated thyme essential oil were 19.50, 24 and 30.33 mm in the concentrations 30, 50 and 70 µl, respectively. While, it was 21, 21.50 and 42 mm in irradiated thyme essential oil at the same concentrations, in the case of Escherichia coli. The data are agreed with Schelz et al. (2006) who found that the inhibition zone was 26 mm. On the other hand, in case of Salmonella typhimurium the obtained inhibition zones in both irradiated and non-irradiated thyme essential oil were 24, 24.33 and 25 mm and 20.50, 25 and 26.50 mm in the concentrations 30, 50 and 70 µl, respectively. The obtained data are similar to the obtained 105
Table (8): Effect of different essential oils in different concentrations (µl) on the inhibition zone (mm) of different bacterial strains Salmonella typhimurium Staphylococcus aureus Escherichia coli Bacillus cereus Conc. (µl) Irradiation doses (kGy) 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 30 20.50B -- 24.00AB -- 17.50C -- 13.50C -- 19.50E -- 21.00D -- 19.50C -- 19.50C -- ± 0.288 ± 1.527 ± 1.443 ± 3.752 ± 0.288 ± 0.577 ± 0.288 ± 0.288
50 25.00A -- 24.33AB -- 29.00B -- 31.00B -- 24.00C -- 21.50D -- 20.50BC -- 21.00C -- ± 2.887 ± 0.882 ± 0.577 ± 1.527 ± 0.577 ± 0.288 ± 0.288 ± 0.577 essential oil 70 26.50A -- 25.00AB -- 32.50AB -- 39.00A -- 30.33B -- 42.00A -- 24.50A -- 23.00AB -- ± 0.866 ± 1.155 ± 1.443 ± 0.577 ± 0.882 ± 0.577 ± 0.866 ± 1.732 10 5 Thyme
30 6.5CD 5.5D 8.5BC -- 4.00C 5.50C 4.67C -- 10.00E 09.50E 13.50D -- 11.50ABC 10.0BC 8.00C -- ± 0.288 ± 0.288 ± 1.443 ± 0.577 ± 0.288 ± 0.882 ± 1.150 ± 0.288 ± 0.866 ± 0.288 ± 2.516 ± 0.577
50 11.0AB 5.5D 11.5A -- 8.50C 6.00C 5.50C -- 13.00D 17.50C 14.50D -- 12.00ABC 11.0ABC 10.30B -- ± 0.577 ± 1.443 ± 0.288 ± 0.866 ± 0.577 ± 0.288 ± 0.577 ± 1.440 ± 0.288 ± 1.155 ± 0.577 ± 1.422 essential oil 70 13.0A 8.5BC 12.5A -- 20.00B 26.50A 29.00A -- 18.50C 30.00A 23.00B -- 14.00AB 14.5A 10.50BC -- ± 0.577 ± 1.041 ± 0.866 ± 6.245 ± 0.288 ± 0.577 ± 0.288 ± 0.577 ± 0.577 ± 1.154 ± 0.288 ± 0.866 Cumin 106
Table (8): Continued
30 2.50B -- -- 3.50B 2.50B -- -- 3.00B 5.00C -- -- 9.00BC 7.50B -- -- 7.50B ±0.288 ±0.288 ± 0.288 ±0.577 ± 2.645 ±0.577 ±0.288 ±0.288
50 2.50B -- -- 7.00A 15.50A -- -- 13.50A 9.50BC -- -- 17.00A 8.50B -- -- 8.50B
±0.288 ±0.577 ± 0.288 ±0.288 ± 0.288 ±0.577 ±0.288 ±0.866 10 6
70 6.50A -- -- 7.00A 17.57A -- -- 19.00A 15.33AB -- -- 21.50A 7.50B -- -- 8.50B Rosemary essential oil ±1.443 ±0.577 ±4.881 ±0.577 ± 2.848 ±0.288 ±0.288 ±0.288
Conc. = concentration of essential oil (µl), Each value (Area under both irradiation doses from 0kGy to 6kGy and concentration from 30µl to 70 µl) represents the mean S.E., The mean value with different superscript alphabets in the same area indicate significant differences (P0.05) using LSD test, -- = Not determined 107
(TE)
(Tm)
(TS)
(Tu)
TE = Thyme essential oil against E. coli, Tm = Thyme essential oil against
S. typhimurium, TS = Thyme essential oil against S. aureus, Tu = Thyme essential oil against B. cereus
Fig. (14): Effect of irradiated thyme essential oil (4kGy) and non-irradiated thyme essential oil on growth inhibition of various bacterial strains 108
45 Salmonella typhimurium Staphylococcus aureus 40 Bacillus cereus Escherichia coli
35
30
25
20 10
15 8
10 Inhibition zone in mm in zone Inhibition
5
0 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl Thyme (0kGy) Thyme (4kGy)
Fig. (15): Antimicrobial potential of both non-irradiated and irradiated thyme essential oils at different concentrations on the bacterial inhibition zone in mm 109
by Teixeira et al. (2013) who found that the inhibition zone of Spain thyme essential oil was 57 mm. The inhibition zones in case of Staphylococcus aureus were 17.50, 29 and 32 mm in non-irradiated thyme essential oil at concentrations 30, 50 and 70 µl, respectively. While, it was 13.50, 31 and 39 mm in irradiated thyme essential oil at the same concentration.
In case of Bacillus cereus the inhibition zones were in both non-irradiated and irradiated essential oil 19.50, 20.50 and 24.50 mm and 19.50, 21 and 23 at concentrations 30, 50 and 70 µl, respectively. The obtained results are defined that the thyme essential oil either irradiated or non-irradiated had strong antimicrobial activity as mentioned by Ait-Ouazzou et al. (2011) who reported that the inhibition zone of essential oil which had strong activity was ≥ 20 mm while, the moderate activity was in inhibition zone < 20 to 12 mm and no inhibition zone was ≤ 12 mm. Regarding to that reporting all concentrations of thyme essential oil either irradiated or non-irradiated had strong activity against selected bacterial strains except at concentration 30 µl in both irradiated and non-irradiated. The mode of action of the antimicrobial activity of thyme essential oil is related to its component. Meanwhile, thymol and carvacrol (the predominate compounds of thyme essential oil) are able to disintegrate the outer membrane of gram-negative bacteria, releasing lipopolysaccharides (LPS) and increasing the permeability of the cytoplasmic membrane to ATP. The presence of magnesium chloride has been shown to have no influence on this action, suggesting a mechanism other than chelation of cations in the outer membrane (Helander et al., 1998). 110
4.3.3.2. Effect of cumin essential oil on growth inhibition of bacterial strains: The data tabulated in Table (8) and illustrated by Figures (16 and 17) showed that, in case of Escherichia coli, the inhibition zones of non- irradiated cumin essential oil were 10, 13 and 18.50 mm at concentrations 30, 50 and 70 µl, respectively.
As classified by Ait-Ouazzou et al. (2011) the concentrations of 50 and 70 µl tends to be a moderate activity while, the concentration 30 µl represents no inhibition. On the hand, irradiated cumin essential oil at doses 2 and 4 kGy showed that the inhibition zones at concentrations 30, 50 and 70 µl were as follow; 9.5, 17.5, 13.5, 14.5 and 23, respectively. Except for 70 µl that had a strong activity against selected bacteria, all concentrations showed moderate activity. Our results come to agreement with previous observations with Singh et al. (2005) who found that cumin essential oil has no inhibition on the gram negative bacteria such as Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium at concentration 6 µl. Furthermore; Hajlaoui et al. (2010) mentioned that the inhibition zone of Tunisian cumin essential oil was 12mm at concentration 10 µl. In case of Salmonella typhimurium the obtained inhibition zones in all concentrations and selected doses represented no inhibition (below 12 mm) but the concentration 70 µl in non-irradiated and irradiated with 4 kGy showed slight moderate activity. Those results are completely agreed with Singh et al. (2005) and Hajlaoui et al. (2010) at concentration 6 and 10 µl, respectively. The inhibition zones in case of Staphylococcus aureus were 4, 8.5 and 20 mm in non-irradiated cumin essential oil at concentrations 30, 50 and 70 µl, respectively. While, it was 5.5, 6 and 26.5 mm in irradiated cumin 111
CE = Cumin essential oil against E. coli, Cm = Cumin essential oil against S.
typhimurium, CS = Cumin essential oil against S. aureus, Cu = Cumin essential oil against B. cereus
Fig. (16): Effect of irradiated cumin essential oil (2 and 4kGy) and non- irradiated cumin essential oil on growth inhibition of various bacterial strains 112
30 Salmonella typhimurium Staphylococcus aureus Bacillus cereus Escherichia coli 25
20
15 11 2 10 Inhibition Zone Zone mmInhibitionin
5
0 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl Cumin (0kGy) Cumin (2kGy) Cumin (4kGy)
Fig. (17): Antimicrobial potential of both non-irradiated and irradiated cumin essential oils at different concentrations on the bacterial inhibition zone in mm 113
essential oil at dose 2kGy while, it was 4.67, 5.50 and 29 mm at dose 4 kGy, at the same concentration. However, the concentrations of 30 and 50 µl represent no inhibition while, the concentration 70 µl showed strong activity. The results are agreed with Hajlaoui et al. (2010) who found the inhibition was 10 mm. In case of Bacillus cereus the inhibition zones in irradiated essential oil at 2 and 4 kGy were 10, 11 and 14.50 mm and 8, 10.3 and 10.5 at concentrations 30, 50 and 70 µl, respectively. While, non-irradiated essential oil represents 11.5, 12 and 14 mm. 4.3.3.3. Effect of rosemary essential oil on growth inhibition of bacterial strains: Results tabulated in Table (8) and illustrated by Figures (18 and 19) showed the inhibition zones of rosemary essential either irradiated or non- irradiated. Thus, the data revealed that, in case of Escherichia coli the inhibition zones obtained in both non-irradiated and irradiated essential oil were 5, 9.5 and 15.33 mm and 9, 17 and 21.5 mm at concentrations 30, 50 and 70 µl. Only one datum noticed a strong activity according to Ait- Ouazzou et al. (2011) at concentration 70 µl treated with 6 kGy. Also, Schelz et al. (2006) mentioned that the inhibition zone of rosemary in case of Escherichia coli was 15 mm. The last data are in the same line with Bousbia et al. (2009) who revealed that Escherichia coli showed 15.5 mm inhibition related to exposing of Algerian rosemary essential oil. After the treatments of either irradiated or non-irradiated rosemary essential oil against the plates inoculated with Salmonella typhimurium, data found that there were no inhibitions (below 12 mm) in all used concentrations as classified by Ait-Ouazzou et al. (2011). 114
(RE)
(Rm)
(RS)
(Ru)
RE = Rosemary essential oil against E. coli, Rm = Rosemary essential oil
against S. typhimurium, RS = Rosemary essential oil against S. aureus, Ru = Rosemary essential oil against B. cereus Fig. (18): Effect of irradiated rosemary essential oil (6kGy) and non-irradiated rosemary essential oil on growth inhibition of various bacterial strains 115
25 Salmonella typhimurium Staphylococcus aureus Bacillus cereus Escherichia coli 20
15
10 5 11
5 Inhibition Zone Zone mmInhibitionin
0 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl Rosemary (0kGy) Rosemary (6kGy)
Fig. (19): Antimicrobial potential of both non-irradiated and irradiated rosemary essential oils at different concentrations on the bacterial inhibition zone in mm 116
In case of Staphylococcus aureus the inhibition zones were in both non- irradiated and irradiated rosemary essential oil 2.5, 15.5 and 17.57 mm and 3, 13.5 and 19 at concentrations 30, 50 and 70 µl, respectively. Bousbia et al. (2009) found that the inhibition zone of Algerian rosemary essential oil was 12.5 mm against Staphylococcus aureus. Finally, rosemary essential oil represents no inhibition (below 12 mm) against Bacillus cereus in both irradiated and non-irradiated. 4.4. Evaluation of interactions between essential oils against bacterial strains: 4.4.1. Evaluate interactions between non-irradiated essential oils mixtures: 4.4.1.1. Evaluate antibacterial activity of mixture made from non-
irradiated cumin and rosemary essential oil (C0 X R0) on bacterial strains: Antibacterial activity of non-irradiated cumin essential combined with rosemary essential oil at different concentrations were tabulated in Table (9) and illustrated by Figure (20). In case of Salmonella typhimurium, the inhibition zones were 6.50, 9.67 and 11 mm at concentrations 30, 50 and 70 µl, respectively. all these inhibition zones means that there were no antimicrobial activity in the C0 X R0 mixture as mentioned by Ait-Ouazzou et al. (2011). The inhibition zones of Escherichia coli were 6.5, 14.33 and 14.40 mm at concentrations 30, 50 and 70 µl, respectively. While, the inhibition zones of Bacillus cereus were 6, 13 and 13.67 mm at the same concentrations. On the other hand, Staphylococcus aureus represent no inhibition (below 12 mm) as declared by Ait-Ouazzou et al. (2011) where, the inhibition zones were 6.40, 11.67 and 12 mm. 117
Table (9): Effect of mixture made from non-irradiated cumin and rosemary essential oils (C0 X R0) in different concentrations on the inhibition zone of selected bacteria
7 Concentration Salmonella Escherichia Bacillus Staphylococcus 11 (µl) typhimurium coli cereus aureus 30 6.50B ± 0.29 6.50B ± 0.29 6.00B ± 2.31 6.40A ± 0.87 C X R 0 0 50 9.67A ± 0.33 14.33A ± 1.20 13.00A ± 0.58 11.67A ± 4.18 mixture 70 11.00A ± 0.58 14.40A ± 0.70 13.67A ± 0.88 12.00A ± 4.58
C0 X R0 = mixture of non-irradiated cumin and rosemary essential oil, Each value represents the mean S.E.; the mean value with different superscript alphabets in the same column indicate significant differences (P0.05) using LSD test. 118
20 Salmonella typhimurium Staphylococcus aureus Bacillus cereus Escherichia coli 18
16
14
12
10
8 11 8 6 Inhibition Zone Zone mmInhibitionin 4
2
0 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl Cumin (0kGy) Rosemary (0kGy) Cumin (0kGy) X Rosemary (0kGy)
Fig. (20): Antimicrobial potential of mixture made from non-irradiated cumin and rosemary essential oils (C0 X R0) at different concentrations on the bacterial inhibition zone in mm 119
The inhibition zones were studied practically and calculated mathematically to all mixtures of both irradiated and non-irradiated essential oils used in our present study as declared by (Lis-Balchin and Deans, 1998a). This trail is done as a potential determination of synergistic activity of essential oils mixtures against selected bacterial strains.
The inhibition zone of practical mixture of C0 X R0 was studied compared to the mathematical mean between both individual cumin and rosemary essential oil. The results found that the mixture of non-irradiated cumin and rosemary essential oils represented higher antibacterial activity against Bacillus cereus compared to other bacterial strains as shown in Table (10). Where, the practical inhibition zone was 13 mm while, the mathematical mean of the individual cumin and rosemary essential oil was 10.25 mm, at the concentration 50 µl (the inhibition zone of individual cumin essential oil was 12mm while, rosemary was 8.5). This finding tends to refer that there was a synergism might be obtained by this mixture. In that declaration, our obtained result is completely agreed with (Davidson and Parish, 1989) who mentioned that an additive effect is observed when the combined effect is equal to the sum of the individual effects. Synergism is observed when the effect of the combined substances is greater than the sum of the individual effects. Antagonism is observed when the effect of one or both compounds is less when they are applied together than when individually applied. Furthermore, Dufour et al. (2003) was chose to distinguish combinations of compounds acting in an additive manner (where the degree of inhibition was greater than could be accounted for by the action of compound 1, or the action of compound 2, acting independently) from those showing a synergistic interaction (where the degree of inhibition was greater than could 120
Table (10): The antibacterial effect of individual non-irradiated cumin and rosemary essential oil and 1:1 mixture of them Inhibition zone (mm) Conc. Essential oil alone (µl) Mathematically Practically * ** Cumin Rosemary Mean Mix 30 6.5 2.5 4.5 6.5
50 11 2.5 6.75 9.6 almonella
S 70 13 6.5 9.75 11 typhimurium
30 4 2.5 3.25 6.4
50 8.5 15.5 12 11.6 aureus 70 20 17.57 18.78 12 taphylococcus S
30 11.5 7.5 9.5 6
50 12 8.5 10.25 13 acillus cereus B 70 14 7.5 10.75 13.6
30 10 5 7.5 6.5
50 13 9.5 11.25 14.3 coli
Escherichia 70 18.5 15.3 16.9 14.4
* ** Mean of the two individual zones of inhibition (mm), The zone of inhibition (mm) of the mixture, Conc. = Concentration 121
be accounted for by the action of compound 1 plus the action of compound 2) because we believe it is the truly synergistic interactions that are of principal interest in food preservation applications. In addition, in case of Escherichia coli the obtained of practical inhibition zone as shown in Table (10) was 14.3 mm while, the mathematical mean of the individual cumin and rosemary essential oil was 11.25 mm at the concentration of 50 µl. the same finding obtained in case of Bacillus cereus at concentration 50
µl where, the obtained inhibition zone of the C0 X R0 mixture was greater than that calculated mathematically and individual essential oils (13 mm in cumin essential oil and 9.5 mm in rosemary essential oil). This datum might be refers to the synergistic effect of that mixture in case of Bacillus cereus at concentration 50 µl. Moreover, Table (10) showed that most of practical inhibition zones that have been done in the present study were higher than mathematical inhibition zones, but not more than the individual essential oil; i.e. in case of C0 X R0 against Salmonella typhimurium at concentration 50 µl. the practical inhibition zone was 9.6 mm while, mathematical inhibition zone was 6.75 mm but, the inhibition zone of individual cumin essential oil was 11 mm. The obtained results are in agreement with (Lis-Balchin and Deans, 1998a), who mentioned that the antibacterial activity of the mixture was also, in most cases, less than that of the most active individual essential oil. Our study also indicates that the essential oils of cumin, which have aldehyde, γ-terpinene and β-pinene, have severe antibacterial activity against gram-positive and gram-negative bacteria (Iacobellis et al. 2005). The mechanisms by which essential oils can inhibit microorganisms have different reasons, and in some part may be due to their hydrophobicity. In this connection, the penetration of the oils into the lipid bilayer of the cell membrane makes the cells more permeable, leading to leakage of vital cell contents (Kim et al. 1995 and Burt 2004). 122
Lipophilic hydrocarbon molecules (γ-terpinene, ρ-cymene and β-pinene) could accumulate in the lipid bilayer and distort the lipid–protein interaction; alternatively, direct interaction of the lipophilic compounds with hydrophobic parts of the protein is possible (Juven et al., 1994 and Sikkema et al., 1995). Some essential oils have been found to stimulate the growth of pseudomycelia (a series of cells adhering end-toend as a result of incomplete separation of newly formed cells) in certain yeasts. This could be an indication that essential oils act on the enzymes involved in the energy regulation or synthesis of structural components (Conner and Beuchat, 1984). Moreover, the antimicrobial activity of essential oils may be due to the presence of synergy between the major components and other constituents of the oils leading to various degrees of antimicrobial activity (Bouhdid et al. 2008). 4.4.1.2. Evaluate antibacterial activity of mixture made from non-
irradiated cumin and thyme essential oil (C0 X T0) on bacterial strains: As antimicrobial activity depends not only on chemical composition but also on lipophilic properties, the potency of functional groups or aqueous solubility (Dorman and Deans, 2000) and the mixture of compounds with different biochemical properties may increase essential oil efficacy. The antibacterial activity of non-irradiated cumin essential combined with thyme essential oil at different concentrations were tabulated in Table (11) and illustrated by Figure (21). In case of Salmonella typhimurium, the inhibition zones were 8.50, 13.50 and 14.67 mm at concentrations 30, 50 and 70 µl, respectively. 123
Table (11): Effect of mixture made from non-irradiated cumin and thyme essential oils (C0 X T0) in different concentrations on the inhibition zone of selected bacteria
Concentration Salmonella Escherichia Bacillus Staphylococcus (µl)
123 typhimurium coli cereus aureus 30 8.50A ± 0.29 13.67A ± 0.88 15.00A ± 0.58 8.00B ± 0.58 C X T 0 0 13.50A ± 2.59 14.00A ± 1.53 15.00A ± 1.53 10.00AB ± 1.16 mixture 50 70 14.67A ± 2.85 17.67A ± 0.88 15.00A ± 3.61 11.50A ± 0.29
C0 X T0 = mixture of non-irradiated cumin and thyme essential oil, Each value represents the mean S.E.; the mean value with different superscript alphabets in the same column indicate significant differences (P0.05) using LSD test. 124
35 Salmonella typhimurium Staphylococcus aureus Bacillus cereus Escherichia coli 30
25
20
15 12 4 10 Inhibition Zone in mm in Zone Inhibition
5
0 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl Cumin (0kGy) Thyme (0kGy) Cumin (0kGy) X Thyme (0kGy)
Fig. (21): Antimicrobial potential of mixture made from non-irradiated cumin and thyme essential oils (C0 X T0) at different concentrations on the bacterial inhibition zone in mm 125
Table (12): The antibacterial effect of individual non-irradiated cumin and thyme essential oil and 1:1 mixture of them Inhibition zone (mm) Conc. Essential oil alone Mathematically Practically (µl) * ** Cumin Thyme Mean Mix 30 6.5 20.5 13.5 8.5
50 11.0 25.0 18.00 13.5
Salmonella 70 13.0 26.5 19.75 14.6 typhimurium
30 4.0 17.5 10.75 8.0
50 8.5 29.0 18.75 10.0 aureus 70 20.0 32.5 26.25 11.5 Staphylococcus
30 10.0 19.5 14.75 15.0
50 11.0 20.5 15.75 15.0 cereus Bacillus 70 14.0 24.5 19.25 15.0
30 10.0 24.0 17.00 13.6
50 13.0 25.0 19.00 14.0 coli
Escherichia 70 18.5 30.3 24.40 17.6
* ** Mean of the two individual zones of inhibition (mm), The zone of inhibition (mm) of the mixture, Conc. = Concentration 126
The concentration of 30 µl showed no inhibition (below 12 mm) as mentioned by Ait-Ouazzou et al. (2011) while, at concentrations 50 and 70 µl the inhibition represented moderate activity against selected bacteria. All inhibition zones of mixture made from non-irradiated cumin and thyme essential oil against Escherichia coli and Bacillus cereus were possess to have moderate activity (the inhibition zone is between 12 and 20 mm). There was no inhibition zone of the essential oil mixture against Staphylococcus aureus in all concentrations used in our study. On the other hand, the application of the declaration mentioned by
Dufour et al. (2003) in case of C0 X T0 mixture as shown in Table (12), there were no synergistic effect noticed in that mixture against selected bacterial strains and at various concentrations in our present study. Moreover, the results are agreed with the previous obtained by Lis-Balchin and Deans (1998a) who mentioned that the antibacterial activity of the mixture was also, in most cases, less than that of the most active individual essential oil. 4.4.1.3. Evaluate antibacterial activity of mixture made from non-
irradiated rosemary and thyme essential oil (R0 X T0) on bacterial strains
As shown in Table (13) and Figure (22), the inhibition zones of R0 X T0 mixture against Salmonella typhimurium are 7.33, 7.50 and 8.50 mm at concentrations 30, 50 and 70 µl, respectively. These inhibition zones refer to no inhibition according to Ait-Ouazzou et al. (2011) who mentioned that below 12 mm of inhibition there are no inhibition activity for the selected antimicrobial used. In case of Escherichia coli, the found inhibition zones were 11.50m 13 and 13 mm at concentrations 30, 50 and 70 µl. these inhibitions showed 127
Table (13): Effect of mixture made from non-irradiated rosemary and thyme essential oils (R0 X T0) in different concentrations on the inhibition zone of selected bacteria
Concentration Salmonella Escherichia Bacillus Staphylococcus 7 (µl) 12 typhimurium coli cereus aureus 30 7.33A ± 0.333 11.50A ± 0.866 13.00B ± 0.577 2.50B ± 0.288 R X T 0 0 50 7.50A ± 0.288 13.00A ± 2.309 21.33A ± 1.202 8.50A ± 0.288 mixture 70 8.50A ± 0.288 13.00A ± 1.527 22.00A ± 1.732 8.50A ± 0.288
R0 X T0 = mixture of non-irradiated rosemary and thyme essential oil, Each value represents the mean S.E.; the mean value with different superscript alphabets in the same column indicate significant differences (P0.05) using LSD test. 128
35 Salmonella typhimurium Staphylococcus aureus Bacillus cereus Escherichia coli 30
25
20
15 12
10 8 Inhibition zone in mm in zone Inhibition
5
0 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl Rosemary (0kGy) Thyme (0kGy) Rosemary (0kGy) X Thyme (0kGy)
Fig. (22): Antimicrobial potential of mixture made from non-irradiated rosemary and thyme essential oils (R0 X T0) at different concentrations on the bacterial inhibition zone in mm 129
Table (14): The antibacterial effect of individual non-irradiated rosemary and thyme essential oils and 1:1 mixture of them Inhibition zone (mm) Conc. Essential oil alone Mathematically Practically (µl) * ** Rosemary Thyme Mean Mix 30 2.5 20.5 11.5 7.3
50 2.5 25 13.75 7.5
Salmonella 70 6.5 26.5 16.5 8.5 typhimurium
30 2.5 17.5 10 2.5
50 15.5 29 22.25 8.5 aureus 70 17.5 32.5 25 8.5 Staphylococcus
30 7.5 19.5 13.5 13
50 8.5 20.5 14.5 21.3 cereus Bacillus 70 7.5 24.5 16 22
30 5 24 14.5 11.5 a
50 9.5 25 17.25 13 coli
Escherichi 70 15.3 30.3 22.8 13
* ** Mean of the two individual zones of inhibition (mm), The zone of inhibition (mm) of the mixture, Conc. = Concentration 130
the antimicrobial activity between no inhibition and moderate activity. On the other hand, the effect of irradiated essential oil when mixed together against Staphylococcus aureus showed no inhibition according to Ait- Ouazzou et al. (2011) because of all obtained inhibitions zones are below 12 mm. While, the concentrations 50 and 70 µl of the same mixture against Bacillus aureus represent strong activity.
However, Table (14) most of the R0 X T0 mixture used against selected bacterial strains showed no synergy according to the (Davidson and Parish,
1989 and Dufour et al., 2003) hypothesis except for the R0 X T0 mixture against Bacillus cereus at concentration 50 µl where, the zone of inhibition was 21.3 mm strong activity, (Ait-Ouazzou et al., 2011) in the practical trail, 14.5 mm in the mathematical calculation while, the individual essential oil was 8.5 mm and 20.5 mm in rosemary and thyme essential oil, respectively. This finding might be showed a synergistic effect against selected bacterial strain. 4.4.2. Evaluate interactions between mixtures made from irradiated essential oils: 4.4.2.1. Evaluate antibacterial activity of irradiated cumin essential oil at
doses 2 and 4 kGy (C2 X C4) mixture on bacterial strains: As shown in Table (15) and Figure (23) the mixture made from irradiated cumin essential oils at doses 2 and 4 kGy, theoretically this trail did not show any further achievements in our investigation, as a point of view. Thus, this trail is done to tray all possibilities changing of irradiation doses to chemical constituents of essential oil - especially after the finding of that 2 and 4 kGy of irradiated cumin essential oil showed a promising antimicrobial activity in the preliminary experiment (Table 6 and Figure 11). The inhibition zones obtained in case of Salmonella typhimurium, Escherichia coli and Staphylococcus aureus were showed no inhibition activity (below 12 mm). Thus, the practical investigation is similar to the 131
theoretical hypothesis where, all obtained data are varying from no inhibition activity to moderate activity (Ait-Ouazzou et al., 2011). As a point of view, the percentage amount of non-oxygenated compounds in irradiated cumin essential oil at dose 4 kGy was 24.60% (Table 38). Moreover, the percentage amount of both γ-terpinene and ρ-cymene were 12.97% and 6.43%, respectively. Thus, that finding might be tens to show slight antagonistic in this mixture
As our pointing of view declared, mechanisms of antimicrobial interaction that produce antagonism are less known, although they include combinations of bactericidal and bacteriostatic agents, use of compounds that act on the same target of the microorganism and chemical (direct or indirect) interactions among compounds. For instance, non-oxygenated monoterpene hydrocarbons such as γ-terpinene and ρ-cymene appear to produce antagonistic effects, since they reduce the aqueous terpene solubility and, therefore, the microbial availability of the active components (Cox et al., 2001). Therefore, specific modes of action of plant constituents with antimicrobial properties on the metabolic activities of microorganisms still need to be clearly defined, even when the antimicrobials are used individually (Alzamora et al., 2003). Despite the previous notifications was in our first importance, Table (16) showed some promising data especially in case the treatment of C2 X C4 mixture against Bacillus cereus at concentration 50 µl. where, the inhibition zone was 15.5 mm (practically), 10.65 mm (mathematically). While, the individual irradiated cumin at doses 2 and 4 kGy were 11 and 10.3 mm. this data could be refers to synergistic effect. 132
Table (15): Effect of mixture made from irradiated cumin essential oil at doses 2 and 4 kGy (C2 X C4) at different concentrations on the inhibition zone of selected bacteria
Concentration Salmonella Escherichia Bacillus Staphylococcus (µl) aureus
typhimurium coli cereus 13
30 7.50B ± 1.44 7.00A ± 0.58 5.00B ± 0.58 5.50A ± 0.27 2 C X C 2 4 50 11.50A ± 0.29 10.50A ± 0.29 15.50A ± 0.29 7.00A ± 1.73 mixture 70 11.50A ± 0.29 11.00A ± 3.51 16.00A ± 0.58 7.10A ± 1.65
C2 X C4= mixture of irradiated cumin essential oil at doses 2 and 4 kGy, Each value represents the mean S.E.; the mean value with different superscript alphabets in the same column indicate significant differences (P0.05) using LSD test. 133
30 Salmonella typhimurium Staphylococcus aureus Bacillus cereus 25 Escherichia coli
20
15 3
13 10 inhibition Zone in mmin inhibitionZone
5
0 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl Cumin (2kGy) Cumin (4kGy) Cumin (2kGy) X Cumin (4kGy)
Fig. (23): Antimicrobial potential of mixture made from irradiated cumin essential oil at doses 2 and 4 kGy (C2 X C4) at different concentrations on the bacterial inhibition zone in mm 134
Table (16): The antibacterial effect of individual irradiated cumin (2 kGy) and irradiated cumin (4 kGy) essential oils and 1:1 mixture of them Inhibition zone (mm) Conc. Essential oil alone Mathematically Practically (µl) Cumin Cumin Mean * Mix ** 2kGy 4kGY 30 5.5 8.5 7 7.5
50 5.5 11.5 8.5 11.5
Salmonella 70 8.5 12.5 typhimurium 10.5 11.5
30 5.5 4.6 5.05 5.5
50 6 5.5 5.75 7 aureus 70 26.5 29 27.75 7.1 Staphylococcus
30 10 8 9 5
50 11 10.3 10.65 15.5 cereus Bacillus 70 14.5 10.5 12.5 16
30 9.5 13.5 11.5 7
50 17.5 14.5 16 10.5 coli
Escherichia 70 30 23 26.5 11
* ** Mean of the two individual zones of inhibition (mm), The zone of inhibition (mm) of the mixture, Conc. = Concentration 135
As mentioned in Table (37) the percentage amount of cumin aldehyde (monoterpene aldehyde, Bettaieb et al., 2011 and Vazin, 2013) was increased related to irradiation dose from 24.93% to 33.65%. This increment might be caused the increment of antimicrobial activity of irradiated cumin essential oil. That hypothesis is in the line with (Arora and Kaur, 1999 and Hajlaoui et al., 2010) who mentioned that the components of essential oil (aldehydes, ketones, and alcohols) showed the strongest antimicrobial activity. 4.4.2.2. Evaluate antibacterial activity of irradiated cumin and rosemary
essential oils at doses 2 and 6 kGy mixture (C2 X R6) on bacterial strains:
The inhibition zones of C2 X R6 mixture are tabulated in Table (17) and Figure (24). The results of this mixture were as follow; in case of Salmonella typhimurium and Staphylococcus aureus were showed no inhibition activity as mentioned by (Ait-Ouazzou et al., 2011) where, all inhibition zones of the mixture were below 12 mm. While the inhibition zones of the mixture illustrated moderate activity in case of Bacillus cereus and Escherichia coli at concentrations 50 and 70 µl. the inhibition zone of 50 µl in case of Bacillus cereus was 14 mm (practically) and 9.75 mm (mathematically). While, it was 14.5 mm (practically) and 11.5 mm (mathematically) at concentration 70 µl. Moreover, the zone of inhibition in case of Escherichia coli at 50 µl was 13 mm (practically) and the same inhibition was in 70 µl (13 mm) while, the mathematical calculation on inhibitions were 17.25 mm and 25.75 mm at concentration 50 and 70 µl, respectively. 136
Table (17): Effect of mixture made from irradiated cumin and rosemary essential oils at doses 2 and 6 kGy, respectively
(C2 X R6) in different concentrations on the inhibition zone of selected bacteria
Concentration Salmonella Escherichia Bacillus Staphylococcus 13 (µl) typhimurium coli cereus aureus 6 30 5.50A ± 0.288 6.00B ± 0.577 5.00B ± 0.577 3.00B ± 0.577 C X R 2 6 50 6.50A ± 0.288 13.00A ± 0.577 14.00A ± 0.577 7.00A ± 0.577 mixture 70 8.00A ± 2.309 13.00A ± 0.577 14.50A ± 0.288 7.00A ± 0.577
C2 X R6= mixture of irradiated cumin and rosemary essential oils at doses 2 and 6 kGy, respectively, Each value represents the mean S.E.; the mean value with different superscript alphabets in the same column indicate significant differences (P0.05) using LSD test. 137
30
25 Salmonella typhimurium Staphylococcus aureus Bacillus cereus Escherichia coli 20
15
7 10 Inhibition Zone Zone mmInhibitionin 13
5
0 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl Cumin (2kGy) Rosemary (6kGy) Cumin (2kGy) X Rosemary (6kGy) Fig. (24): Antimicrobial potential of mixture made from irradiated cumin and rosemary essential oils at doses 2 and 6
kGy (C2 X R6) at different concentrations on the bacterial inhibition zone in mm 138
Table (18): The antibacterial effect of individual irradiated cumin (2 kGy) and irradiated rosemary (6 kGy) essential oils and 1:1 mixture of them Inhibition zone (mm) Conc. Essential oil alone Mathematically Practically (µl) Cumin Rosemary * ** 2kGy 6kGY Mean Mix 30 5.5 3.5 4.5 5.5
50 5.5 7 6.25 6.5
Salmonella 70 8.5 7 typhimurium 7.75 8
30 5.5 3 4.25 3
50 6 13.5 9.75 7 aureus 70 26.5 19 22.75 7 Staphylococcus
30 10 7.5 8.75 5
50 11 8.5 9.75 14 cereus Bacillus 70 14.5 8.5 11.5 14.5
30 9.5 9 9.25 6
50 17.5 17 17.25 13 coli
Escherichia 70 30 21.5 25.75 13
* ** Mean of the two individual zones of inhibition (mm), The zone of inhibition (mm) of the mixture, Conc. = Concentration 139
4.4.2.3. Evaluate antibacterial activity of mixture made from irradiated
cumin and thyme essential oil (C2 X T4) at doses 2 and 4 kGy on bacterial strains: As shown in Table (19) and Figure (25), the practical zone of inhibition was 8, 12 and 12 mm in case of Salmonella typhimurium at concentrations 30, 50 and 70 µl, respectively. While, the zone of inhibition in case of Escherichia coli was 13, 14 and 14 mm at concentrations 30, 50 and 70 µl, respectively. The noticed observation was that, the concentration 50 and 70 µl in both bacteria have the same inhibition zone and in case of Staphylococcus aureus, that observation was closely happened where the zone of inhibition at the same concentrations were 7 and 7.5 mm, respectively. As appoint of view, there were no relationship between the increment of essential oil quantity and the antimicrobial activity especially in the presence of strong antimicrobials together. Furthermore, the zones of in inhibition in case of Staphylococcus aureus were possess to have no inhibition activity according to (Ait-Ouazzou et al., 2011). That finding is in the line with the previous founding by Gutierrez et al., (2008a) who defined that the mechanism of action as well as essential oil composition deserves to be studied in more detail in order to elucidate why combinations of essential oils with a strong individual antimicrobial efficacy such as oregano and thyme, did not show synergistic effects, and why on the other hand combinations of two essential oils with individually moderate activity resulted in enhanced effects in combination, such as marjoram, basil, rosemary or sage. Lambert et al. (2001) reported that carvacrol and thymol in combination were additive against S. aureus and Pseudomonas aeruginosa. 140
Table (19): Effect of mixture made from irradiated cumin and thyme essential oils at doses 2 and 4 kGy, respectively (C2
X T4) in different concentrations on the inhibition zone of selected bacteria
Concentration Salmonella Escherichia Bacillus Staphylococcus
(µl) aureus 1 typhimurium coli cereus 40 30 8.00A ± 1.732 13.00A ± 2.886 12.00A ± 0.577 6.50A ± 0.288 C X T 2 4 50 12.00A ± 1.154 14.00A ± 0.577 13.00A ± 0.577 7.00A ± 0.577 mixture 70 12.00A ± 1.154 14.00A ± 1.527 13.00A ± 4.163 7.50A ± 0.288
C2 X T4= mixture of irradiated cumin and thyme essential oils at doses 2 and 4 kGy, respectively, Each value represents the mean S.E.; the mean value with different superscript alphabets in the same column indicate significant differences (P0.05) using LSD test. 141
45 Salmonella typhimurium
40 Staphylococcus aureus Bacillus cereus 35 Escherichia coli 30
25
20
1 15 14
Inhibition Zone Zone mmInhibitionin 10
5
0 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl Cumin (2kGy) Thyme (4kGy) Cumin (2kGy) X Thyme (4kGy)
Fig. (25): Antimicrobial potential of mixture made from irradiated cumin and thyme essential oils at doses 2 and 4 kGy
(C2 X T4) at different concentrations on the bacterial inhibition zone in mm 142
Table (20): The antibacterial effect of individual irradiated cumin (2 kGy) and irradiated thyme (4 kGy) essential oils and 1:1 mixture of them Inhibition zone (mm) Conc. Essential oil alone Mathematically Practically (µl) Cumin Thyme * ** 2kGy 4kGY Mean Mix 30 5.5 24 14.75 8
50 5.5 24.3 14.9 12
Salmonella 70 8.5 25 16.75 12 typhimurium
30 5.5 13.5 9. 5 6.5
coccus 50 6 31 18.5 7 aureus 70 26.5 39 32.75 7.5 Staphylo
30 10 19.5 14.75 12
50 11 21 16 13 cereus Bacillus 70 14.5 23 18.75 13
30 9.5 21.5 15.5 13
50 17.5 22.5 20 14 coli
Escherichia 70 30 42 36 14
* ** Mean of the two individual zones of inhibition (mm), The zone of inhibition (mm) of the mixture, Conc. = Concentration 143
Nazer et al. (2005) found that thymol in combination with other aromatic compounds led to improved inhibition, but no real synergistic effect was demonstrated between compounds against Salmonella. As plant essential oils possess similar composition, their combinations may exhibit addition rather than a synergistic effect. 4.4.2.4. Evaluate antibacterial activity of mixture made from irradiated
cumin and rosemary essential oil (C4 X R6) at doses 4 and 6 kGy on bacterial strains: Table (21) and Figure (26) illustrated that the inhibition zone of the mixture made from irradiated cumin essential oil at dose 4 kGy and irradiated rosemary essential oil at dose 6 kGy were recorded no inhibition activity as hypothesis of (Ait-Ouazzou et al., 2011). Where, all obtained data of the mixture against selected bacterial strains were below 12 mm. this obtaining was might be related to the antimicrobial activity of cumin (moderate antimicrobial) and rosemary (weak antimicrobial). The results recorded in Table (22) showed that there were no probability to any synergism could be happened in this mixture (hypothesis). This hypothesis came from the obtained practical inhibition zones are below even the calculating inhibition zones and each individual irradiated essential oil. The obtained data are in agreement with Lis-Balchin and Deans (1998a) who found that the antibacterial activity of the 1:1:1 mixtures (cinnamon, coriander and rosewood; cassis, aniseed and caraway and dill, orange and nutmeg) were also, in most cases, less than that of the most active individual essential oil. 144
Table (21): Effect of mixture made from irradiated cumin and rosemary essential oils at doses 4 and 6 kGy, respectively
(C4 X R6) in different concentrations on the inhibition zone of selected bacteria
Concentration Salmonella Escherichia Bacillus Staphylococcus 14 (µl) typhimurium coli cereus aureus 4 30 6.00A ± 1.732 6.50A ± 0.288 6.00A ± 2.309 5.50A ± 0.288 C X R 4 6 50 6.00A ± 1.732 7.00A ± 1.732 8.66A ± 0.882 6.50A ± 0.288 mixture 70 8.50A ± 0.500 7.50A ± 2.021 9.00A ± 0.577 6.50A ± 2.021
C4 X R6 = mixture of irradiated cumin and rosemary essential oils at doses 4 and 6 kGy, respectively, Each value represents the mean S.E.; the mean value with different superscript alphabets in the same column indicate significant differences (P0.05) using LSD test. 145
30 Salmonella typhimurium Staphylococcus aureus Bacillus cereus Escherichia coli 25
20
15
5 10 Inhibition Zone Zone mmInhibitionin 14
5
0 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl Cumin (4kGy) Rosemary (6kGy) Cumin (4kGy) X Rosemary (6kGy) Fig. (26): Antimicrobial potential of mixture made from irradiated cumin and rosemary essential oils at doses 4 and 6
kGy (C4 X R6) at different concentrations on the bacterial inhibition zone in mm 146
Table (22): The antibacterial effect of individual irradiated cumin (4 kGy) and irradiated rosemary (6 kGy) essential oils and 1:1 mixture of them Inhibition zone (mm) Conc. Essential oil alone Mathematically Practically (µl) Cumin Rosemary * ** 4kGy 6kGY Mean Mix 30 8.5 3.5 6 6
50 11.5 7 9.25 6
Salmonella 70 12.5 7 9.75 8.5 typhimurium
30 4.6 3 3.8 5.5
50 5.5 13.5 9.5 6.5 aureus 70 29 19 24 6.5 Staphylococcus
30 8 7.5 7.75 6
50 10.3 8.5 9.4 8.6 cereus Bacillus 70 10.5 8.5 9.5 9
30 13.5 9 11.75 6.5
50 14.5 17 15.25 7 coli
Escherichia 70 23 21.5 22.25 7.5
* ** Mean of the two individual zones of inhibition (mm), The zone of inhibition (mm) of the mixture, Conc. = Concentration 147
4.4.2.5. Evaluate antibacterial activity of mixture made from irradiated
cumin and thyme essential oil (C4X T4) at doses 4 and 4 kGy on bacterial strains: As shown in Table (23) and Figure (27), the results represented in case of Salmonella typhimurium the zone of inhibition was 6, 11 and 11.5 mm at concentrations 30, 50 and 70 µl, respectively. While, it was 3, 12.5 and 13 mm in case of Escherichia coli at the same concentrations.
In addition, Bacillus cereus and Staphylococcus aureus were showed inhibition zones 10, 11.5 and 12 mm and 7, 11.5 and 11.5 mm at concentrations 30, 50 and 70 µl, respectively. These obtaining results are in similar with that discussed in the previous section. As a result, combinations with other compounds containing different chemical structures may improve the essential oil efficacy. For example, synergism between carvacrol and its precursor ρ-cymene has been noted (Ultee et al., 2000). It appears that ρ-cymene, a very weak antibacterial, swells bacterial cell membranes to a greater extent than carvacrol does, so ρ- cymene probably enables carvacrol to be more easily transported into the cell. 4.4.2.6. Evaluate antibacterial activity of mixture made from irradiated
rosemary and thyme essential oil (R6 X T4) at doses 6 and 4 kGy on bacterial strains: As show by Figure (28) and tabulated in Table (25) the antimicrobial activity of mixture made from irradiated rosemary (6kGy) and irradiated thyme (4kGy) were measured as the inhibition of zone. The inhibition zones of the mixture in case of Salmonella typhimurium and Escherichia 148
Table (23): Effect of mixture made from irradiated cumin and thyme essential oils at doses 4 and 4 kGy, respectively (C4
X T4) in different concentrations on the inhibition zone of selected bacteria
Concentration Salmonella Escherichia Bacillus Staphylococcus (µl) aureus 14 typhimurium coli cereus 8 30 6.00B ± 1.732 3.00B ± 0.577 10.00A ± 1.155 7.00B ± 1.732
C4 X T4 A A A A mixture 50 11.00 ± 0.577 12.50 ± 0.288 11.50 ± 0.288 11.50 ± 0.288 70 11.50A ± 1.443 13.00A ± 1.527 12.00A ± 1.000 11.50A ± 0.764
C4 X T4 = mixture of irradiated cumin and thyme essential oils at doses 4 and 4 kGy, Each value represents the mean S.E.; the mean value with different superscript alphabets in the same column indicate significant differences (P0.05) using LSD test. 149
45 Salmonella typhimurium Staphylococcus aureus 40 Bacillus cereus Escherichia coli 35
30
25
20 9
14 15 Inhibition Zone Zone mmInhibitionin
10
5
0 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl Cumin (4kGy) Thyme (4kGy) Cumin (4kGy) X Thyme (4kGy) Fig. (27): Antimicrobial potential of mixture made from irradiated cumin and thyme essential oils at doses 4 and 4 kGy
(C4 X T4) at different concentrations on the bacterial inhibition zone in mm 150
Table (24): The antibacterial effect of individual irradiated cumin (4 kGy) and irradiated thyme (4 kGy) essential oils and 1:1 mixture of them Inhibition zone (mm) Conc. Essential oil alone Mathematically Practically (µl) Cumin Thyme * ** 4kGy 4kGY Mean Mix 30 8.5 24 16.25 6
50 11.5 24.3 18.4 11
70 12.5 25 18.25 11.5 Salmonella typhimurium
30 4.6 13.5 9.05 7
50 5.5 31 30 11.5
aureus 70 29 39 22.25 11.5 Staphylococcus 30 8 19.5 13.75 10
50 10.3 21 15.65 11.5 cereus Bacillus 70 10.5 23 16.75 12
30 13.5 21.5 17.5 3
50 14.5 22.5 18.5 12.5 richia coli 70 23 42 32.5 13 Esche
* ** Mean of the two individual zones of inhibition (mm), The zone of inhibition (mm) of the mixture, Conc. = Concentration 151
coli were 7.5, 10.5 and 13 mm and 9, 12, 50 and 13 mm at concentrations 30, 50 and 70 µl, respectively. Meanwhile, Bacillus cereus represents 8.5, 12 and 12.5 of inhibition and finally in case of Staphylococcus aureus the zone of inhibitions were 5.5 , 8.5 and 8.5 mm. The main components of rosemary and sage, which are camphor and eucalyptol, possess oxygen functions in their structure and these functions are known to increase the antimicrobial properties of terpenoids (Naigre et al., 1996). 4.5. Determination of both minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC): The bacteria demonstrating the largest inhibition zones by diffusion method were not always the ones that present the lowest MIC and MBC values. Perhaps, the diameter of the growth inhibition zone is often affected by the oil solubility and volatility (Bouhdid et al. 2008). Thus, the minimum inhibitory concentration (MIC) is cited by most researchers as a measure of the antibacterial performance of essential oils. The definition of the MIC differs between publications and this is another obstacle to comparison between studies. In some cases, the minimum bactericidal concentration (MBC) or the bacteriostatic concentration is stated, both terms agreeing closely with the MIC (Burt, 2004). 4.5.1. Determination of MIC and MBC of non-irradiated essential oil: The effect of minimum inhibitory concentration (MIC) on bacterial viable cells was tested in liquid medium. The effect of the solvent used to dilute essential oils was also determined against selected bacterial strains. Dimethyl sulfuoxide (DMSO) was used in our trail. DMSO showed no inhibition in all dilutions under investigation. The results are in agreement with (Mahboubi et al., 2007 and Teixeira et al., 2013). 152
Table (25): Effect of mixture made from irradiated rosemary and thyme essential oils at doses 6 and 4 kGy, respectively
(R6 X T4) in different concentrations on the inhibition zone of selected bacteria
Concentration Salmonella Escherichia Bacillus Staphylococcus (µl) typhimurium coli cereus aureus 15 2 30 7.50B ± 0.288 9.00A ± 4.618 8.50A ± 0.288 5.50B ± 0.288 R X T 6 4 10.50A ± 0.763 12.50A ± 1.443 12.00A ± 1.732 8.50A ± 0.288 mixture 50 70 13.00A ± 1.154 13.00A ± 1.527 12.50A ± 3.753 8.50A ± 0.288
R6 X T4 = mixture of irradiated rosemary and thyme essential oils at doses 6 and 4 kGy, Each value represents the mean S.E.; the mean value with different superscript alphabets in the same column indicate significant differences (P0.05) using LSD test. 153
45 Salmonella typhimurium Staphylococcus aureus 40 Bacillus cereus Escherichia coli 35
30
25
20
3 15 15
10 Inhibition zone in mm in zone Inhibition 5
0 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl 30 µl 50 µl 70 µl Rosemary (6kGy) Thyme (4kGy) Rosemary (6kGy) X Thyme (4kGy) Fig. (28): Antimicrobial potential of mixture made from irradiated rosemary and thyme essential oils at doses 6 and 4
kGy (R6 X T4) at different concentrations on the bacterial inhibition zone in mm 154
Table (26): The antibacterial effect of individual irradiated rosemary (6 kGy) and irradiated thyme (4 kGy) essential oils and 1:1 mixture of them Inhibition zone (mm) Conc. Essential oil alone Mathematically Practically (µl) Rosemary Thyme * ** 6kGy 4kGY Mean Mix 30 3.5 24 13.75 7.5
50 7 24.3 15.65 10.5 lmonella
Sa 70 7 25 16 13 typhimurium
30 3 13.5 8.25 5.5
50 13.5 31 22.25 8.5 aureus 70 19 39 29 8.5 Staphylococcus
30 7.5 19.5 13.5 8.5
50 8.5 21 14.75 12 cereus Bacillus 70 8.5 23 15.75 12.5
30 9 21.5 15.25 9
50 17 22.5 19.75 12.5 coli scherichia
E 70 21.5 42 31.75 13
* ** Mean of the two individual zones of inhibition (mm), The zone of inhibition (mm) of the mixture, Conc. = Concentration 155
As shown in Table (27) and illustrated by Figure (29) MIC of non- irradiated cumin essential oil was 0.256, 1.024, 0.256 and 1.024 mg/ml against S. typhimurium, E. coli, S. aureus and B. cereus, respectively. The same results are in the previous study by (Allahghadri et al., 2010 and Ozcan and Erkmen, 2001) who mentioned that the cumin essential oils had antimicrobial activity against both E. coli O157:H7 and E. coli, respectively. While, non-irradiated rosemary have MIC 0.256, 2.048, 0.512 and 2.048 mg/ml against S. typhimurium, E. coli, S. aureus and B. cereus, respectively. The finding results are higher than that obtained by Teixeira et al. (2013) who found that the MIC for rosemary essential oil against S. typhimurium was 90.8 mg/ml. and also Rosato et al. (2007) who found that the MIC of rosemary essential oil was about 1.4 mg/ml. These results are agreement with those published by Zaouali et al., (2010), who also found that rosemary oils rich in eucalyptol exhibited moderate or no action against S. aureus. This affirmation supports the theory that a major proportion of α-pinene and camphor induces the antibacterial activity against S. aureus. Moreover, the obtained results showed high amount of rosemary essential oil to make MIC where, 2.048 mg/ml was the MIC against E. coli. This finding is in the line with Zaouali et al., (2010) and Jordán et al., (2013) who mentioned that the moderate antibacterial activity for rosemary essential oils from the var typicus (eucalyptol chemotype) and var troglodytorum (camphor chemotype) were against this Gram-negative bacterium (E. coli). In addition to this, terpenes have the ability to disrupt and penetrate the lipid structure of the cell wall of bacteria, leading to the denaturing of proteins and the destruction of cell membranes, leading to cytoplasmic leakage, cell lysis and eventually cell death. Thus, our present study revealed that the essential oil of rosemary have α-pinene, ρ-cymene, camphor and limonene which achieve the (Bajpai et al., 2012) notification. 156
Furthermore, non-irradiated thyme essential oil represents MIC 0.256, 0.512, 0.256 and 0.512 mg/ml against the same bacterial strains. Thus, thyme essential oil showed the highest essential oil in the antimicrobial activity measured as MIC, which means the concentrations of essential oil in case of thyme lower than other essential oils used in our present study. The differences in the MIC activity might be related with distinct chemical composition of the essential oils tested as mentioned in Tables (2, 3 and 4).
These results are in agreement with Prabuseenivasan et al. (2006) and Rota et al. (2004) who mentioned that thyme essential oil and its predominant compounds (thymol and carvacrol) have MIC between 0.2 and 25.6 mg/ml. While, Al-Bayati et al. (2008) found that the MICs of thyme essential oil were 31.2, 15.6, 62.5 and 125 µg/ml against S. aureus, B. cereus, E. coli and S. typhimurium respectively. Schelz et al. (2006) mentioned that the MICs of thyme and rosemary essential oils against E. coli F ̓ lac were 1.5 and 11.3 mg/ml, respectively. On the other hand, minimum bactericidal concentration (MBC) was determined in our present study. However, MBC was tabulated in Table (27) and Illustrated by Figure (30), the MBC of cumin essential oil against selected bacterial strains were 0.768, 2.048, 0.768 and 4.096 mg/ml. while in case of rosemary and thyme essential oil, the MBC were 0.768, 4.096, 1.024 and 4.096 mg/ml and 0.512, 1.024, 0.768 and 2.048 mg/ml, respectively. In order to elucidate if the observed antimicrobial effect of the cumin, rosemary and thyme essential oils were microbicide or micro-biostatic, we decided to calculate the minimal bactericidal concentration (MBC/MIC) relation. The presence of alive and active bacteria was detected at all the concentrations of the essential oil capable of inhibiting bacterial growth. For this reason, the MBC would be higher than the MIC; the MBC/ MIC relation would be clearly greater than one, which would indicate a bacteriostatic effect of all selected essential oils against selected bacterial strains. 157
Table (27): Minimum inhibitory concentrations (MIC) and Minimum bactericidal concentrations (MBC) of both irradiated and non-irradiated different essential oils Non-irradiated essential oils Irradiated essential oils Bacterial strains C0 R0 T0 C2 C4 R6 T4 Salmonella typhimurium 0.256 0.256 0.256 0.512 0.512 0.512 0.512 MIC Escherichia coli 1.024 2.048 0.512 0.512 0.512 1.024 0.512 (mg/ml) Staphylococcus aureus 0.256 0.512 0.256 0.512 0.384 0.512 0.512 Bacillus cereus 1.024 2.048 0.512 0.512 0.512 2.048 0.512
Salmonella typhimurium 0.768 0.768 0.512 1.024 1.024 1.024 1.024 7 Escherichia coli 2.048 4.096 1.024 1.024 1.024 2.048 1.024 15 MBC (mg/ml) Staphylococcus aureus 0.768 1.024 0.768 1.024 0.768 1.024 1.024 Bacillus cereus 4.096 4.096 2.048 1.024 1.024 4.096 1.024 MIC = Minimum Inhibitory Concentration; MBC = Minimum Bactericidal Concentration; C0 = non-irradiated cumin essential oil; R0 = non-irradiated rosemary essential oil; T0 = non-irradiated thyme essential oil; C2 = irradiated cumin essential oil at dose 2 kGy; C4 = irradiated cumin essential oil at dose 4 kGy; R6 = irradiated rosemary essential oil at dose 6 kGy; T4 = irradiated thyme essential oil at dose 4 kGy. 158
2.5 Salmonella typhimurium Escherichia coli
2 Staphylococcus aureus Bacillus cereus
1.5 15
1 8
0.5 Minimum Inhibitory Concentration (mg/ml) Concentration Inhibitory Minimum
0 Cumin (0) kGy Rosemary (0) kGy Thyme (0) kGy
Fig. (29): Minimum Inhibitory Concentrations (MIC) of non-irradiated cumin (C0), rosemary (R0) and thyme (T0) essential oil 159
4.5 Salmonella typhimurium Escherichia coli 4 Staphylococcus aureus 3.5 Bacillus cereus
3
2.5
2 9 15 1.5
1
Minimum Minimum Bacterecidal Concentration (mg/ml) 0.5
0 Cumin (0) kGy Rosemary (0) kGy Thyme (0) kGy
Fig. (30): Minimum Bactericidal Concentrations (MBC) of non-irradiated cumin (C0), rosemary (R0) and thyme (T0) essential oil 160
This declaration was accompanied with Pérez et al. (1999) who mentioned the relation between MBC and MIC in classifying of the bacteriostatic. 4.5.2. Determination of MIC and MBC of mixtures made from non- irradiated essential oils: Mixture of non-essential oils were collecting together to find the best and the highest mixture that could be represented synergistic antibacterial activity against selected bacterial strains. These mixtures tabulated in Table (28) and illustrated by Figure (31) represented the MICs. Where, MIC of non- irradiated cumin and rosemary essential oil (C0 X R0) was 0.256 mg/ml in all selected bacterial strains (S. typhimiurium, E. coli, S. aureus and B. cereus). This obtaining also appeared in the MBC as shown by Figure (32) where, the MBCs of the same bacteria were 0.512, 0.512, 0.512 and 0.512 mg/ml. the obtained results are in the line with Jordán et al. (2013) who found that the MIC and MBC in all case studied were the same against all selected bacteria.
It's clearly to say that the MIC of C0 X R0 in case of E. coli (0.256 mg/ml) was lower than that obtained by their individual essential oil where, the MIC of C0 was 1.024 mg/ml and the MIC of R0 was 2.048 mg/ml. thus the mixture of C0 X R0 might be represent a synergistic activity. In addition, this finding appeared in case of B. cereus with the same mixture. These statements underline the affirmation that the antibacterial activity of cumin and rosemary essential oil against E. coli can be attributed to the combination of various minor components present in the oil, which act synergistically with the rest of the volatile compounds present Zaouali et al., (2010). According to Bajpai et al. (2012), it is possible that synergistic interactions exist between the essential oil components and the antibacterial activity detected for some oils against different strains of S. typhimurium. 161
This is in agreement with the affirmation that the antibacterial activity of an essential oil may be related to the chemical configuration of its components, the proportions in which they are present and the interactions between them (Bajpai et al., 2012; Dorman and Deans, 2000 and Jordán et al., 2013).
The C0 X T0 and R0 X T0 mixtures were showed that the MIC of them almost higher than that obtained from their individual essential oils. Where, i.e. the MIC of C0 X T0 was 0.512 mg/ml while the MIC of C0 and T0 was
0.256 and 0.256 mg/ml, respectively. On the other hand, the C0 X T0 mixture was higher in the MIC 0.384 mg/ml in case of B. cereus than its components where, C0 have 1.024 mg/ml and T0 have 0.512 mg/ml. The MBCs also as mentioned before are higher than the MIC of the same mixture. These findings are close to Pérez et al. (1999).
Our study found obviously that the C0 X R0 mixture was lower than the C0
X T0 and R0 X T0 mixtures. That means the mixture made from non- irradiated cumin and rosemary essential oils have the highest antimicrobial activity compared to other mixtures. Furthermore, cumin and rosemary possess to have moderate antimicrobial activity (Zaouali et al., 2010 and Jordán et al., 2013) related to their chemical composition. Rosemary essential oil showed weak antimicrobial activity in our present study according to the hypothesis of (Ait-Ouazzou et al., 2011). The last obtaining results are in line with the previous founding by Gutierrez et al., (2008a) who defined that the mechanism of action as well as essential oil composition deserves to be studied in more detail in order to elucidate why combinations of essential oils with a strong individual antimicrobial efficacy such as oregano and thyme, did not show synergistic effects, and why on the other hand combinations of two essential oils with individually moderate activity resulted in enhanced effects in combination, such as marjoram, basil, rosemary or sage. 162
Table (28): Minimum inhibitory concentrations (MIC) and Minimum bactericidal concentrations (MBC) of both irradiated and non-irradiated different essential oils mixtures Mixtures made from Mixtures made from irradiated essential oils Bacterial non-irradiated essential oils strains C0XR0 C0XT0 R0XT0 C2XC4 C2XR6 C2XT4 C4XR6 C4XT4 R6XT4 S. typhimurium 0.256 0.512 0.512 0.384 0.512 0.512 0.256 0.512 0.512 E. coli 0.256 0.384 0.512 0.512 0.512 0.384 0.384 0.512 0.512 S. aureus 0.256 0.512 0.256 0.384 0.384 0.384 0.384 0.384 0.512 MIC
(mg/ml) B. cereus 0.256 0.384 0.512 0.384 0.384 0.384 0.384 0.384 0.384
S. typhimurium 0.512 1.024 1.024 0.768 1.024 1.024 0.512 1.024 1.024 16
E. coli 0.512 0.768 1.024 1.024 1.024 0.768 0.768 1.024 1.024 2 S. aureus 0.512 1.024 0.512 0.768 0.768 0.768 0.768 0.768 1.024
MBC B. cereus 0.512 0.768 0.768 0.768 0.768 0.768 0.768 0.768 0.768 (mg/ml) MIC = Minimum Inhibitory Concentration; MBC = Minimum Bactericidal Concentration; C0 = non-irradiated cumin essential oil; R0 = non-irradiated rosemary essential oil; T0 = non-irradiated thyme essential oil; C2 = irradiated cumin essential oil at dose 2 kGy; C4 = irradiated cumin essential oil at dose 4 kGy; R6 = irradiated rosemary essential oil at dose 6 kGy; T4 = irradiated thyme essential oil at dose 4 kGy; 163
0.6 Salmonella typhimurium Escherichia coli Staphylococcus aureus Bacillus cereus
0.5
0.4
0.3
163 0.2
0.1 Minimum Inhibitory Concentration (mg/ml) Concentration Inhibitory Minimum
0 C0 X R0 C0 X T0 R0 X T0
Fig. (31): Minimum Inhibitory concentrations (MIC) of mixtures made from non-irradiated cumin (C0), rosemary (R0) and thyme (T0) essential oil 164
1.2 Salmonella typhimurium Escherichia coli Staphylococcus aureus Bacillus cereus
1
0.8
0.6 16 4
0.4
0.2 Minimum Bacterecidal Concentration (mg/ml) Concentration BacterecidalMinimum
0 C0 X R0 C0 X T0 R0 X T0
Fig. (32): Minimum Bactericidal concentrations (MBC) of mixtures made from non-irradiated cumin (C0), rosemary (R0) and thyme (T0) essential oil 165
4.5.3. Determination of MIC and MBC of irradiated essential oil: The chemical composition of essential oils is critical for their antibacterial activities (Zaouali et al., 2010). For these reason a quantitative evaluation of the antibacterial activity of irradiated cumin, rosemary and thyme essential oils were determined by the micro-dilution bioassay against the Gram- positive bacteria, S. aureus and B. cereus, and against the Gram-negative bacteria, E. coli and S. typhimurium.
As shown in Table (27) and illustrated by Figure (31), the MICs of irradiated cumin essential oil at doses 2 (C2) and 4 (C4) kGy were almost similar (0.512 mg/ml) except for the MIC of C4 against S. aureus was 0.384 mg/ml while; the MIC of C2 was 0.512 mg/ml. Moreover, the MIC of irradiated thyme essential oil (T4) against all selected bacteria was 0.512 mg/ml.
On the contrary, the MIC of irradiated rosemary ate dose 6 kGy (R6) showed irregular MICs against selected bacteria where, the MICs of S. typhimurium and S. aureus were 0.512 mg/ml. While, the MICs of R6 against E. coli and B. cereus were 1.024 and 2.048 mg/ml, respectively. On the other hand, MBC of irradiated essential oils showed increasing rate almost two fold of MIC of irradiated essential oil. Where, i.e., the MBC of R6 against E. coli was 1.024 mg/ml while, the MIC of R6 against the same bacteria was 2.048 mg/ml. As mentioned by Pérez et al. (1999) who mentioned the relation between MBC and MIC in classifying of the bacteriostatic. Our results confirmed that the MCB/MIC relation showed that all irradiated essential oil classified as bacteriostatic because the MBC of these essential oils are higher that MIC of the same essential oils. 166
2.5 Salmonella typhimurium
Escherichia coli
2 Staphylococcus aureus
Bacillus cereus
1.5 16 6 1
0.5 Minimum Inhibitory Concentration (mg/ml) Concentration Inhibitory Minimum
0 Cumin (2) kGy Cumin (4) kGy Rosemary (6) kGy Thyme (4) kGy
Fig. (33): Minimum Inhibitory Concentrations (MIC) of irradiated cumin, rosemary and thyme essential oil 167
4.5 Salmonella typhimurium Escherichia coli
4 Staphylococcus aureus Bacillus cereus
3.5
3
2.5
2 7
16 1.5
1 Minimum Bactericidal Concentration (mg/ml) Concentration Bactericidal Minimum 0.5
0 Cumin (2) kGy Cumin (4) kGy Rosemary (6) kGy Thyme (4) kGy
Fig. (34): Minimum Bactericidal Concentrations (MBC) of irradiated cumin, rosemary and thyme essential oil 168
4.5.4. Determination of MIC and MBC of mixtures made from irradiated essential oils: Table (28) and Figure (35) showed the MICs of the mixtures made form irradiated essential oils under investigation. Theoretically, the mixture made from irradiated cumin at doses 2 (C2) and 4 (C4) kGy did not present new in the antimicrobial activity but, in case of S. typhimurium, S. aureus and B. cereus the MICs were 0.384, 0.384 and 0.384 mg/ml. that means the MICs of the mixture represent antimicrobial activity compared to the individual essential oils in the mixture. The obtained data might be logically approved because of some authors explain the mode of action of the essential oils is related to its chemical composition itself. However, Harris (2002) found that the minor and major components are acting together to significantly increased antimicrobial activity.
The MIC of C2 X R6 was prosing mixture in case of B. cereus where, the
MIC of the mixture was 0.384 mg/ml while; the MIC of C2 was 0.512 mg/ml and R6 was 2.048 mg/ml. The mixture of C4 X R6 showed the highest antimicrobial activity in case of S. typhimurium compared to other mixtures where, the MIC of C4 X R6 mixtures was 0.512 mg/ml. that finding achieved the Gutierrez et al., (2008a) founding. In addition, the irradiation of both cumin and rosemary essential oil might be enhance their antimicrobial activity when they combining together. Burt (2004) reviewed the antimicrobial activity of essential oils, reporting a multiplicity of factors ranging from the culture of the plant to variations in the experimental conditions for the measurement of the antibacterial activity, as well as differences in the methodology as the type of Tween and DMSO used to dissolve the hydrophobic components.
C2 X T4 mixture showed an improvement in the synergism between its individual components where, the mixture MICs in case of E. coli and B. 169
cereus showed 0.384 and 0.384 mg/ml compared to the individual components which have The MICs 0.512 mg/ml in all cases.
The MIC of the mixture R6 X T4 might be not possessing synergy because of the obtained data in the MIC of the mixture almost higher than the individual components of the mixture. For example the MIC of the R6 X T4 against S. typhimurium was 0.512 mg/ml while, the MICs of R6 and T4 were 0.512 and 0.512 mg/ml. on the contrary, the MIC of the same mixture was
0.384 mg/ml against B. cereus while, the MICs of R6 and T4 were 2.048 and 0.512 mg/ml, respectively. On the MBCs obtaining results, Table (28) and Figure (36) showed the same trend in the MICs where, the MBC almost represent two fold of the MIC concentration used against selected bacterial strains.
Furthermore, the MIC of mixtures C2 X R6 and C4 X R6 showed promising antimicrobial activity and synergistic activity as the MIC of the same mixtures did. 4.6. Determination of Synergistic by Checker board method: 4.6.1. Fraction Inhibitory Concentration (FIC): The quantitative effects of cumin, rosemary and thyme in combination with the each other are described in terms of fraction inhibitory concentration (FIC) indices. The FICs of all essential oils combinations either irradiated or non-irradiated are shown in Table (29 and 30). The FICs classified as follow: Synergy(S, FIC < 0.5), Addition (A, 0.5 ≤ FIC ≤ 1), Indifference (I, 1 < FIC ≤ 4) and antagonism (FIC > 4) according to (Schelz et al., 2006 and Houghton, 2009). 170
0.6 Salmonella typhimurium Escherichia coli Staphylococcus aureus Bacillus cereus
0.5
0.4
0.3 1 70
0.2
0.1 Minimum Inhibitory Concentration (mg/ml) Concentration Inhibitory Minimum
0 C2 X C4 C2 X R6 C2 X T4 C4 X R6 C4 X T4 R6 X T4
Fig. (35): Minimum Inhibitory concentrations (MIC) of mixtures made from irradiated cumin (C2), cumin (C4) rosemary (R6) and thyme (T4) essential oil 171
1.2 Salmonella typhimurium Escherichia coli Staphylococcus aureus 1 Bacillus cereus
0.8
0.6 1 17 0.4
0.2 Minimum Bacterecidal Concentration (mg/ml) Concentration BacterecidalMinimum
0 C2 X C4 C2 X R6 C2 X T4 C4 X R6 C4 X T4 R6 X T4
Fig. (36): Minimum Bactericidal concentrations (MBC) of mixtures made from irradiated cumin (C2), cumin (C4) rosemary (R6) and thyme (T4) essential oil 172
4.6.2. Isobolgraphic methods: This method supplies a graphic demonstration with linearly arranged x and y axes reflecting the dose rates of the single individual components (Figure 37). The dose combinations are represented by geometric points with coordinates matching the dose rate of the separate components in the combination. An isobole is understood to be a line or curve between points of the same effect. The construction of isoboles requires the knowledge of the amounts of the individual components in the combination for one dose combination per effect level, at least, if the mechanism of interaction can be seen as independent of the amount of the single components. Data on different effect levels are necessary for the identification of the interaction, because quality and quantity of the interaction can depend on the effect grade (Wagner and Ulrich-Merzenich, 2009). According to that introduction all possibilities mixture of selected essential oils either irradiated or non-irradiated were studied and the kind of synergetic effect were obtained in Figures (38, 39, 40, 41, 42, 43, 44, 45 and 46).
As shown by Figure (38) the mixture of C0 X R0 introduced FICs 2, 1.5, 0.375 and 0.375 in the case of S. typhimurium, S. aureus, B. cereus and E. coli. These results represented a synergistic effect clearly occurred against B. cereus and E. coli where, the FICs were below 0.5 (Schelz et al., 2006 and
Houghton, 2009). Frankly speaking, this finding in the FIC of C0 X R0 against both B. cereus and E. coli give a great confidence and promising data to how can moderate or weak antimicrobial might be show antimicrobial activity when combining together (this finding are agreed with that obtained in the preceding sections in our investigation) (Bajpai et al., 2012; Dorman and Deans, 2000, Jordán et al., 2013 and Zaouali et al., 2010) and also we could obtain mixture have antimicrobial activity without affecting the organoleptic and sensory properties. 173
Table (29): Fraction inhibitory concentration of either irradiated or non- irradiated essential oils mixtures against selected bacterial strains Fraction Inhibitory Concentration (FIC) S. typhimurium S. aureus B. cereus E. coli C0 X R0 2.0 (I) 1.50 (I) 0.375 (S) 0.375 (S)
C0 X T0 4.0 (I) 4.00 (I) 1.125 (I) 1.125 (I)
R0 X T0 4.0 (I) 1.50 (I) 0.937 (A) 1.250 (I)
C2 X C4 1.5 (I) 1.75 (I) 1.500 (I) 2.000 (I)
C2 X R6 2.0 (I) 1.50 (I) 0.937 (A) 1.500 (I)
C2 X T4 2.0 (I) 1.50 (I) 1.500 (I) 1.500 (I)
C4 X R6 1.0 (A) 1.75 (I) 0.937 (A) 1.125 (I)
C4 X T4 2.0 (I) 1.75 (I) 1.500 (I) 2.000 (I)
R6 X T4 2.0 (I) 2.00 (I) 0.937 (A) 1.500 (I)
Synergy(S, FIC < 0.5), Addition (A, 0.5 ≤ FIC ≤ 1), Indifference (I, 1 < FIC ≤ 4), antagonism (FIC > 4) 174
Table (30): Fraction inhibitory concentrations (FICs) of both irradiated and non-irradiated different essential oils and their mixtures Salmonella typhimurium Staphylococcus aureus Bacillus cereus Escherichia coli
MICa MICb MICc FIC MICa MICb MICc FIC MICa MICb MICc FIC MICa MICb MICc FIC C0XR0 0.256 0.256 0.256 2.000(I) 0.256 0.512 0.256 1.500(I) 1.024 2.048 0.256 0.375(S) 1.024 2.048 0.256 0.375(S)
C0XT0 0.256 0.256 0.512 4.000(I) 0.256 0.256 0.512 4.000(I) 1.024 0.512 0.384 1.125(I) 1.024 0.512 0.384 1.125(I)
R0XT0 0.256 0.256 0.512 4.000(I) 0.512 0.256 0.256 1.500(I) 2.048 0.512 0.384 0.937(A) 2.048 0.512 0.512 1.250(I)
C2XC4 0.512 0.512 0.384 1.500(I) 0.512 0.384 0.384 1.750(I) 0.512 0.512 0.384 1.500(I) 0.512 0.512 0.512 2.000(I) 17 (I) (I) (A) (I)
C2XR6 0.512 0.512 0.512 2.000 0.512 0.512 0.384 1.500 0.512 2.048 0.384 0.937 0.512 1.024 0.512 1.500 4
C2XT4 0.512 0.512 0.512 2.000(I) 0.512 0.512 0.384 1.500(I) 0.512 0.512 0.384 1.500(I) 0.512 0.512 0.384 1.500(I)
C4XR6 0.512 0.512 0.256 1.000(A) 0.384 0.512 0.384 1.750(I) 0.512 2.048 0.384 0.937(A) 0.512 1.024 0.384 1.125(I)
C4XT4 0.512 0.512 0.512 2.000(I) 0.384 0.512 0.384 1.750(I) 0.512 0.512 0.384 1.500(I) 0.512 0.512 0.512 2.000(I) Treated essential oils (kGy) R6XT4 0.512 0.512 0.512 2.000(I) 0.512 0.512 0.512 2.000(I) 2.048 0.512 0.384 0.937(A) 1.024 0.512 0.512 1.500(I)
C0 = non-irradiated cumin essential oil; MICb= MIC of the second essential oil individually (mg/ml); R0 = non-irradiated rosemary essential oil; MICc= MIC of practical essential oil mixture of a and b (mg/ml); T0 = non-irradiated thyme essential oil; S = Synergy (FIC < 0.5); A = Addition (0.5 ≤ FIC ≤ 1); I = C2 = irradiated cumin essential oil at dose 2 kGy; Indifference (1 < FIC ≤ 4); AN = Antagonism (FIC > 4). C4 = irradiated cumin essential oil at dose 4 kGy; R6 = irradiated rosemary essential oil at dose 6 kGy; T4 = irradiated thyme essential oil at dose 4 kGy; MICa= MIC of the first essential oil individually (mg/ml); 175
Fig. (37): Isoboles for zero-interaction, synergism and antagonism 176
On the other hand, the FICs the C0 X R0 in the case of S. typhimurium and S. aureus showed an indifference activity.
As illustrated in Table (39) R0 X T0 the FICs showed indifference in case of S. typhimurium, S. aureus and E. coli where, the FICs were 4, 1.5 and 1.25 while, the FIC in case of B. cereus was 0.937 that means addition.
In the case of FIC of C0 X T0 mixture, data shown by Figure (40) illustrated that the FICs of the mixtures against all selected bacterial showed indifference where, the FICs were 4, 4, 1.125 and 1.125 in S. typhimurium, S. aureus, B. cereus and E. coli, respectively. Moreover, the FIC of S. typhimurium and S. aureus could be explained in other studies as antagonism (Didry et al., 1993b and Fadi et al., 2012) who was defined the antagonistic effect when FIC is more than 2. Regarding to the gamma irradiation, irradiation of essential oils also studied in comparing with non-irradiated essential oils. As shown by Figure
(41) the FICs of C2 X C4 mixture were 1.5, 1.75, 1.5 and 2 in S. typhimurium, S. aureus, B. cereus and E. coli, respectively. This finding might be logically approved because of this mixture chemically contains the same components but in different percentage.
The C2 X R6 mixture as shown by Figure (42) possesses to have indifference in the FICs in the bacterial strains S. typhimurium, S. aureus and E. coli. But in B. cereus the FIC represents addition effect. As mentioned in the preceding section the relationship between C2 and R6 showed a synergistic effect this notification could be achieved by the Figure (42). In the mixture made from irradiated cumin essential oil at dose 2 kGy and thyme essential oil at dose 4 kGy (C2 X T4) as shown by Figure (43), all FICs showed indifference in all selected bacterial strains.
As mentioned in other sections, the mixture of C4 X R6 showed the possibility in the synergistic effect might be happened. Figure (44) 177
represented that the FICs of C4 X R6 mixture were 1, 1.75, 1.125 and 0.937 in S. typhimurium, S. aureus, E. coli and B. cereus. The addition effect was observed in B. cereus where, the FIC was 0.937. While, indifference effect were observed in the rest of bacterial strains in the same mixture.
The FICs of C4 X T4 mixture was shown by Figure (45) the effect of the mixture on the selected bacteria was indifference in all cases.
Last but not least, Figure (46) showed the relationship between irradiated rosemary essential oil at dose 6 kGy and irradiated thyme essential oil at dose 4 kGy (R6 X T4). All FICs represented indifference in S. typhimurium, S. aureus, E. coli but the addition effect appeared in the case of B. cereus. The promising observation in our investigation was none of these combinations displayed a synergistic activity against the bacteria used in this study except for the C0 X R0 mixtures in case of B. cereus and E. coli. 4.7. Antioxidant activity of essential oils: Owing to the complex reactive facets of phytochemicals, the antioxidant activities of plant extracts (essential oils) cannot be evaluated by only a single method, but at least two test systems have been recommended for the determination of antioxidant activity to establish authenticity (Schlesier et al., 2002). There are many different methods for determining antioxidant function each of which depends on a particular generator of free radicals, acting by different mechanisms (Huang et al., 2005). The antioxidant activity of essential oil is believed to be mainly due to their redox properties, which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Amensour et al., 2009). For this reason the antioxidant activity of thyme, cumin and rosemary essential oil was determined by DPPH radical scavenging method and Rancimat test. 178
(FIC = 2) 0 .3 0 Indifference (FIC = 0.375) 2 .0 Synergistic (C 0 X R 0 ) 0 .2 5
1 .5 0 .2 0
0 .1 5 1 .0
0 .1 0 B . c e r e u s
S. typhim urium 0 .5 0 .0 5 (C 0 X R 0 ) Concentraction of Cumin E.O. (mg/ml) Concentration of Rosemary E.O. (mg/ml) 0 .0 0 0 .0 0 .0 0 0 .0 5 0 .1 0 0 .1 5 0 .2 0 0 .2 5 0 .3 0 0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 Concentraction of Rosemary E.O. (mg/ml) Concentration of Cum in essential oil (m g/m l)
(FIC = 1.5) Synergistic (FIC = 0.375) 17 0 .6 Indifference 2 .0 8
0 .5 1 .5
0 .4
1 .0 0 .3 ( C 0 X R 0 ) E . c o li S . a u r e u s 0 .2 0 .5
0 .1 (C 0 X R 0 )
0 .0 0 .0 Concentration of rosemary essential oil (mg/ml) 0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 Concentration0 of Non-Irradiated .0 0 rosem ary essential0 ol .0(m g/m5 l) 0 .1 0 0 .1 5 0 .2 0 0 .2 5 0 .3 0 Concentration of Non-Irradiated cum in essential oil (m g/m l) Concentration of cumin essential oil (mg/ml)
Fig. (38): The isobole method describing the effect of non-irradiated cumin and rosemary essential oil mixtures against selected bacterial strains 179
(FIC = 4) 0 .6 Indifference 0 .6 A d d itio n (FIC = 1.125) (R 0 X T 0 ) 0 .5 0 .5
0 .4 0 .4 (R 0 X T 0 ) 0 .3 0 .3 B . c e r e u s 0 .2 0 .2 S. typhimurium 0 .1 0 .1
Concentration0 .0 of Thyme E.O. (mg/ml)
Concentration0 .0 of Rosemary E.O. (mg/ml) 0 .0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 0 .0 0 .5 1 .0 1 .5 2 .0 Concentration of Thyme E.O. (mg/ml) Concentration of Rosemary E.O. (mg/ml)
9 0 .3 0 Indifference Indifference (FIC = 4) 0 .6 (FIC = 1.125) 17 (R 0 X T 0 ) 0 .2 5 (R 0 X T 0 ) 0 .5
0 .2 0 0 .4 E.O. (mg/ml)
0 .1 5 0 .3 Thyme E . c o li S . a u r e u s 0 .1 0 0 .2
0 .0 5 0 .1 Concentration of 0 .0 0 Concentration0 .0 of Rosemary E.O. (mg/ml) Concentration of thyme0 .0 essential oil (mg/ml) 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 0 .0 0 .5 1 .0 1 .5 2 .0 Concentration of Rosemary essential oil (mg/ml) Concentration of Rosemary E.O. (mg/ml)
Fig. (39): The isobole method describing the effect of non-irradiated cumin and thyme essential oil mixtures against selected bacterial strains 180 (FIC = 4) (FIC = 0.937) 0 .6 Indifference 0.6 Indifference (C 0 X T 0 ) 0 .5 0.5
0 .4 0.4 (C 0 X T 0 )
0 .3 0.3 B . c e re u s
0 .2 0.2
0 .1 S. typhimurium 0.1 Concentration of Cumin E.O. (mg/ml) Concentration of Thyme E.O. (mg/ml)
Concentration of Cumin E.O. (mg/ml) 0.0 0 .0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 .0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 Concentration of cuimn essential oil (mg/ml) Concentration of Thyme E.O. (mg/ml)
0 .6 Indifference (FIC = 1.25) 1 (FIC = 1.5) 80 0 .6 Indifference
(C 0 X T 0) 0 .5 0 .5 (C 0 X T 0 ) 0 .4 0 .4
0 .3
0 .3 E . c o li
0 .2 S . a u re u s 0 .2
0 .1 0 .1
0 .0 0 .0 Concentration of thyme0 .0 essential oil (mg/ml) 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2
Concentration of Non-Irradiated0 .0 Cumin E.O. (mg/ml) 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 Concentration of Non-Irradiated Thyme E.O. (mg/ml) Concentration of cumin essential oil (mg/ml)
Fig. (40): The isobole method describing the effect of non-irradiated rosemary and thyme essential oil mixtures against selected bacterial strains 181
0.6 Indifference (FIC = 1.5) 0 .6 Indifference (FIC = 1.5)
0.5 0 .5
0.4 (C 2 X C 4) 0 .4 (C 2 X C 4)
0.3 0 .3
0.2 B . c e re u s 0 .2 S. typhimurium
0.1 0 .1
0.0
Concentratin of Irradited0.0 Cumin E.O. -2kGy- (mg/ml) 0.1 0.2 0.3 0.4 0.5 0.6 0 .0 0 .0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6
Concentratin of Irradited Thyme E.O. -4kGy- (mg/ml) Concentration of Irradiated Cumin E.O. - 2kGy - (mg/ml) Concentration of Irradiated Cumin E.O. - 4kGy - (mg/ml) 1
18 Indifference 0 .4 0 (C 2 X C 4) (FIC = 2) (FIC = 1.75) 0 .6 Indifference 0 .3 5 (C 2 X C 4) 0 .5 0 .3 0 0 .4 0 .2 5
0 .2 0 0 .3 S . a u re u s 0 .1 5 E . c o li 0 .2 0 .1 0 0 .1 0 .0 5
0 .0 0 0 .0 0 .0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 0 .0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 Concentration of Irradiated Cuimn E.O. - 2kGy - (mg/ml) Concentration of Irradiated Cumin E.O. - 4kGy - (mg/ml) Concentration of Irradiated Cumin E.O. - 2kGy - (mg/ml) Concentration of Irradiated Cuimn E.O. - 4kGy - (mg/ml)
Fig. (41): The isobole method describing the effect of irradiated cumin (2kGy) and cumin (4kGy) essential oil mixtures against selected bacterial strains 182
Indifference (FIC = 2) 0.6 2 .0 A d d itio n (FIC = 0.937) (C 2 X R 6 ) 0.5
1 .5 0.4
0.3 1 .0
0.2 B . c e re u s S. typhimurium 0 .5 0.1 (C 2 X R 6 ) 0.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration of irradiated Rosemary E.O. (6kGy) (mg/ml) Concentration of Irradiated Cumin E.O. - 2kGy - (mg/ml) 0 .0 Concentration of Irradiated Rosemary E.O. - 6kGy - (mg/ml) 0 .0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 Concentration of irradiated Cumin E.O. (2kGy) (mg/ml) 18 0 .6 Indifference (FIC = 1. 5) 1 .2 Indifference (FIC = 1.5) 2
0 .5 1 .0
0 .4 (C 2 X R 6 ) 0 .8
0 .3 0 .6 (C 2 X R 6 ) S . a u re u s E . c o il
0 .2 0 .4
0 .1 0 .2
0 .0 0 .0 0 .0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 0 .0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 Concentration of Irradiated Rosemary E.O. - 6 kGy - (mg/ml) Concentration of Irradiated CuminConcentration E.O. - 2kGy - (mg/ml) of Irradiated Rosemary E.O. - 6kGy - (mg/ml) Concentration of Irradiated Cumin E.O. - 2 kGy - (mg/ml)
Fig. (42): The isobole method describing the effect of irradiated cumin (2kGy) and rosemary (6kGy) essential oil mixtures against selected bacterial strains 183
(FIC = 2) 0.6 Indifference (FIC = 1.5) 0.6 Indifference
(C2 X T4) 0.5 0.5
0.4 (C 2 X T4) 0.4
0.3 0.3 B . c e re u s 0.2 0.2
0.1 0.1 S. typhimurium
0.0 Concentration of Irradiated0.0 Cumin E.O. - 2kGy - (mg/ml) 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration of Irradiated Thyme E.O. - 4kGy - (mg/ml) Concentration of Irradiated Cumin E.O. - 2kGy - (mg/ml) Concentration of Irradiated Thyme E.O. - 4kGy - (mg/ml) 3
18 0.6 Indifference (FIC = 1.5) 0.6 Indifference (FIC = 1. 5)
0.5 0.5
0.4 (C 2 X T4) 0.4 (C 2 X T4)
0.3 0.3 E . co li S . a u re u s 0.2 0.2
0.1 0.1
0.0 0.0 Concentration of Irradiated Cumin E.O. - 2kGy - (mg/ml)
Concentration of irradiated0.0 Cuimn E.O. - 2kGy - (mg/ml) 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration of irradiated Thyme E.O. - 4kGy - (mg/ml) Concentration of Irradiated Thyme E.O. - 4kGy - (mg/ml)
Fig. (43): The isobole method describing the effect of irradiated cumin (2kGy) and thyme (4kGy) essential oil mixtures against selected bacterial strains 184
0.6 (FIC = 1) A d d itio n A ddition (FIC = 0.937) 2 .0 0.5
0.4 1 .5
0.3 (C 4 X R 6 ) 1 .0 B . c e re u s
0.2 S. typhimurium
0.1 0 .5 (C 4 X R 6) 0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0 .0 Concentration of Irradiated Cumin E.O. - 4kGy - (mg/ml) Concentration of Irradiated Rosemary E.O. - 6kGy - (mg/ml) 0 .0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6
Concentration of Irradiated RosemaryConcentration E.O. - 6kGy - (mg/ml) of Irradiated Cumin E.O. - 4kGy - (mg/ml) 18 4 (FIC = 1. 75) 0 .6 Indifference (FIC = 1.125) 1 .2 Indifference
0 .5 1 .0
0 .4 (C 4 X R 6) 0 .8
0 .3 0 .6 E . c o li
0 .2 0 .4 (C 4 X R 6) S . a u re u s
0 .1 0 .2
0 .0 0 .0 0 .0 0 0 .0 5 0 .1 0 0 .1 5 0 .2 0 0 .2 5 0 .3 0 0 .3 5 0 .4 0 0 .0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 Concentration of Irradiated Rosemary E.O. - 6kGy - (mg/ml)
Concentration of irradiated RosemaryConcentration E.O. - 6kGy - (mg/ml) of irradiated Cumin E.O. - 4kGy - (mg/ml) Concentration of Irradiated Cumin E.O. - 4kGy - (mg/ml)
Fig. (44): The isobole method describing the effect of irradiated cumin (4kGy) and rosemary (6kGy) essential oil mixtures against selected bacterial strains 185
0.6 Indifference (FIC = 1.5) 0.6 Indifference (FIC = 2)
(C 4 X T4) 0.5 0.5
0.4 (C 4 X T4) 0.4
0.3 0.3 B . c e re u s 0.2 0.2
S. typhimurium 0.1 0.1
0.0
Concentration of Irradiated0.0 Cumin E.O. - 4kGy - (mg/ml) 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration of Irradiated Thyme E.O. - 4kGy - (mg/ml) Concentration of Irradiated CuminConcentration E.O. - 4kGy - (mg/ml) of Irradiated Thyme E.O. - 4kGy - (mg/ml) 5 0.6 Indifference (FIC = 2) 18 0.6 Indifference (FIC = 1. 75) (C 4 X T4) 0.5 0.5
0.4 0.4 (C 4 X T4)
0.3 E . c o li 0.3
0.2 0.2 S . a u re u s 0.1 0.1
0.0 0.0 Concentration of Irradiated Cumin E.O. - 4kGy - (mg/ml)
Concentration of Irradiated0.00 Thyme E.O. - 4kGy - (mg/ml) 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration of Irradiated Cumin E.O. - 4kGy - (mg/ml) Concentration of Irradiated Thyme E.O. - 4kGy - (mg/ml)
Fig. (45): The isobole method describing the effect of irradiated cumin (4kGy) and thyme (4kGy) essential oil mixtures against selected bacterial strains 186
Addition (FIC = 0.937) Indifference (FIC = 2) 0.6 0.6 (R6 X T4) 0.5 0.5
0.4 0.4 (R6 X T4) 0.3
0.3 B . cereus 0.2 0.2 0.1 0.1 S. typhimurium 0.0
0.0 Concentration of Irradiated Thyme0.0 E.O. - 4kGy - (mg/ml) 0.5 1.0 1.5 2.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Concentration of Irradiated Rosemary E.O. - 6kGy - (mg/ml)
Concentration of Irradiated Rosemary ConcentrationE.O. - 6kGy - (mg/ml) of Irradiated Thyme E.O. - 4kGy - (mg/ml) 18 6
0.6 Indifference (FIC = 2) 0.6 Indifference (FIC = 1.5) (R6 X T4) (R6 X T4) 0.5 0.5
0.4 0.4
0.3 0.3 E. coli S . aureus 0.2 0.2
0.1 0.1
0.0
0.0 Concentration of Irradiated Thyme E.O. -4kGy - (mg/ml) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Concentration of Irradiated Roaemary E.O.Concentration - 6kGy - (mg/ml) of Irradiated Thyme E.O. - 4kGy - (mg/ml) Concentration of Irradiated Rosemary E.O. - 6kGy - (mg/ml)
Fig. (46): The isobole method describing the effect of irradiated rosemary (6kGy) and thyme (4kGy) essential oil mixtures against selected bacterial strains 187
4.7.1. Preliminary selection experiment: A set of experiments were conducted to detect the proper concentration of cumin, rosemary and thyme essential oil which mixed with sunflower oil. The acceptable level for this system is shown by Figures (47, 48 and 49) and tabulated in Table (31). Statistical analysis demonstrated that the odor of all of sunflower oil supplemented with cumin , rosemary and thyme essential oil used in this experiment were significantly different compared to control sunflower oil (without essential oil) but, the odor of cumin essential oil appears in sunflower oil at concentration 0.3 % and more (0.4 % and 0.5 %) with significant differences. Thus, the concentration of cumin essential used in our study is 0.4%, this concentration represented higher acceptable odor.
Cuminaldehyde, menthane derivatives, γ-terpinene, ρ-cymene and β- pinene are major components of many essential cumin oils and are mainly responsible for the aroma and biological effects (Lis-Balchin et al., 1998b). On the other hand, the concentrations of rosemary essential oil represented significantly differences compared to sunflower oil without adding any essential oil. But the concentration of rosemary essential oil 0.2% showed very weak odor according to (El-Baroty, 1988). In addition, thyme essential oil represented very weak odor at concentration of 0.1%. Thus, the final concentration of adding cumin, rosemary and thyme essential oil were 0.4%, 0.2% and 0.1%, respectively. According to Table (32) which presented the olfactive note of the main essential oil constituents, it could be summarized that the odor of all essential oil produced from the complex chemical structure. Meanwhile, cumin essential oil has cuminaldehyde, γ-terpinene, α-terpinen-7-al, γ-terpinene-7- al and β-pinene as main components in the present study. Cuminaldehyde (24.93%) present a smell of sharp and oily. These results are in agreement 188
with (Sigma-Aldrich, 2001 and Lucchesi et al., 2004a). This note might by acceptable and common for the Egyptian panelists.
Rosemary essential oil have 1,8-cineol, α-pinene, camphene, limonene and camphor in concentrations 33.2%, 17.1%, 6.35%, 5.93% and 29.74%. These compounds tend to have herbaceous aroma especially, in the presence of 1,8-cineol and camphor, the finding is in the similar with (Lucchesi et al., 2004b). Concerning with thyme, the main components of it essential oil are ρ-cymene, γ-terpinene and thymol, which have concentration of 33.6%, 13.1% and 31.3%, respectively. These main compounds have herbaceous note and might be warm and sweaty, the
Table (31): Mean of acceptable levels and standard errors for cumin, rosemary and thyme essential oils added to sunflower oil Concentration Threshold score (%) Cumin essential Rosemary Thyme essential oil essential oil oil
C B C 0.0 0.50 ± .019 0.78 ± 0.15 0.44 ± 0.18
BC A B 0.1 1.00 ± 0.27 1.89 ± 0.39 1.44 ± 0.29
BC A B 0.2 0.75 ± 0.25 1.78 ± 0.22 1.44 ± 0.29
AB A B 0.3 1.50 ± 0.19 2.22 ± 0.28 1.67 ± 0.33
AB A A 0.4 1.50 ± 0.27 2.22 ± 0.28 2.56 ± 0.18
A A AB 0.5 2.00 ± 0.19 2.44 ± 0.24 2.11 ± 0.26
Each value represents the mean ± S.E., The mean value with different superscript alphabets in a row indicate significant differences (P< 0.05) using LSD test, Threshold value refers to the minimum concentration at which stimulus is easily characterized, The aroma was sniffed by 13 judgers, The intensity of odor was described according to the follow scale (0 = no odor, 1 = very weak odor, 2 = higher acceptable odor and 3 = non acceptable odor) 189
2 1.8 1.6 1.4 1.2 2.0A ± 0.189 1 0.5C ± 0.189 0.8 1.0BC ± 0.267 0.6 0.4 0.2 0 9
18 0.75BC ± 0.250
1.5AB ± 0.267
1.5AB ± 0.189
Fig. (47): Acceptable level of adding cumin essential oil to sunflower oil 190
2.5
2
1.5 2.444A ± 0.242 B 1.889A ± 0.389 0.7778 ± 0.147 1
0.5
0 1 90
1.778A ± 0.222 2.222A ± 0.278
2.222A ± 0.278
Fig. (48): Acceptable level of adding rosemary essential oil to sunflower oil 191
3
2.5
2
1.5 2.111AB ± 0.261 1.444B ± 0.294 0.4441 C ± 0.176
0.5
0 1 19 1.444B ± 0.294
2.556A ± 0.176
1.667B ± 0.333
Fig. (49): Acceptable level of adding thyme essential oil to sunflower oil 192
Table (32): Main components and olfactive notes of cumin, rosemary and thyme essential oil either irradiated or non-irradiated Compounds Cumin Rosemary Thyme Olfactive note** 0 kGy 4 kGy 0 kGy 6kGy 0 kGy 4 kGy α-Thujene 0.26 -- -- 0.48 2.52 0.3 Weak minty, camphoraceous α-Pinene 0.98 0.18 17.1 13.33 1.92 0.21 Warm, resinous and refreshing Camphene -- -- 6.35 -- 0.76 0.08 Camphoraceous β-Pinene 17.44 3.82 0.92 -- 0.5 0.57 Smell of dry wood, resinous, slightly clinging Sabinene 0.43 0.13 0.92 ------Fresh, citrus-note, spicy
Myrcene 0.68 0.28 0.97 0.82 2 -- Citrus-note, spicy 19 α-Phellandrene -- 0.35 -- 0.35 0.76 -- Minty, herbaceous 2 α-Terpinene 0.42 0.09 0.53 0.58 0.17 0.47 Weak herbal ρ-Cymene 6.72 6.43 0.63 7.52 33.6 12.11 Citrus fruit note, reminiscent of lemon and bergamot o-Cymene 0.09 ------Herbal Limonene -- -- 5.93 15.37 2.06 -- Fresh, citrus, sweet, lemon Δ-3-Carene -- -- 0.22 -- 0.33 -- Sweet, weak lime-like 1,8-Cineol -- -- 33.2 22.33 -- -- Fresh, light, freshly camphorated γ-Terpinene 19.49 12.97 0.27 0.26 13.1 6.98 Herbaceous, smell of citrus fruit Terpinolene -- -- 0.12 0.58 -- -- Plastic-like α-Linalool -- -- 1.38 -- 0.87 2.41 Fresh and floral with a slight note of citrus 193
Table (32): Continued Terpineol 0.17 -- 2.91 1.16 1.24 0.83 Delicately floral and sweet Camphor -- -- 14.5 29.74 -- -- Camphor Citronelol 1.6 1.61 -- 0.62 -- -- Rose-like Borneol -- -- 0.48 -- 0.29 2.78 Camphor-like Cuminaldehyde 24.93 33.65 ------Sharp, acid, woody, oily α-Terpinene-7-al 10.55 ------Fresh, spicy 37.98 γ-Terpinene-7-al 14.88 ------Fresh, spicy Geraniol -- -- 2.79 -- 0.35 0.2 Citrus- and rose-like Thymol ------31.7 64.06 Strong, herbaceous, warm, ferly clinging, 3
19 medicinal and sweet Carvacrol 0.15 ------3.16 0.1 Medicinal, herbal, sweet ** Odour description using GC-sniffing technique, partly in correlation with published data (Sigma-Aldrich, 2001 and Lucchesi et al., 2004a). 194
Egyptian panelists might be rejected the grassy or herbaceous aroma found in the rosemary and thyme essential oil. Our results come to agreement with previous observation with Bousbia et al. (2009) who mentioned that the essential oil of rosemary obtained by hydro-distillation had freshly camphorated and citrus, boiled odor and different from fresh herb.
Previous studies are consistent with present findings; they have also shown that high concentrations of plant essential oil, which are required to achieve antibacterial activity against food-borne pathogens, could not be applicable to foods due to adverse organoleptic properties (Burt, 2004 and Holley and Patel, 2005). 4.7.2. Rancimat test: The oxidative stability is a direct evidence for changes in oxidation resistance (Rossel, 1989). The results in Table (33) revealed that the oxidative stability of sunflower oil without any antioxidants neither natural nor synthetic (control) was 8.31 hr (100 % stability). While, BHT as a synthetic antioxidant raises the oxidative stability to 162.5%. on the other hand, addition of 0.2% rosemary essential oil to sunflower oil at different irradiation doses caused slight increment in the relative stability of sunflower essential oil and that increments were 101.8%, 105.8%, 107.3% and 107.3% at irradiated dose 0, 2, 4 and 6 kGy, respectively. Moreover, addition of 0.4% cumin essential oil to sunflower oil increased the oxidative stability to 112.7% and 114.1% at irradiated doses 2 kGy and 4 kGy, respectively compared to control one (addition of non- irradiated cumin essential oil) which represented the oxidative stability 106.6%. But the irradiation dose 6 kGy revealed that the oxidative stability 105.8%. 195
Thyme essential oil at dose 0.1% showed that the oxidative stability of sunflower oil increased due to increase irradiation doses. However, the oxidative stability was 112.3%, 113.5% and 122.7% at doses of 0, 2, and 4 kGy while, irradiation dose 6 kGy decrease the oxidative stability (111.7%) compared to other used irradiation doses. The reason to high oxidative stability of adding thyme essential oil may be related to the chemical composition of thyme essential oil which has thymol caused preventing the heat-induced loss of α-tocopherol contained in sunflower oil, thus inhibiting or retarding its oxidation and/or polymerization (Bensmira et al., 2007) or may be related to thymol content which play an important free radical scavenger (Vardar-Ünlü et al., 2003). Thus, the addition of essential oil either irradiated or non-irradiated at the previous concentrations caused detectable increments in the relative stability but not like 0.02% BHT, which represents the highest relative oxidative stability. Arici et al. (2007) found a negative correlation between rancimat value and irradiation doses but up to 10 kGy. Rancimat value decreased non- significantly up to irradiation dose 2.5 kGy. According to this data, at the beginning, while the oil of the sample which is not exposed to irradiation had a resistance time of 7.72 hours, the highest value; it was found that related to the irradiation doses, induction time of the sample’s oils decreased and the lowest value of the oil of the sample of 10 kGy was recorded as 0.62 hours. It was found that the effect of irradiation exposure on the rancimat rate of black cumin seed samples was statistically significant (p<0.01). 196
Table (33): Effect of different concentrations of rosemary, thyme and cumin essential oils either irradiated or non- irradiated on the oxidative stability of sunflower oil assessed by Rancimat test Irradiation Treatments Control Sunflower oil SFO + 0 kGy 2 kGy 4 kGy 6 kGy (SFO) 0.02 % BHT Stability Relative Stability Relative Stability Relative Stability Relative Stability Relative Stability Relative (hrs) Stability (hrs) Stability (hrs) Stability (hrs) Stability (hrs) Stability (hrs) Stability
(%) (%) (%) (%) (%) (%) 19 6 SFO + 0.2% IREO 8.46 101.8 8.80 105.8 8.92 107.3 8.92 107.3
SFO + 0.1% ITEO 9.33 112.3 9.43 113.5 10.20 122.7 9.28 111.7 8.31 100 13.5 162.5
SFO + 0.4% ICEO 8.86 106.6 9.37 112.7 9.48 114.1 8.80 105.9
SFO = Sunflower oil, IREO = Irradiated Rosemary Essential Oil, ITEO = Irradiated Thyme Essential Oil, ICEO = Irradiated Cumin Essential Oil 197
4.7.3. DPPH radical scavenging method: The molecule of 2,2-diphenyl-1-picrylhydrazyl (DPPH): Figure (50) is characterized as a stable free radical by virtue of the delocalization of the spare electron over the molecule as a whole, so that the molecules do not dimerise, as would be the case with most other free radicals. The delocalization also gives rise to the deep violet colour, characterized by an absorption band in ethanol solution centered at about 520 nm.
When a solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, then this gives rise to the reduced form Figure (51) with the loss of this violet colour (although there would be expected to be a residual pale yellow colour from the picryl group still present). Representing the DPPH radical by Z• and the donor molecule by AH (antioxidant), the primary reaction is: Z• + AH = ZH + A• …………………. [1]
Fig. (50): Diphenyl-2-picrylhydrazyl Fig. (51): Diphenyl-2-picrylhydrazyl (Free Radical) (Non-Radical)
Where ZH is the reduced form and A• is free radical produced in this first step. This latter radical will then undergo further reactions which control the overall stoichiometry, that is, the number of molecules of DPPH reduced (decolorized) by one molecule of the reductant. The reaction [1] is therefore intended to provide the link with the reactions taking place in an oxidizing system, such as the autoxidation of a lipid or other unsaturated substance; the DPPH molecule Z• is thus intended to represent the free radicals formed in 198
the system whose activity is to be suppressed by the substance AH, (Molyneux, 2004). 4.7.3.1. Individual essential oils: 4.7.3.1.1. Scavenge activity of cumin essential oil: All of the assessed samples were able to reduce the stable, purple-colored radical DPPH into yellow-colored DPPH-H. The obtained data are represented by Figures (52, 53 and 54) and tabulated in Table (34). Figure (52) and Table (34) show the DPPH scavenging activity changes of non-irradiated and irradiated cumin essential oil. Irradiation resulted in a slight increase (76.49% and 76.16%) in the DPPH radical-scavenging ability of the cumin essential oil at doses 2 and 4 at concentration 200 ppm compared to 6 kGy (66.75%) which represented slight decrease, compared with non-irradiated essential oil. The same finding was found in the concentration 500 ppm. These finding was in the same line with Kim et al., 2009 who found that the scavenging activity was increased slightly after irradiation with 1, 3, 5, and 5kGy. On the other hand, at concentration of 300 and 400 ppm slight decrease was observed after irradiation doses 2, 4, and 6 kGy. Comparing with BHT, all previous results obtained in both irradiated and non-irradiated cumin essential oil are less than in scavenging activity of BHT where, represented 95.14%, 95.86%, 96.23 and 97.70% in the concentration 200, 300, 400 and 500 ppm, respectively. However, the highest scavenging activity of non-irradiated cumin essential oil was found at 500 ppm concentration, while the highest concentration of 199
Table (34): Effect of gamma irradiation on the DPPH scavenging activity of cumin, rosemary and thyme essential oil Concentration in ppm
Irradiation 200ppm 300ppm 400ppm 500ppm doses % of DPPH Scavenging activity
BHT 95.14 95.86 96.23 97.70
0 kGy 74.78 79.25 80.41 81.20
Cumin 2 kGy 76.49 77.10 78.14 83.45 essential oil 4 kGy 76.16 78.47 79.85 88.24 6 kGy 66.75 77.99 82.98 84.79
0 kGy 72.23 76.96 88.26 89.01
Rosemary 2 kGy 86.61 87.13 88.59 89.52 essential oil 4 kGy 81.60 88.54 92.24 93.07 6 kGy 78.28 90.98 94.03 97.41
0 kGy 90.77 93.09 94.07 95.59
Thyme 2 kGy 93.22 93.94 95.90 96.24 essential oil 4 kGy 96.34 97.15 97.83 98.40 6 kGy 90.51 90.54 92.38 95.86
DPPH = 1,1-diphenyl-2-picrylhydrazyl, BHT = Buteylated Hydroxyl Toluene, ppm = part per million, kGy = kilo Gray 200
1 0 0
9 5
9 0
8 5
8 0 200
7 5
Scavenging effect (% ) Cum in (0 kGy) Cum in (2 kGy) 7 0 Cum in (4 kGy) Cum in (6 kGy) B H T 6 5 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 Concentration (ug/m l) Fig. (52): The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of non-irradiated and irradiated cumin essential oils and of synthetic antioxidant (BHT) 201
irradiated cumin essential oil was found at 4 kGy at 500 ppm concentration. Furthermore, Singh et al. (2005) found that the scavenging activity of cumin essential oil at 25µl was about 64% and it was lower than BHT which represents scavenging activity at the same concentration about 85%. The obtained data are in agreement with Fakoor and Rasooli (2008) reported that Cuminum cyminum and Rosmarinus officinalis essential oils notably reduced the concentration of DPPH free radical, with an efficacy lower than that of reference oil Thymus x-porlock (69.29% inhibition) and slightly lower than that of Trolox. The performance of the rosemary oil was better than that of Cuminum cyminum. In addition, Hajlaoui et al. (2010) found that Cuminum cyminum essential oils gave interesting results in terms of both antimicrobial activity and ability to neutralize free radicals and prevent unsaturated fatty acid oxidation. 4.7.3.1.2. Scavenge activity of rosemary essential oil: As shown in Table (34), rosemary essential oil represented that the scavenging activity of irradiated rosemary essential oil is gradually increased compared to non-irradiated essential oil. In addition, essential oil irradiated with 6 kGy represented the highest scavenging activity 90.98%, 94.03% and 97.41% at concentrations 300, 400 and 500 ppm, respectively. Concentration 200 ppm of irradiated essential oil at dose 6 kGy showed the lowest scavenging activity (78.28%) of all irradiated samples. Meanwhile, the scavenging activity of irradiated rosemary essential oil at concentration 500 ppm (97.41%) is almost as in BHT at the same concentration 500 ppm (97.70%). Regarding the chemical composition of both non-irradiated essential oil and irradiated essential oil as shown in Table (37) and illustrated by Figure (53). Some chemical constituents are increased and other decreased. 202
Camphor is one of the chemical components which represent a great increase in irradiated essential oil at dose 6 kGy (from 14.5% to 29.74%). As appoint of view, camphor possess to be an oxygenated compound which has an antioxidant activity. The obtained results are completely agreed with (Wang et al., 2008) who found the significant high contents of compounds, such camphor and borneol in extracts from rosemary taken at the flowering stage might be in part in the elevation of the antioxidant capacity.
Moreover, the scavenging activity of Ecuadorian rosemary essential oil was about 64.5% this percentage was higher twice than trolox (28.2%) (Sacchetti et al., 2005) that increasing was the reason of the higher percentage of α-pinene, 1,8-cineol and camphor. In addition, Yosr et al., (2013) found that the essential oil of rosemary cultivated Tunisia were characterized by high amount of camphor (14.5%), 1,8-cineol (35.8%) and α- pinene (10.6%) this percentage caused increased antioxidant activity of rosemary essential oil. Thus, 6 kGy represent the highest scavenging activity of all irradiation doses used at concentration of 300, 400 and 500 ppm. 4.7.3.1.3. Scavenge activity of thyme essential oil: Thyme essential oil showed the greatest antioxidant activity in all essential oil used in our present study. Data are tabulated in Table (34) and Figure (54). The scavenging activity of non-irradiated thyme essential oil was increased from 90.77% at 200 ppm to 93.09%, 94.07% and 95.59% at concentrations 300ppm, 400ppm and 500ppm, respectively. In addition, irradiation doses 2 kGy and 4 kGy represent a gradually increase in the scavenging activity. Where, the scavenging activity was 93.22%, 93.94%, 95.90 and 96.24% at dose 2 kGy in concentrations 200ppm, 300ppm, 400ppm and 500ppm, respectively. While it was 96.34%, 97.15% 97.83% and 98.40% at dose 4 kGy in concentrations 200ppm, 300ppm, 400ppm and 500ppm, respectively. 203
On the contrary, irradiation dose 6 kGy reduce the scavenging activity to 90.51%, 90.54%, 92.38 and 95.86% in concentrations 200ppm, 300ppm, 400ppm and 500ppm, respectively. Chemical composition of the thyme essential oil as shown in Table (37) is showed noticeable increments and decrements in the chemical composition of thyme essential oil related to irradiation dose. As appoint of view, the increments of some chemical constituents – which classified as oxygenated compounds - due to irradiation such as; linalool (from 0.87% at 0 kGy to 2.41% at 4kGy), borneol (from 0.29% at 0kGy to 2.78% at 4kGy), the reason of increase scavenging activity constituents of rosemary essential oil is due to the high percentage of borneol and other chemicals (Wang et al., 2008) and thymol (from 31.7% at 0 kGy to 64.06% at 4kGy). The last component and carvacrol were the predominant compounds and the antioxidant and antimicrobial properties of thyme and many essential oils are related to the these compounds (Sacchetti et al., 2005; Teixeira et al., 2013; Turek and Stintzing, 2012 and Viuda-Martos et al., 2011). On the other hand, irradiation dose 4 kGy showed the highest scavenging activity even rather than BHT at the same concentrations. Where, the scavenging activity of BHT at 200ppm, 300ppm, 400ppm and 500ppm were 95.14%, 95.86%, 96.23% and 97.70%, respectively. While, the scavenging activity of irradiated thyme at 4 kGy was 96.34%, 97.15% 97.83% and 98.40%, respectively. The scavenging activity of Thymus vulgaris essential oil (75.6%) were higher than those obtained by Cananga odorata, Cymbopogon citratus, Rosmarinus officinalis and Curcuma longa (Sacchetti et al., 2005). The mode of action of carvacrol, one of the major components of oregano and thyme oils, appears to have received the most attention from researchers. Thymol is structurally very similar to carvacrol, having the hydroxyl group at a different location on the phenolic ring. Both substances appear to have antioxidant activity (Lambert et al., 2001). 204
9 8 9 6 9 4 9 2 9 0 8 8 8 6
8 4 20
8 2 4 8 0 7 8 Rosemary (0 kGy)
Scavenging7 6 effect (% ) Rosemary (2 kGy) Rosemary (4 kGy) 7 4 Rosemary (6 kGy) 7 2 B H T 7 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 Concentration (ug/ml)
Fig. 53. The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of non-irradiated and irradiated rosemary essential oils and of synthetic antioxidant (BHT) 205
9 9 9 8 9 7 9 6 9 5 9 4 9 3 9 2 5 9 1 20 9 0 8 9 Thyme (0 kGy) Scavenging8 8 effect (% ) Thyme (2 kGy) Thyme (4 kGy) 8 7 Thyme (6 kGy) 8 6 B H T 8 5 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 Concentration (ug/m l)
Fig. (54): The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of non-irradiated and irradiated thyme essential oils and of synthetic antioxidant (BHT) 206
Miguel (2010) reported that the phenolic compounds (thymol and carvacrol) are known for their properties to scavenge free radicals and to inhibit lipid oxidation. These compounds exhibit in vitro and in vivo antioxidant activity, inhibiting lipid peroxidation by acting as chain-breaking peroxyl-radical scavengers. In addition, phenols directly scavenge reactive oxygen species (hydroxyl radicals, peroxynitrite and hypochlorous acid). 4.7.3.2. Mixtures essential oils:
4.7.3.2.1. Mixtures made from non-irradiated essential oils: Data tabulated in Table (35) and illustrated by Figure (55) showed that the scavenging activity of mixtures made from non-irradiated cumin essential oils and non-irradiated rosemary essential oil were less than the scavenging activity of individual essential oil. Where, the scavenging activity of the
C0 X R0 mixture was 67.25%, 71.01%, 75.41% and 76.16% in the concentrations 200ppm, 300ppm, 400ppm and 500ppm, respectively. On the contrary, the scavenging activity of cumin essential oil was lower than that obtained by the C0 X T0 mixture (Figure, 56). But, the scavenging activity of thyme essential oil was higher than the C0 X T0 mixture. The antioxidant effect is due to the presence of thymol and carvacrol, but a possible synergistic effect among oxygen containing compounds can be suggested too. These results indicate that the thyme essential oil could be in use as potential resource of natural antioxidants for food industry so that it is interesting to examine its application as natural antioxidant additive in some final food products (Kulisic et al., 2004).
While, the scavenging activity of the R0XT0 mixture was 80.24%, 87.89%, 89.74% and 89.9% in the concentrations 200 ppm, 300 ppm, 400 ppm and 500 ppm, respectively. Regarding to the Table (35) and Figure (57), the scavenging activity of rosemary essential oil is lower than that 207
obtained by the mixture while the thyme essential oil has higher scavenging activity compared to the mixture. But both individual essential oil and their mixture have lower scavenging activity than the scavenging obtained by BHT.
Table (35): Effect of gamma irradiation on the DPPH scavenging activity of mixture made from cumin, rosemary and thyme essential oils Concentration in ppm
200ppm 300ppm 400ppm 500ppm
% of DPPH Scavenging activity
BHT 95.14 95.86 96.23 97.70
C0 X R0 67.25 71.01 75.41 76.16
C0 X T0 81.5 83.87 88.23 93.51
R0 X T0 80.24 87.89 89.74 89.9
C0 = non-irradiated cumin essential oil, R0 = non-irradiated rosemary essential oil,
T0 = non-irradiated thyme essential oil, DPPH = 1,1-diphenyl-2-picrylhydrazyl, BHT = Buteylated Hydroxyl Toluene, ppm = part per million, kGy = kilo Gray 208
1 0 0
9 5
9 0
8 5 20 8 8 0
7 5
Scavenging effect (% ) Cumin (0kGy) C0 7 0 Rosemary (0kGy) R0 C 0 X R 0
6 5 BHT 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 Concentration (ug/ml)
Fig. (55): The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of non-irradiated cumin and rosemary essential oils combination and of synthetic antioxidant (BHT) 209
1 0 0
9 5
9 0
8 5 9 20
8 0 Cumin (0kGy) C0
Scavenging effect (% ) Thyme (0kGy) T0 7 5 C 0 X T 0 BHT
2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 Concentration (ug/m l)
Fig. (56): The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of non-irradiated cumin and thyme essential oils combination and of synthetic antioxidant (BHT) 210
9 8 9 6 9 4 9 2 9 0 8 8 8 6
8 4 2 10 8 2 8 0 7 8 Rosemary (0kGy) R0 7 6 Scavenging effect (% ) Thym e (0kGy) T0 7 4 R 0 X T 0 7 2 B H T 7 0 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 Concentration (ug/m l)
Fig. (57): The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of non-irradiated rosemary and thyme essential oils combination and of synthetic antioxidant (BHT) 211
4.7.3.2.2. Mixtures made from irradiated essential oils: The scavenging activity of irradiated essential oils of cumin, rosemary and thyme and their mixtures were tabulated in Table (36) and illustrated by Figures (58, 59, 60, 61, 62 and 63). As shown in Table (36) and Figures (58, 59, 60, 61, 62 and 63), data showed that the scavenging activity of mixture made from irradiated (2 kGy) cumin essential oil (C2) and irradiated (4 kGy) cumin essential oil (C4), mixture made from irradiated (2 kGy) cumin essential oil (C2) and irradiated (6kGy) rosemary essential oil (R6), mixture made from irradiated (4 kGy) cumin essential oil (C4) and irradiated (6 kGy) rosemary essential oil (R6) and mixture made form irradiated (6 kGy) rosemary essential oil (R6) and irradiated (4 kGy) thyme essential oil (T4), was lower than all individual irradiated essential oils. In addition, all previous essential oils either individual or in mixtures have lower scavenging activity than BHT except irradiated thyme essential oil where, tends to have a higher scavenging activity than BHT. Only mixtures, Table (36) and Figures (60 and 62) made from both irradiated (2kGy) cumin essential oil (C2) and irradiated (4kGy) thyme essential oil (C4), and irradiated (4kGy) cumin essential oil (C4) and irradiated (4kGy) thyme essential oil (T4), represent a higher scavenging activity than both irradiated cumin essential oil at dose 2kGy and 4kGy. But, lower than the irradiated thyme essential oil which possess to have a higher scavenging activity even above BHT. All above mentioned data could be summarized in, the irradiation doses which used in the present study possess to be promising doses in that essential oil especially in thyme which represents a great stability to the effect of irradiation doses as mentioned by (Fazakerley et al., 1961). Concerning with the mixtures made from irradiated thyme essential oil, the mixture of irradiated thyme essential oil and irradiated essential oil is 212
better than the mixture of irradiated thyme essential oil and rosemary where, at concentration of 500 ppm the scavenging activity of the C4 X T4 and C2 X
T4 mixtures were 92.5% and 94.71% compared to the mixture of R6 X T4 which has scavenging activity 87.08%. As a point of view, the chemical composition of irradiated thyme essential oil as shown in Table (4) showed that the percentage of oxygenated compounds was 70.71% while the hydrocarbon percentage was 20.83%.
Meanwhile, irradiated cumin essential oil at dose 4 kGy has an oxygenated compound percentage 73.73% and hydrocarbon compound percentage 24.60%. On the other hand, irradiated rosemary essential oil showed lower percentage of oxygenated compound 25.77% and higher percentage in hydrocarbon content 69.29%. That finding might be responsible for a synergistic effect between the essential oils that have a high content of oxygenated compounds. The obtained data are in agreement with (Kulisic et al., 2004) who found that the fractions CHO (oxygenated compounds) were the most potent antioxidant suggesting that the antioxidant activity of oregano essential oil is due to more polar constituents. Among these, the fact that CHO fractions are more effective as antioxidant than phenolic fraction or its pure constituents thymol and carvacrol suggests that the synergy among minor oxygen containing compounds has determining role. On the other hand, the less effectiveness of total oil in comparison with CHO fraction may be due to smaller concentration of oxygen containing compounds in total oil and because of the presence of the hydrocarbons, which showed the lowest antioxidant activity when isolated as CH (Hydrocarbon compounds) fraction. 213
Table (36): Effect of gamma irradiation on the DPPH scavenging activity of mixture made from cumin, rosemary and thyme essential oils Concentration in ppm
200ppm 300ppm 400ppm 500ppm
% of DPPH Scavenging activity
BHT 95.14 95.86 96.23 97.70
C2 X C4 64.48 67.62 71.65 73.34
C2 X R6 64.69 68.3 71.27 71.95
C2 X T4 80.02 83.94 84.14 94.71
C4 X T4 82.33 83.55 89.74 92.5
C4 X R6 64.32 68.13 73.86 86.3
R6 X T4 78.52 81.61 83.95 87.08
C2 = irradiated cumin essential oil at dose 2kGy, C4 = irradiated cumin essential oil at dose 4kGy, T4 = irradiated thyme essential oil at dose 4kGy, R6 = irradiated rosemary essential oil at dose 6kGy, DPPH = 1,1-diphenyl-2-picrylhydrazyl, BHT = Buteylated Hydroxyl Toluene, ppm = part per million, kGy = kilo Gray 214
1 0 0
9 5
9 0
8 5
8 0 21 4 7 5
Scavenging7 0 effect (% ) Cumin (2kGy) C2 Cumin (4kGy) C4 6 5 C 2 X C 4 BHT 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 CnecntrationConcentration (µg/ml) (ug/m l) Fig. (58): The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of irradiated cumin and cumin essential oils combination and of synthetic antioxidant (BHT) 215
1 0 0
9 5
9 0
8 5
5 8 0 21
7 5
Scavenging7 0 effect (% ) Cumin (2kGy) C2 Rosemary (6kGy) R6 6 5 C 2 X R 6 B H T 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 Concentration (ug/ml) Fig. (59): The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of irradiated cumin and rosemary essential oils combination and of synthetic antioxidant (BHT) 216
100
95
90 21 6 85
Scavenging80 effect (%) Cumin (2kGy) C2 Thyme (4kGy) T4 C 2 X T 4 B H T 75 200 250 300 350 400 450 500 Concentration (ug/ml) Fig. (60): The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of irradiated cumin and thyme essential oils combination and of synthetic antioxidant (BHT) 217
9 8 9 6 9 4 9 2 9 0 8 8 8 6 8 4 8 2 8 0 7 7 8 21 7 6 7 4 7 2
Scavenging7 0 effect (%) Cumin (4kGy) C4 6 8 Rosemary (6kGy) R6 6 6 C 4 X R 6 6 4 B H T 6 2 2 0 0 2 5 0 3 0 0 3 5 0 4 0 0 4 5 0 5 0 0 Concentration (ug/ml) Fig. (61): The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of irradiated cumin and rosemary essential oils combination and of synthetic antioxidant (BHT) 218
98 96 94 92 90 88
86 21 8 84 82 80 Scavenging effect (%) Cumin (4kGy) C4 78 Thyme (4kGy) T4 C 4 X T4 76 B H T 200 250 300 350 400 450 500 Concentration (ug/ml) Fig. (62): The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of irradiated cumin and thyme essential oils combination and of synthetic antioxidant (BHT) 219
98
96
94
92
90
88
9 86 21 84
82
Scavenging effect (%) Rosemary (6kGy) R6 80 Thyme (4kGy) T4 R 6 X T4 78 B H T 200 250 300 350 400 450 500 Concentration (ug/ml)
Fig. (63): The reduction of alcoholic DPPH solutions: the rate of hydrogen donation ability of irradiated rosemary and rosemary essential oils combination and of synthetic antioxidant (BHT) 220
4.8. Chemical composition of irradiated essential oil:
After all previous preliminary experiments made to select the recommended gamma irradiation doses either in antimicrobial trail or antioxidant trail, the chemical constituents of cumin, rosemary and thyme essential oils after irradiation were fractionated and identified by gas chromatography and mass spectroscopy technique (GC/MS). 4.8.1. Chemical composition of irradiated cumin essential oil (4 kGy): The essential oil obtained from cumin seeds by steam distillation was irradiated at dose 4 kGy. In comparison with the chemical composition of non-irradiated essential oil, ten chemical groups were fractionated and identified. The obtained data are tabulated in Table (38) and illustrated by Figure (64). From these results it could be indicated that 18 compounds were isolated from irradiated cumin essential oil. The identified compounds (14) accounted for 98.33% of the composition of cumin essential oil. The remaining portion 1.67% was representing 4 unknown constituents. By the comparison of the data given in Tables (37) it could be indicated that the chemical composition of the oil of cumin essential oil (control) differs from irradiated cumin essential oil in a few changes. Some chemical compounds completely disappeared form irradiated essential oil such as; α- thujene and carvacrol. While, a few chemical constituents decreased from non-irradiated cumin essential oil (0 kGy) to irradiated cumin essential oil (4 kGy); γ-terpinene (– 6.52%) and β-pinene (– 13.62%) as shown in Table (37). On the other hand, irradiation of cumin essential oil led to great increase in the content of cuminaldehyde which is defined as a predominant compound of cumin essential oil from 24.93% (0 kGy) to 37.98% (4 kGy) by 221 Table (37): Chemical components of cumin, rosemary and thyme essential oil before and after irradiation Compounds Cumin Percentage of Rosemary Percentage of Thyme Percentage of 0 kGy 4 kGy increase (+) or 0 kGy 6kGy increase (+) or 0 kGy 4 kGy increase (+) or decrease (-) decrease (-) decrease (-) α-Thujene 0.26 -- – 0.26 -- 0.48 + 0.48 2.52 0.3 – 2.22 α-Pinene 0.98 0.18 – 0.80 17.1 13.33 – 3.77 1.92 0.21 – 1.71 Camphene ------6.35 -- – 6.35 0.76 0.08 – 0.68 β-Pinene 17.44 3.82 – 13.62 0.92 -- –0.92 0.5 0.57 + 0.07 Sabinene 0.43 0.13 – 0.30 0.92 -- – 0.92 ------Myrcene 0.68 0.28 – 0.40 0.97 0.82 – 0.15 2 -- – 2.00 22
α-Phellandrene -- 0.35 + 0.35 -- 0.35 + 0.35 0.76 -- – 0.76 1 α-Terpinene 0.42 0.09 – 0.33 0.53 0.58 + 0.05 0.17 0.47 + 0.30 ρ-Cymene 6.72 6.43 -- 0.63 7.52 + 6.89 33.6 12.11 – 21.49 o-Cymene 0.09 -- – 0.09 ------Limonene ------5.93 15.37 + 9.44 2.06 -- – 2.06 Δ-3-Carene ------0.22 -- – 0.22 0.33 -- – 0.33 1,8-Cineol ------33.2 22.33 – 10.9 ------γ-Terpinene 19.49 12.97 – 6.52 0.27 0.26 – 0.01 13.1 6.98 – 6.12 222
Table (37): Continued Terpinolene ------0.12 0.58 + 0.46 ------α-Linalool ------1.38 -- – 1.38 0.87 2.41 + 1.54 Terpineol 0.17 -- – 0.17 2.91 1.16 – 1.75 1.24 0.83 – 0.41 Camphor ------14.5 29.74 + 15.24 ------Citronelol 1.60 1.61 + 0.01 -- 0.62 + 0.62 ------Borneol ------0.48 -- – 0.48 0.29 2.78 + 2.49
Cuminaldehyde 24.93 33.65 + 8.72 ------22 2 α-Terpinene-7-al 10.55 ------37.98 + 12.55 γ-Terpinene-7-al 14.88 ------Geraniol ------2.79 -- – 2.79 0.35 0.2 – 0.15 Thymol ------31.7 64.06 + 32.36 Carvacrol 0.15 -- – 0.15 ------3.16 0.1 – 3.06 223
Table (38): Chemical composition of irradiated cumin (Cuminum cyminum) essential oil (4kGy) Compounds Peak Number by Figure (64) % Aliphatic terpenes -Myrcene 4 0.28 Total 0.28 Monocyclic terpenes α-Phellandrene 5 0.35 Sabinene 2 0.13 -Terpinene 6 0.09 -Terpinene 8 12.97 Total 13.54 Bicyclic terpenes -Pinene 1 0.18 -Pinene 3 3.82 Total 4 Aliphatic alcohols Citranellol 10 1.61 Total 1.61 Cyclic terpene alcohols ρ-menthen-4-ol 9 0.22 Total 0.22 Monotcyclic terpene aldehyde phellandral 12 0.27 Total 0.27 Aromatic hydrocarbons ρ-Cymene 7 6.43 Total 6.43 Monocyclic terpene aldehyde Cuminaldehyde 11 33.65 Total 33.65 Monoterpenes aldehyde Terpinene-7-al 13 37.98 Total 37.98 Sesquiterpene hydrocarbone β-Bisabolene 14 0.35 Total 0.35 Total known 98.33 Total unknown 15, 16, 17, 18 01.67 Total oxygenated compounds 73.73 Total non-oxygenated compounds 24.60 224
11 13
8
7 22 4
3
10
15 17 4 5 1 9 12 14 2 6 16 18
Fig. (64). Chemical components of irradiated cumin essential oil at dose 4 kGy 225
increase of (+ 8.72%) as shown in Table (37). The effect of irradiation process could be resulted from chemical modification of terpenes including the oxidative effect; this effect of irradiation process was degreed according to the content of terpenes and other hydrocarbons in the essential oil. On the olfactory evidence, the order of resistance to irradiation was aldehydes > alcohols > ester > hydrocarbon > phenol. Regarding the effect of gamma irradiation on the essential oil components, the decrease of hydrocarbons (γ- terpinene and β-pinene) and the increase of oxygenated compounds (cuminaldehyde) might be related to the resistant to irradiation where, aldehydes are more stable to irradiation than hydrocarbons (Fazakerley et al., 1961). In addition, irradiation with gamma rays at dose 10 and 25 kGy in caraway essential oil led to increase the percentage amount of cuminaldehyde from 20.90% to (23.53% and 22.08%), respectively as mentioned by Fatemi et al. (2011). 4.8.2. Chemical composition of irradiated rosemary essential oil (6 kGy): Rosemary essential oil is irradiated at dose 6 kGy then fractionated and identified by GC/MS. In comparison with the chemical composition of non- irradiated essential oil, ten chemical groups were fractionated and identified. The obtained data are tabulated in Table (39) and illustrated by Figure (65). From the data tabulated in Table (39) it could be indicated that 18 compounds were isolated from irradiated rosemary essential oil. The identified compounds (15) accounted for 95.06% of the composition of 226
Table (39). Chemical composition of irradiated rosemary (Rosmarinus officinalis) essential oil (6 kGy) Compounds Peak Number in Fig (65) % Aliphatic terpenes -Myrcene 3 0.82 Total 0.82 Monocyclic terpenes α-thujene 1 0.48 α-phellandrene 4 0.35 Limonene 6 15.37 -Terpinene 7 0.26 -Terpinolene 8 0.58 Total 17.04 Bicyclic terpenes -Pinene 2 13.33 Total 13.33 Cyclic ether monoterpene 1,8-Cineole 12 22.33 Total 22.33 Aliphatic alcohols α-Citranellol 10 0.62 Total 0.62 Cyclic terpene alcohols α-Terpineol 9 1.16 Total 1.16 Cyclic terpene ketone Camphor 13 29.74 Total 29.74 Cycylic terpene alcohol ρ-Menthen-8-Ol 11 1.66 Total 1.66 Aromatic hydrocarbons ρ-Cymene 5 7.52 Total 7.52 Sesquiterpene hydrocarbons Caryophyllene 17 0.66 α-Farnesene 18 0.18 Total 0.84 Total known 95.06 Total unknown 14, 15, 16 4.94 Total oxygenated compounds 25.77 Total non-oxygenated compounds 69.29 227 7 22 Relative Abundance Relative
Fig. (65): Chemical components of irradiated rosemary essential oil at dose 6 kGy 228
rosemary essential oil. The remaining portion 4.94% was representing four unknown constituents. Concerning minor chemical compounds in irradiated rosemary essential oil as shown in Table (39), there were complete loss of some chemical compounds such as; sabinene, α-terpinen, pinocarvone, camphene, β-pinene and Δ-3-carene, borneol. Moreover, the fifth chemical group which consists of linalool and geraniol completely disappeared. Also, the group namely bicyclic ketone terpene (verbenone) and oxygenated sequiterpene (Caryophyllene oxide) disappeared. As shown in Table (39), prior to irradiated rosemary essential oil at dose 6 kGy, the linalool and α-terpineol content were decreased by 1.38% and 2.75%, respectively. The previous results are in the same line with Sjövall et al., (1990) who found that irradiation with 10 and 50 kGy led to decrease of the lialool and α-terpineol content by (4 - 13%). In addition, Fan and Sokorai (2002) found that irradiated fresh cilantro leaves at doses 1, 2 and 3 kGy caused significantly decrease in the dodecanal, linalool and (E)-2- cocecenal amount after 14 day.
The great increase of the content of ρ-cymene by 6.89% and limonene by 9.44% is due to the changes induced in terpenes by irradiation that could be explained by oxidation or hydroxylation of aromatic rings in terpene molecules (Sádecká, 2007). On the other hand, camphene and 1,8-cineol decrease by 6.35% and 10.9%, respectively. Camarago et al. (2008) found that the content of cineol in the essential oil of Turnera diffusa Wild was decreased after exposure to different irradiation doses. 4.8.3. Chemical composition of irradiated thyme essential oil (4 kGy): The essential oil obtained from thyme by steam distillation was irradiated at dose 4 kGy. In comparison with the chemical composition of non- irradiated thyme essential oil, nine chemical groups were fractionated and identified. The obtained data are tabulated in Table (40) and illustrated by Figure (66). 229
From the obtained results it could be indicated that 23 compounds were isolated from irradiated thyme essential oil. The identified compounds (16) accounted for 91.54% of the composition of thyme essential oil. The remaining portion 8.46% was representing 7 unknown constituents.
The first group namely aliphatic terpenes (β-myrcene and β-ocimene) which are founded in non-irradiated thyme essential oil completely disappeared after irradiation of thyme essential oil at dose 4 kGy. That might be related to oxidation or hydroxylation of aromatic rings in terpene molecules (Sádecká, 2007). Data represented in Table (40), illustrated in the second group namely monocyclic terpenes, the content of α-Thujene, α-phellandrene, γ-terpinene and limonene decreased by (– 2.22%, – 0.76%, – 6.12% and – 2.06%, respectively) while, α-terpinene increased by + 0.30%. Other minor chemical constituents represented few changes where, α - pinene, campene, myrcene, Δ-3-carene, terpineol and geraniol decreased by – 1.71%, – 0.68%, – 2%, – 0.33%, – 0.41% and – 0.15%. While, β-pinene, α- linalool and borneol increased by + 0.07%, + 1.54% and + 2.49%. The great decrease illustrated in the chemical group namely aromatic hydrocarbons which consists of ρ-cymene where, ρ-cymene decreased from 33.6% to 12.11% by decreasing of – 21.49%. On the other hand, thymol which is defined as predominant compound of thyme essential oil was increased by + 32.36% from 31.7% in non-irradiated thyme essential oil to 64.06% in irradiated thyme essential oil at dose 4 kGy. 4.9. Application of adding cumin, rosemary and thyme essential oils and their mixtures in food system: Food is a rich environment for most bacteria. Food-borne illnesses caused by consumption of food contaminated with pathogenic bacteria and/or their toxins are a great problem in public health. Essential oils represent a source 230
Table (40): Chemical composition of irradiated thyme (Thymus vulgaris) essential oils (4 kGy) Compounds Peak number in Fig (66) % Monocyclic terpenes -Thujene 1 0.3 -Terpinene 5 0.47 -Terpinene 7 6.98 Total 7.75 Bicyclic terpenes -Pinene 2 0.21 Camphene 3 0.08 -Pinene 4 0.57 Total 0.86 Aliphatic alcohols Linalool 9 2.41 Geraniol 14 0.2 Total 2.61 Aliphatic aldehyde Geranial 14 0.21 Total 0.21 Cyclic terpene alcohols Borneol 10 2.78 α-Terpineol 8 0.83 Total 3.61 Monoterpene phenol Thymol 15 64.06 Carvacrol 16 0.1 Total 64.16 Monoterpene ester Geranyl acetate 17 0.12 Total 0.12 Aromatic hydrocarbons ρ-Cymene 6 12.11 Total 12.11 Sesquiterpene hydrocarbone β-Caryophyllene 18 0.11 Total 0.11 Total known 91.54 Total unknown 11, 12, 19, 20, 21, 22, 23 8.46 Total oxygenated compounds 70.71 Total non-oxygenated compounds 20.83 231
1515 1 23
Relative Abundance Relative 7
16 19
11 18 22 5 23 1 2 4 12 3 13 14 20 1 2 17 21
2
Fig. (66): Chemical components of irradiated thyme essential oil at dose 4 kGy 232
of natural antimicrobial substances and have the potential to be used in the food industry as a preservative to prevent spoilage and to increase the shelf life of products. The essential oils could also reduce side effects by their replacement of chemical preservatives (Solórzano-Santos and Miranda- Novales, 2012). The essential oils have high efficiency against the food-borne pathogen and spoilage microorganisms in liquid phase and in agar plates but this effect in food is only achieved with higher concentration of essential (Burt, 2004, Hulin et al., 1998 and Yamazaki et al., 2004). This fact may imply an organoleptic impact, caused by altering the natural taste of the food by exceeding the acceptable flavour thresholds (Nazer et al., 2005). Hence, for reducing the sensory effect, one of the alternative approaches may be the use of synergism between essential oils. According to the preliminary experiment have been done in the preceding section. The essential oil concentration used in the mayonnaise trail was as follow; 0.5, 0.7 and 0.9% of cumin essential oil added to mayonnaise while, rosemary essential oil in concentrations 0.3, 0.5 and 0.7% added to mayonnaise. Thyme essential oil was added at concentrations 0.4, 0.6 and 0.8%. A set of experiments were conducted to detect the proper concentration of cumin, rosemary and thyme essential oil which mixed with mayonnaise. The threshold level of odor for this system is shown by Figures (67, 68 and 69) and tabulated in Table (41). In addition, the threshold level of the taste of adding cumin, rosemary and thyme essential oils were illustrated by Figures (70, 71 and 72) Statistical analysis demonstrated that the odor of all of mayonnaise supplemented with cumin, rosemary and thyme essential oil used in this experiment were significantly different compared to each other. All concentrations of essential oils were rejected by the panelist. However, the 233
taste and odor used were higher and not acceptable. For instance, rosemary and cumin essential oil has a bitter, astringent taste, which complements a wide variety of foods (Jiang et al., 2011). The ways in which essential oils are applied and the concentrations at which they are used are important factors related to their effectiveness, and in some circumstances, essential oils could be the cause of changes in flavour, odour and other characteristics of food products. Past studies with plant essential oil components used for flavouring show that some bacterial species could be inhibited by direct application without affecting the flavour of the food products (Du et al., 2011). 4.9.1. Effect of adding essential oils mixtures on the microbial total count of mayonnaise: Table (42) showed total microbial count of handmade mayonnaise prepared from non-irradiated ingredients supplemented with essential oils mixture at concentration 0.2%. The microbial load of mayonnaise was measured as 5.6 X 104 cfu/g (colony forming unit/g). While, the addition of 1 C0 X R0 mixture reduce the microbial count to 42 X 10 cfu/g.
1 On the other hand, C0 X T0 mixture reduce the microbial load to 18 X 10 cfu/g this reduction of the total count might be related to the mode of action of thyme essential oil especially in the acidic media. Where, thymol was found to be more inhibitive at pH 5.5 than 6.5. At low pH the thymol molecule would be undissociated and therefore more hydrophobic, and so may bind better to the hydrophobic areas of proteins and dissolve better in the lipid phase (Juven et al., 1994). The last finding was appeared in the case 2 of T4 X R6 and T0 X R0 mixtures where, the total count was 55 X 10 cfu/gm and 23 X 102 cfu/gm, respectively. 234
Table (41): Mean threshold values and standard errors for the odor of adding cumin, rosemary and thyme essential oils to mayonnaise Threshold score Concentration Cumin essential oil Rosemary essential oil Thyme essential oil (%) Aroma Taste Aroma Taste Aroma Taste B C B B B B
0.0 3.75 ± 0.31 4.33 ± 0.33 3.71 ± 0.42 4.00 ± 0.41 3.50 ± 0.43 4.00 ± 0.45 234 0.3 -- -- 5.14A ± 0.59 6.25A± 0.48 -- -- 0.4 ------6.83A ± 0.48 6.00A± 0.32 0.5 6.75A ± 0.59 6.33B± 0.33 5.43A ± 0.65 6.25A± 0.85 -- -- 0.6 ------7.08A ± 0.37 7.00A± 0.63 0.7 6.88A ± 0.29 7.67AB± 0.67 5.71A ± 0.68 7.00A± 0.41 -- -- 0.8 ------7.50A ± 0.56 6.80A± 0.86 0.9 7.13A ± 0.58 8.00A± 0.001 ------Each value represents the mean ± S.E., The mean value with different superscript alphabets in a row indicate significant differences (P< 0.05) using LSD test, Threshold value refers to the minimum concentration at which stimulus is easily characterized, the aroma and taste were sniffed and tasted by 13 judgers. 235
8
7
6
5 3.75B ± 0.31 4
3
2
6.75A ± 0.59 1 7.13A ± 0.58 0 235
6.88A ± 0.29
Fig. (67): Threshold level of the odor of adding cumin essential oil to mayonnaise recipe 236
6
5 3.71B ± 0.42 4
3
2
1 5.71A ± 0.67
5.43A ± 0.65 0 23 6
5.14A ± 0.95
Fig. (68): Threshold level of the odor of adding rosemary essential oil to mayonnaise recipe 237
8 7 6 5 B 4 3.50 ± 0.43 3 2 7.50A ± 0.56 1 6.83A ± 0.48
7 0 23
7.08A ± 0.37
Fig. (69): Threshold level of the odor of adding thyme essential oil to mayonnaise recipe 238
8 7 6 B 5 4.33 ± 0.33 4 3 2 A AB 8.00 ± 0.001 1 7.67 ± 0.67
0 23 8
6.33B± 0.33
Fig. (70): Threshold level of the taste of adding cumin essential oil to mayonnaise recipe 239
7
6 B 5 4.00 ± 0.41
4
3
2 A 6.25A± 0.85 1 6.25 ± 0.48 0 9 23
7A± 0.41
Fig. (71): Threshold level of the taste of adding rosemary essential oil to mayonnaise recipe 240
7
6 5 4.00B± 0.45 4
3
2 A 7.00A± 0.63 6.80 ± 0.86 1 2
0 40
6.00A± 0.32
Fig. (72): Threshold level of the taste of adding thyme essential oil to mayonnaise recipe 241
1 Mayonnaise supplemented with C4 X R6 mixture has total count 29 X 10 cfu/gm. Cumin essential oil has cuminaldehyde as a predominate compound. However, antimicrobial activity of the cumin essential oil has been related to cumin aldehyde and a-terpinen-7-al. In general, essential oils can exert their antimicrobial activity via destructing the cell membranes (Tassou et al., 2000). As mentioned by Pajohi et al., 2011 the essential oil alone could not inhibit the bacterial growth of B. cereus at 25°C in the barley soup. But it could be appeared in the combination of cumin essential oil and nicin. The cumin essential oil could not damage bacterial cell wall, but extensive destruction was detected in the cell walls and cytoplasm of the bacteria treated with the cumin essential oil in combination with nicin.
Table (42): Total count of the handmade mayonnaise supplemented with essential oils mixture 101 102 103 104 105 Control 81 73 56 5.6 N.D. C0 X R0 42 N.D. N.D. N.D. N.D. C0 x T0 18 N.D. N.D. N.D. N.D. T0 X R0 31 55 N.D. N.D. N.D. C4 X R6 29 N.D. N.D. N.D. N.D. C4 X T4 30 N.D. N.D. N.D. N.D. T4 X R6 20 23 N.D. N.D. N.D. N.D. = not detected
4.9.2. Effect of adding essential oils mixtures to sterilized mayonnaise inoculated with Salmonella typhimurium: The ingredients of mayonnaise were irradiated at different doses to achieve the sterility of the mayonnaise recipe. Then, handmade mayonnaise was supplemented with 0.2% mixtures made from both irradiated and non- 242
irradiated essential oil. Mayonnaise was inoculated with 105 cfu/g. S. typhimurium under sterile condition. All obtained data are showed that the mixtures at the previous concentration completely inhibit the growth of S. typhimurium even at the first dilution up to the fifth dilution except for the mixture T4 X R6 and C4 X 1 1 T4 where, the growth was 3 X 10 and 6 X 10 cfu/g. while, the growth in the control sample was 5.6 X 104 cfu/g.
However, essential oils antimicrobial efficacy in foods is usually achieved at higher concentrations, which many times entail a sensory impact, caused by altering the natural taste and/or odor of the food by exceeding the acceptable flavor and/or odor thresholds (Nazer et al., 2005).
5. SUMMARY The present investigation was carried out in an effort to open a new area for the possibility of making synergism among cumin, rosemary and thyme essential oils either irradiated or non-irradiated against pathogenic microbial strains as well as, determining the antioxidant activity of these essential oil mixtures. Our findings reinforced that the mixtures of essential oils both irradiated and non-irradiated with different chemical composition at sufficient low concentration could arise as an alternative to replace synthetic sanitizers classically applied in some foods, and to reach the balance between the demand for the microbial safety and organoleptic acceptability. Thus, our data could be summarized as follow: 1. The results showed that the essential oils of cumin, rosemary and thyme did not show any differences in the physiochemical properties according to irradiation doses 2, 4 and 6 kGy, respectively compared to non- irradiated essential oil. 2. GC/MS analysis of non-irradiated cumin, rosemary and thyme essential oils, individually indicated that; cumin essential oil contains 24.93% cuminaldehyde as a predominate compound while, the content of - pinene and -pinene was 0.98 and 17.44 %, respectively. While, ρ- cymene contains 6.72%. The content of monoterpenes aldehydes group was 25.43%. Totally, the amount of total oxygenated compounds was 52.82% while, the amount of total non-oxygenated compounds 46.92%. On the other hand, the chemical composition of rosemary essential oil was mainly limonene (5.93%), camphene (6.35%), 1,8-cineol (33.2%) and camphor (14.5%). Totally, the amount of oxygenated compounds in the rosemary essential oil was 58.63% while, the amount of non- oxygenated was 38.50%.Thyme essential oil mainly contains -terpinene (13.1%), thymol (31.7%), carvacrol (3.16%) and ρ-cymene (33.6%). Finally, the amount of the oxygenated compounds in the thyme essential 244
oil was 38.5% while, the amount of non-oxygenated compounds was 61.5%. 3. From the preliminary selection experiments that assess the recommended doses for antibacterial against; S. typhimurium, S. aureus, B. cereus and E. coli, it could be concluded that the irradiation dose combatable with cumin essential oil was 2 and 4 kGy. While, the recommended irradiation dose for rosemary essential oil was 6 kGy. Finally the recommended dose for thyme essential oil was 4 kGy. The preliminary experiment to assess the antifungal recommended irradiation dose was the same as the preceding mentioned in the antibacterial where, both irradiated and non- irradiated cumin and thyme essential oil completely inhibit the growth of A. flavus. While, the completely inhibition in case of rosemary essential oil appeared at dose 4 kGy. 4. Regarding to the amount t of cumin, rosemary and thyme essential oils into the well, the inhibition zones are increased gradually according to the increase of the essential oil amount till 70 µl. 5. The evaluation of interactions between essential oils both irradiated and
non-irradiated, the C0 X R0 mixture showed that the practical inhibition zones against both E. coli and B. cereus at concentration 50 µl were 13.6 mm and 14.3 mm, respectively. This inhibition zones are higher than the mathematical inhibition zones of the mixture against the same bacterial
strains as well as, the individual essential oils included in the C0 X R0
mixture. C0 X T0 mixture represents the highest antibacterial activity against B. cereus where the inhibition zone was 14 mm at concentration 50 µl. All practical inhibition zones were lower than the mathematical
one. In the R0 X T0 mixture, the same as appeared in the C0 X T0 mixture where, the highest inhibition zone was 21.3 mm at the same concentration against B. cereus. 6. Evaluation of the mixtures made from irradiated essential oils was
studied C2 X C4 mixture does not show any differences. While the C2 X
R6 mixture represents a promising differences in case of B. cereus where, 245
the practical inhibition zone was (14 mm) higher than mathematical one (11.5 mm) and both individual irradiated cumin at 2 kGy (11 mm) and
irradiated rosemary at 6 kGy (8.5 mm). In the C2 X T4 mixture, in spite of the highest antimicrobial activity of both thyme and cumin essential oil either irradiated or non-irradiated individually, the mixture made from them possess to be low or moderate antimicrobial activity. All practical inhibition zones of this mixture were lower than mathematical one or
their individual essential oils. C4 X R6 mixture almost represents the
practical inhibition zones similar to mathematical one. The mixture C4 X
T4 is almost similar to the C2 X T4 en the antibacterial activity. In case of
R6 X T4, the mixture shows almost the same results in case of 50 and 70 µl against E. coli, B. cereus and S. aureus where, the inhibition zones were 12.5 and 13.00 mm in E. coli, 12 and 12.5 mm in B. cereus and 8.5 and 8.5 mm in S. aureus. 7. The minimum inhibitory concentration (MIC) was determined in the individual essential oils and in their mixtures. In case of non-irradiated essential oil, the MIC of non-irradiated cumin, rosemary and thyme, respectively was 0.256 mg/ml against S. typhimurium. While, it was 1.024, 2.048 and 0.512 mg/ml against E. coli. 0.256, 0.512 and 0.256 mg/ml against S. aureus. The MIC of essential oils was 1.024, 2.048 and 0.512 mg/ml against B. cereus. In case of irradiated essential oils, the MIC was almost similar especially in the MIC of irradiated cumin (2kGy) and irradiated thyme (4kGy). On the other hand, the minimum bactericidal concentration (MBC) was determined. The data revealed that the MBCs of all both irradiated and non-irradiated were almost two fold of the MICs. 8. The MICs of the mixtures made from non-irradiated essential oils were
similar (0.256 mg/ml) in case of C0 X R0 in all selected bacteria. While,
the C0 X T0 mixture showed MICs 0.512, 0.384, 0.512 and 0.384 mg/ml against S. typhimiurium, E. coli, S. aureus and B. cereus, respectively. In addition, the MICs of the same arranged bacteria were 0.512, 0.512, 246
0.256 and 0.512 mg/ml, respectively. The MICs in case of mixtures made from irradiated essential oils were arranged from 0.256 to 0.512 mg/ml
where, the lowest MIC appeared in C4 X R6 mixture. On the other hand, the MBCs of all both irradiated and non-irradiated essential oils were increased two fold of MICs. 9. Synergistic activity of all mixtures made from essential oils both irradiated or not under investigations was determined by the fraction inhibitory concentration (FIC) indices. The FIC of all mixtures represents
indifference but, the C0 X R0 mixture against B. cereus (0.375) and E. coli (0.375) was synergy (below 0.5). Furthermore, the FIC shows
addition in case of R0 X T0, C2 X R6, C4 X R6 and R6 X T4 against B.
cereus. And in case of C4 X R6 against S. typhimurium. 10. The preliminary experiments assess to determine the acceptability levels of sunflower oil odor supplemented with non-irradiated essential oil indicated that, the acceptability level of cumin essential oil was significantly appeared in the concentration of 0.2% and the higher acceptable dose was 0.4%. On the contrary, the acceptability levels of both rosemary and thyme essential oil were appeared after 0.1% but the doses of 0.2 in rosemary and 0.1 % in thyme were higher and acceptable. 11. The antioxidant activity was measured by Rancimat test and 2,2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method; according to Rancimat test revealed that the oxidative stability of the sunflower oil without essential oils was 100% (measured as percentage of relative stability). In case of supplemented sunflower oil with 0.02% BHT, the relative stability was 162.5% while, the highest relative stability (107.3%) was appeared in the addition of 0.2% irradiated rosemary essential oil at dose 4 and 6 kGy. The highest relative stability (122.7%) was appeared in the addition of 0.1% irradiated thyme essential oil at dose 4 kGy. The addition of 0.4% irradiated cumin essential oil at dose 4 kGy caused the highest relative stability (114.1%). 247
12. Regarding to the DPPH radical scavenging activity, the scavenging activity of BHT at concentrations 200, 300, 400 and 500 ppm was 95.14, 95.86, 96.23 and 97.70%, respectively. The highest scavenging activity of cumin essential oil appeared at the concentration 500 ppm in irradiated cumin essential oil at dose 4 kGy. In the irradiated rosemary essential oil at dose 6 kGy at concentration 500 ppm, the scavenging activity (97.41%) is near by the scavenging activity of BHT at the same concentration. The promising data could be noticed in case of irradiated thyme essential oil at dose 4 kGy, where the scavenging activity is greater than BHT. The mixtures made from non-irradiated essential oils illustrated that the
scavenging activity of C0 X T0 was 93.51% at concentration of 500 ppm, this percentage the scavenging activity is greater than cumin essential oil and lower than thyme essential oil. On the contrary, mixtures made from irradiated essential oils were possessed to have lower scavenging activity than that obtained by the same mixtures in case of non-irradiation. 13. As our investigation previously appeared, the recommended doses of irradiated essential oil due to the antimicrobial activity and the antioxidant activity were 4 kGy in case of cumin and thyme while, 6 in case of rosemary. Thus, the chromatographic analysis of these doses were s follow; irradiated cumin essential oil at dose 4 kGy was produced; the decrement in content of β-pinene from 17.44% in control to 3.82% in irradiated essential oil also, the decrement of γ-terpinene from 19.49% to 12.97%. While, the increments of cuminaldehyde from 24.93% to 33.65% and the group of monoterpene aldehyde from 25.43% to 37.98%. Thus, the amount of oxygenated compounds increased from 52.82% to 73.73%. While, the GC/MS of irradiated rosemary at dose 6 kGy revealed that α-pinene reduced from 17.1% in non-irradiated rosemary essential oil to 13.33% in the irradiated essential oil, the content of 1,8- cineol was decreased from 33.2% to 22.3%. While, the content of limonene was increased from 5.93% to 15.37% and the content of 248
camphor was increased from 14.5% to 29.74%. Thus, the content of oxygenated compounds was decreased from 58.63% to 25.77%. Finally in case of irradiated thyme essential oil, the amount of hydrocarbons in the i.e. α-thujene, α-pinene, ρ-cymene and γ-terpinene was totally decreased according to the irradiation dose while, the oxygenated compounds i.e. α-linalool, borneol and thymol was increased meanwhile, the amount of oxygenated compounds increased from 38.5% to 70.71%. 14. In case of application in food system, the handmade mayonnaise was tested organoleptically (odor and taste) to assess the acceptability level of adding non-irradiated essential oils. Essential oils was adding in concentrations 0.5, 0.7 and 0.9% of cumin essential oil, 0.4, 0.6 and 0.8% of rosemary essential oil and 0.3, 0.5 and 0.7% of thyme essential oil. The obtained data in cases studied were different significantly compared to control samples in odor and taste. 15. The effect of adding mixtures made from essential oils to the mayonnaise recipe was studied and the obtained data showed that the total count of 4 control mayonnaise was 5.6 X 10 cfu/g. while, the C0 X R0 mixture 1 1 reduce the microbial load to 42 X 10 cfu/g, C0 X T0 was 18 X 10 cfu/g. On the other hand, the effect of adding essential oils mixtures to sterilized handmade mayonnaise (all ingredients are irradiated separately) inoculated with S. typhimurium revealed that all obtained data showed that the mixtures at 0.2% concentration completely inhibit the growth of S. typhimurium even at the first dilution up to the fifth dilution except for 1 the mixture T4 X R6 and C4 X T4 where, the growth was 3 X 10 and 6 X 101 cfu/gm. Thus, the mixtures of essential oils either irradiated or non-irradiated
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and anti-inflammatory potential of Lavandula stoechas and Thymus herba-barona essential oils. Industrial Crops and Products, 44: 97 – 103. ۷. اﻟﻣﻠﺧـص اﻟﻌــرﺑﻰ دراﺳﺎت ﻋﻠﻰ اﻟﺗﺄﺛﯾر اﻟﺗﻌﺎوﻧﻰ ﻟﺑﻌض اﻟزﯾوت اﻟﻌطرﯾﺔ اﻟﻣﺷﻌﻌﺔ ﻓﻰ ﺑﻌض اﻟﻣﻧﺗﺟﺎت اﻟﻐذاﺋﯾﺔ
أﺟرﯾت ﻫذﻩ اﻟدراﺳﺔ ﺑﻬدف دراﺳﺔ إﻣﻛﺎﻧﯾﺔ اﻟﺣﺻول ﻋﻠﻰ ﻧﺷﺎط ﺗﻌﺎوﻧﻰ ﻟﺑﻌض اﻟزﯾوت اﻟﻌطرﯾﺔ ﻣﺛل اﻟزﯾت اﻟﻌطرى" اﻟﻛﻣون (C)، ﺣﺻﺎﻟﺑﺎن (R) واﻟزﻋﺗر (T) "، ﺳواء اﻟزﯾوت اﻟﻣﺷﻌﻌﺔ أو ﻏﯾر اﻟﻣﺷﻌﻌﺔ وﻫذا ﺿد ﺑﻌض اﻟﻣﯾﻛروﺑﺎت اﻟﻣرﺿﯾﺔ ﺑﺎﻹﺿﺎﻓﺔ ﻟدراﺳﺔ اﻟﺗﺄﺛﯾر اﻟﺗﻌﺎوﻧﻰ ﻟﺗﻠك اﻟزﯾوت اﻟﻣﺷﻌﻌﺔ وﻏﯾر اﻟﻣﺷﻌﻌﺔ ﻛﻣﺿﺎدات أﻛﺳدﻩ. وﻣن ﺛم ﯾﻣﻛن ﺗﻠﺧﯾص اﻟﻧﺗﺎﺋﺞ اﻟﺗﻰ ﺗوﺻﻠت إﻟﯾﻬﺎ اﻟدراﺳﺔ ﻓﯾﻣﺎ ﯾﻠﻰ: 1. أظﻬرت اﻟدراﺳﺔ أن اﻟزﯾوت اﻟﻌطرﯾﺔ ﻟﻠﻛﻣون وﺣﺻﺎﻟﺑﺎن واﻟزﻋﺗر ﻟم ﯾﺣدث ﻟﻬﺎ أى ﺗﻐﯾرات ﻓﻰ اﻟﺧﺻﺎﺋص اﻟﻔﯾزﯾﺎﺋﯾﺔ اﻟﻛﯾﻣﯾﺎﺋﯾﺔ ﻧﺗﯾﺟﺔ اﻟﻣﻌﺎﻣﻠﺔ اﻹﺷﻌﺎﻋﯾﺔ ﺑﺟرﻋﺎت 2، 4 و 6 ﻛﺟراى ﻣﻘﺎرﻧﺔ ﺑﺎﻟﻌﯾﻧﺎت ﻏﯾر اﻟﻣﺷﻌﻌﺔ. 2. أظﻬرت ﻧﺗﺎﺋﺞ اﻟﺗﺣﻠﯾل اﻟﻛروﻣﺎﺗوﺟراﻓﻰ ﻟﻌﯾﻧﺎت اﻟزﯾت اﻟﻌطرى ﻏﯾر اﻟﻣﺷﻌﻊ ﻟﻛل ﻣن اﻟﻛﻣون، ﺣﺻﺎﻟﺑﺎن واﻟزﻋﺗر ﻣﺎ ﯾﻠﻰ: اﻟزﯾت اﻟﻌطرى ﻟﻠﻛﻣون ﯾﺣﺗوى ﻋﻠﻰ ﻧﺳﺑﺔ 24.93٪ ﻣن ﻣرﻛب اﻟــ cuminaldehyde وﻫو اﻟﻣرﻛب اﻟﻔﻌﺎل اﻟرﺋﯾﺳﻰ ﻟزﯾت اﻟﻛﻣون، ﺑﯾﻧﻣﺎ ﯾﺣﺗوى ﻋﻠﻰ اﻟــ α-pinene ﺑﻧﺳﺑﺔ 0.98٪ و اﻟــ β-pinene ﺑﻧﺳﺑﺔ 17.44٪. أﻣﺎ ﻣرﻛب اﻟــ ρ-cymene ﻓﻧﺳﺑﺗﺔ 6.72٪. ﻋﻣوﻣﺎ ﻛﺎﻧت ﻧﺳﺑﺔ اﻟﻣرﻛﺑﺎت اﻷﻛﺳﯾﺟﯾﻧﯾﺔ 52.82٪ ﺑﯾﻧﻣﺎ ﻛﺎﻧت ﻧﺳﺑﺔ اﻟﻣرﻛﺑﺎت اﻟﻬﯾدروﻛرﺑوﻧﯾﺔ 46.92٪. ﻋﻠﻰ اﻟﺟﺎﻧب اﻷﺧر، أوﺿﺢ اﻟﺗرﻛﯾب اﻟﻛﯾﻣﺎوى ﻟزﯾت ﺣﺻﺎﻟﺑﺎن ﻏﯾر اﻟﻣﺷﻌﻊ أﻧﺔ ﯾﺣﺗوى ﺑﺻﻔﺔ رﺋﯾﺳﯾﺔ ﻋﻠﻰ اﻟــ limonene ﺑﻧﺳﺑﺔ 5.93٪، اﻟـــ camphene ﺑﻧﺳﺑﺔ 6.35٪، اﻟــــ cineol-1,8 ﺑﻧﺳﺑﺔ 33.2٪ أﻣﺎ اﻟـــــ camphor ﻓﻛﺎﻧت ﻧﺳﺑﺗﺔ 14.5٪. وﻗد ﻛﺎﻧت ﻧﺳﺑﺔ اﻟﻣرﻛﺑﺎت اﻷﻛﺳﯾﺟﯾﻧﯾﺔ ﻓﻰ زﯾت ﺣﺻﺎﻟﺑﺎن اﻟﻌطرى ﻏﯾر اﻟﻣﺷﻌﻊ 58.63٪ ﺑﯾﻧﻣﺎ اﻟﻣرﻛﺑﺎت اﻟﻬﯾدروﻛرﺑوﻧﯾﺔ ﻛﺎﻧت 2
38.50٪. زﯾت اﻟزﻋﺗر اﻟﻌطرى إﺣﺗوى ﺑﺻﻔﺔ أﺳﺎﺳﯾﺔ ﻋﻠﻰ ﻣرﻛﺑﺎت اﻟـــــ ρ-cymene ،carvacrol ،thymol ،γ-terpinene ﺑﻧﺳﺑﺔ ٪13.1، 31.7٪، 3.16٪ و 33.6٪ ، ﻋﻠﻰ اﻟﺗواﻟﻰ. 3. ﺗم ﻋﻣل ﺗﺟرﺑﺔ ﻣﺑدﺋﯾﺔ ﻟﺗﺣدﯾد اﻟﺟرﻋﺔ اﻹﺷﻌﺎﻋﯾﺔ اﻟﻣﺛﻠﻰ ﻟﻠزﯾت اﻟﻌطرى وذﻟك ﻣن وﺟﻬﺔ ﻧظر اﻟﺧواص اﻟﻣﺿﺎدة ﻟﻠﺑﻛﺗرﯾﺎ ﺿد Salmonella typhimurium، Bacillus cereus ،Staphylococcus aureus و Escherichia coli وﻛﺎﻧت اﻟﻧﺗﺎﺋﺞ ﻛﺎﻟﺗﺎﻟﻰ: اﻟﺟرﻋﺔ اﻹﺷﻌﺎﻋﯾﺔ اﻟﻣﺛﻠﻰ ﻟزﯾت اﻟﻛﻣون اﻟﻌطرى ﻛﺎﻧت 2 و 4 ﻛﺟراى ﺑﯾﻧﻣﺎ ﻛﺎﻧت اﻟﺟرﻋﺔ اﻟﻣﺛﻠﻰ ﻟﺣﺻﺎﻟﺑﺎن 6 ﻛﺟراى وﻓﻰ اﻟﻧﻬﺎﯾﺔ اﻟﺟرﻋﺔ اﻟﻣﺛﻠﻰ ﻟﻠزﻋﺗر ﻛﺎﻧت 4 ﻛﺟراى. وإ ﺟرﯾت ﻧﻔس اﻟﺗﺟرﺑﺔ ﻋﻠﻰ ﻓطر Aspergillus flavus وأوﺿﺣت اﻟﻧﺗﺎﺋﺞ أن ﻛﻼ ﻣن زﯾت اﻟﻛﻣون واﻟزﻋﺗر اﻟﻌطرى اﻟﻣﺷﻌﻊ وﻏﯾر اﻟﻣﺷﻌﻊ ﻗد أﺣدث ﺗﺛﺑﯾطﺎ ﻛﺎﻣﻼ ﻓﻰ ﻧﻣو اﻟﻔطر ﺑﯾﻧﻣﺎ ﻧﻣﺎ اﻟﻔطر ﻓﻰ اﻷطﺑﺎق اﻟﻣﻌرﺿﺔ ﻟزﯾت ﺣﺻﺎﻟﺑﺎن اﻟﻌطرى اﻟﻣﺷﻌﻊ ﺑﺟرﻋﺔ 4 ﻛﺟراى ﺛم إﺧﺗﻔﻰ اﻟﻧﻣو ﺗﻣﺎﻣﺎ ﻋﻧد اﻟﺟرﻋﺎت اﻷﻋﻠﻰ وﺣﺗﻰ اﻟﺟرﻋﺔ 10 ﻛﺟراى. 4. أظﻬرت اﻟﻧﺗﺎﺋﺞ أن ﻣﻧطﻘﺔ اﻟﺗﺛﺑﯾط ﺗزداد ﺑزﯾﺎدة ﻛﻣﯾﺔ اﻟزﯾت اﻟﻌطرى داﺧل اﻟـــ well ﻣن 30 ﻣﯾﻛروﻟﯾﺗر وﺣﺗﻰ 70 ﻣﯾﻛروﻟﯾﺗر ﻓﻰ اﻟــــ well. 5. أظﻬرت ﻧﺗﺎﺋﺞ ﺗﻘﯾﯾم اﻟﺗﺄﺛﯾرات اﻟﻣﺗداﺧﻠﺔ ﺑﯾن اﻟزﯾوت وذﻟك ﻓﻰ ﺣﺎﻟﺔ اﻟﻣﺧﺎﻟﯾط
اﻟﻣﻛوﻧﺔ ﻣن زﯾوت ﻋطرﯾﺔ ﻏﯾر ﻣﺷﻌﻌﺔ ﻣﺎ ﯾﻠﻰ: اﻟﻣﺧﻠوط C0 X R0 أﻋطﻰ ﻣﺳﺎﺣﺔ ﺗﺛﺑﯾط ﻓﻌﻠﻰ ﺿد ﻛﻼ ﻣن اﻟــ E. coli و B. cereus 13.6 ﻣم و 14.3 ﻣم ﻋﻠﻰ اﻟﺗواﻟﻰ. ﻫذﻩ اﻟﻣﺳﺎﺣﺔ ﻣن اﻟﺗﺛﯾﺑط ﻛﺎﻧت أﻋﻠﻰ ﻣن اﻟﻣﺳﺎﺣﺔ اﻟﺣﺳﺎﺑﯾﺔ وﻛذﻟك ﻛﺎﻧت
أﻋﻠﻰ ﻣن ﻣﺳﺎﺣﺔ اﻟﺗﺛﺑﯾط ﻟﻛل زﯾت ﻋطرى ﻋﻠﻰ ﺣد. اﻟﻣﺧﻠوط C0 X T0 أﻋطﻰ أﻋﻠﻰ ﻣﺳﺎﺣﺔ ﻣن اﻟﺗﺛﺑﯾط ﺿد اﻟــــ B. cereus ﺣﯾث ﻛﺎﻧت 14 ﻣم ﻋﻧد اﻟﺗرﻛﯾز
50 ﻣﯾﻛروﻟﯾﺗر. أﻣﺎ ﺑﺎﻟﻧﺳﺑﺔ ﻟﻠﻣﺧﻠوط R0 X T0 ﻓﻘد أظﻬرت اﻟﻧﺗﺎﺋﺞ أن أﻋﻠﻰ ﻣﺳﺎﺣﺔ ﺗﺛﺑﯾط ﻛﺎﻧت ﻋﻧد اﻟﺗرﻛﯾز 50 ﻣﯾﻛروﻟﯾﺗر ﻟﺑﻛﺗرﯾﺎ اﻟــــ B. cereus وﻛﺎﻧت 21 ﻣم. 3
6. أظﻬرت اﻟدراﺳﺔ أن إﺳﺗﺧدام ﻣﺧﺎﻟﯾط اﻟزﯾوت اﻟﻌطرﯾﺔ اﻟﻣﻛوﻧﺔ ﻣن زﯾت ﻋطرى
ﻣﻌﺎﻣل إﺷﻌﺎﻋﯾﺎ ﻣﺎ ﯾﻠﻰ: اﻟﻣﺧﻠوط C2 X C4 ﻟم ﯾﺑدى إى إﺧﺗﻼﻓﺎت ﻣﻌﻧوﯾﺔ ﻋن
اﻟﻣﺧﻠوط اﻟﻣﻛون ﻣن زﯾوت ﻏﯾر ﻣﻌﺎﻣﻠﺔ إﺷﻌﺎﻋﯾﺎ. ﻓﻰ ﺣﯾن أن اﻟﻣﺧﻠوط C2 X R6 أظﻬر ﻧﺗﺎﺋﺞ واﻋدة ﺧﺻﯾﺻﺎ ﺗﺟﺎﻩ ﺑﻛﺗرﯾﺎ اﻟــــ B. cereus ﺣﯾث ﻛﺎﻧت ﻣﺳﺎﺣﺔ اﻟﺗﺛﺑﯾط اﻟﻔﻌﻠﯾﺔ 14 ﻣم واﻟﺗﻰ ﻛﺎﻧت أﻛﺛر ﻣن اﻟﻣﺳﺎﺣﺔ اﻟﺣﺳﺎﺑﯾﺔ وﻛذﻟك ﻛﺎﻧت أﻛﺑر ﻣن ﻣﺳﺎﺣﺔ اﻟﺗﺛﺑﯾط ﻟﻛل زﯾت ﻋطرى ﻣﻧﻔردا ﺣﯾث أﻋطﻰ اﻟﻛﻣون اﻟﻣﻌﺎﻣل إﺷﻌﺎﻋﯾﺎ وﺣدة ﻣﺳﺎﺣﺔ ﺗﺛﺑﯾط 11 ﻣم أﻣﺎ ﺣﺻﺎﻟﺑﺎن اﻟﻣﺷﻌﻊ 8.5 ﻣم. ﻓﻰ ﺣﺎﻟﺔ اﻟﻣﺧﻠوط
C2 X T4 ﻓﺑﺎﻟرﻏم ﻣن أن ﻛﻼ ﻣن اﻟزﯾﺗﯾن ﯾﺗﻣﯾزان ﺑﺈرﺗﻔﺎع ﻣﺳﺎﺣﺔ اﻟﺗﺛﺑﯾط اﻟﻣﯾﻛروﺑﯾﺔ ﻛل ﻋﻠﻰ ﺣد، إﻻ أن اﻟﻣﺧﻠوط ﻣﻧﻬﻣﺎ أﻋطﻰ ﻣﺳﺎﺣﺔ ﺗﺛﺑﯾط أﻗل ﻣﻘﺎرﻧﺔ ﺑﻛل زﯾت ﻋطرى ﻣﻧﻔردا، ﺣﯾث ﻛﺎﻧت ﻛل ﻣﺳﺎﺣﺎت اﻟﺗﺛﺑﯾط اﻟﻣﺗﺣﺻل ﻋﻠﯾﻬﺎ ﻣن ﻫذا اﻟﻣﺧﻠوط ﺗﺟﺎﻩ اﻟﻣﯾﻛروﺑﺎت ﻣﺣل اﻟدراﺳﺔ أﻗل ﻣن اﻟﻣﺳﺎﺣﺔ اﻟﺣﺳﺎﺑﯾﺔ. اﻟﻣﺧﻠوط
C4 X R6 ﺗﻘرﯾﺑﺎ أﻋطﻰ ﻣﺳﺎﺣﺔ ﺗﺛﺑﯾط ﻓﻌﻠﯾﺔ ﻗرﯾﺑﺔ ﻣن اﻟﻣﺳﺎﺣﺔ اﻟﺣﺳﺎﺑﯾﺔ.
أﻣﺎ اﻟﻣﺧﻠوط C4 X T4 ﻓﻘد ﻛﺎﻧت ﻧﺗﺎﺋﺟﻪ ﻗرﯾﺑﻪ إﻟﻰ ﺣد ﻛﺑﯾر ﻣن ﻧﺗﺎﺋﺞ اﻟﻣﺧﻠوط
C2 X T4. وأﺧﯾرا، ﻓﻰ ﺣﺎﻟﺔ اﻟﻣﺧﻠوط R6 X T4 ﺣﯾث أظﻬرت اﻟﻧﺗﺎﺋﺞ ﺗﺳﺎوى ﻣﺳﺎﺣﺔ اﻟﺗﺛﺑﯾط ﺗﻘرﯾﺑﺎ ﻓﻰ ﺣﺎﻟﺔ اﻟﺗرﻛﯾزان 50 و 70 ﻣﯾﻛروﻟﯾﺗر ﺿد ﺑﻛﺗرﯾﺎ .B E. coli ،cereus و S. aureus ﺣﯾث ﻛﺎﻧت ﻣﺳﺎﺣﺔ اﻟﺗﺛﺑﯾط ﻓﻰ ﻛل ﻣﺧﻠوط ﻛﺎﻟﺗﺎﻟﻰ: 12 ﻣم، 12.5 ﻣم ﻓﻰ B. cereus و 12.5 ﻣم و 13 ﻣم ﻓﻰ E. coli و 8.5 ﻣم و 8.5 ﻣم ﻓﻰ S. aureus. 7. ﺗم ﺗﻘدﯾر اﻟﺗرﻛﯾز اﻟﻣﻧﺎﺳب ﻟﻠﻘﺿﺎء ﻋﻠﻰ 90٪ ﻣن اﻟﻣﯾﻛروﺑﺎت (Minimum Inhibitory Concentration, MIC) ﻓﻰ اﻟزﯾوت اﻟﻌطرﯾﺔ وﻛذﻟك ﻣﺧﺎﻟﯾطﻬﺎ ﺳواء اﻟﻣﺷﻌﻌﺔ وﻏﯾر اﻟﻣﺷﻌﻌﺔ. ﻓﻘد ﻛﺎﻧت اﻟـــ MIC ﻓﻰ ﺣﺎﻟﺔ اﻟزﯾوت اﻟﻌطرﯾﺔ ﻏﯾر ﻣﺷﻌﻌﺔ اﻟﻛﻣون، ﺣﺻﺎﻟﺑﺎن و اﻟزﻋﺗر 0.256 ﻣﻠﺟرام/ﻣل ﺗﺟﺎﻩ ﺑﻛﺗرﯾﺎ اﻟـــ S. typhimurium، ﺑﯾﻧﻣﺎ ﻛﺎﻧت 1.024، 2.048 و 0.512 ﻣﻠﺟرام/ﻣل ﻋﻠﻰ اﻟﺗواﻟﻰ ﺗﺟﺎﻩ ﺑﻛﺗرﯾﺎ E. coli. وﻛﺎﻧت 0.256، 0.512 و 0.256 ﻣﻠﺟرام/ﻣل ﻋﻠﻰ اﻟﺗواﻟﻰ ﺗﺟﺎﻩ ﺑﻛﺗرﯾﺎ S. aureus. و ﻛﺎن اﻟـــ MIC ﻟﻠزﯾوت 4
اﻟﻌطرﯾﺔ 1.024، 2.048 و 0.512 ﻣﻠﺟرام/ﻣل ﺗﺟﺎﻩ اﻟــ B. cereus. أﻣﺎ ﻓﻰ ﺣﺎﻟﺔ اﻟزﯾوت اﻟﻌطرﯾﺔ اﻟﻣﺷﻌﻌﺔ، ﻓﻘد ﻛﺎﻧت اﻟــــ MIC ﺗﻘرﯾﺑﺎ ﻣﺷﺎﺑﻬﺔ ﻟﻧﺗﺎﺋﺞ اﻟــ MIC اﻟﺧﺎﺻﺔ ﺑﺎﻟزﯾوت ﻏﯾر اﻟﻣﺷﻌﻌﺔ ﺧﺻوﺻﺎ ﻓﻰ ﺣﺎﻟﺔ زﯾت اﻟﻛﻣون واﻟزﻋﺗر اﻟﻣﻌﺎﻣل إﺷﻌﺎﻋﯾﺎ ﺑﺟرﻋﺔ 2 و 4 ﻛﺟراى، ﻋﻠﻰ اﻟﺗواﻟﻰ. وﻋﻠﻰ اﻟﺟﺎﻧب اﻷﺧر، ﺗم ﺗﻘدﯾر اﻟﺗرﻛﯾز اﻟﻣﻧﺎﺳب ﻟﻘﺗل اﻟﺑﻛﺗرﯾﺎ ﻣﺣل اﻟدراﺳﺔ (Minimum Bactericidal Concentration, MBC) وأظﻬرت اﻟﻧﺗﺎﺋﺞ أن ﻗﯾم اﻟــــ MBC ﻟﻛل ﻣن اﻟزﯾوت اﻟﻌطرﯨﺔ اﻟﻣﺷﻌﻌﺔ وﻏﯾر اﻟﻣﺷﻌﻌﺔ ﺗﻘرﯾﺑﺎ ﻣﻘﺎرﺑﺔ ﻟﺿﻌف ﺗرﻛﯾز اﻟـــ MIC. 8. ﻛﺎﻧت اﻟــــ MICs ﻟﻠﻣﺧﺎﻟﯾط اﻟﻣﻛوﻧﺔ ﻣن زﯾوت ﻋطرﯾﺔ ﻏﯾر ﻣﺷﻌﻌﺔ ﻣﺗﺷﺎﺑﻬﺔ
ﺧﺻوﺻﺎ ﻓﻰ اﻟﻣﺧﻠوط C0 X R0 واﻟذى ﻛﺎن 0.256 ﻣﻠﺟرام/ﻣل ﺗﺟﺎﻩ اﻟﺑﻛﺗرﯾﺎ
ﻣﺣل اﻟدراﺳﺔ. ﺑﯾﻧﻣﺎ اﻟـــ C0 X T0 أﻋطﻰ MIC 0.512، 0.384 ،0.512 و 0.384 ﻣﻠﺟرام/ﻣل ﺗﺟﺎﻩ ﺑﻛﺗرﯾﺎ S. aureus ،E. coli ،S. typhimurium و B. cereus ﻋﻠﻰ اﻟﺗواﻟﻰ. اﻟــ MICs ﻓﻰ اﻟﻣﺧﺎﻟﯾط اﻟﻣﻛوﻧﺔ ﻣن زﯾوت ﻋطرﯾﺔ ﻣﺷﻌﻌﺔ أﻋطت أﻗل MIC ﻟﻬﺎ ﻋﻧد 0.256 ﻣﻠﺟرام / ﻣل وﻛﺎﻧت ﻓﻰ
اﻟﻣﺧﻠوط C4 X R6 ﺑﯾﻧﻣﺎ أﻋﻠﻰ ﻗﯾﻣﺔ ﻟﻠــــ MIC ﻫﻰ 0.512 ﻣﻠﺟرام / ﻣل ﻓﻰ أﻛﺛر ﻣن ﻣﺧﻠوط ﻣﺧﺗﻠف. وﻋﻠﻰ اﻟﺟﺎﻧب اﻷﺧر ﻗﯾم اﻟـــ MBCs ﻟﻛل اﻟزﯾوت اﻟﻌطرﯾﺔ ﺳواء اﻟﻣﺷﻌﻌﺔ وﻏﯾر اﻟﻣﺷﻌﻌﺔ زادت ﺑﻣﻌدل اﻟﺿﻐف ﺗﻘرﯾﺑﺎ. 9. اﻟﺗﺄﺛﯾر اﻟﺗﻌﺎوﻧﻰ ﻟﻛل ﻣﺧﺎﻟﯾط اﻟزﯾوت اﻟﻌطرﯾﺔ ﻣﺣل اﻟدراﺳﺔ (اﻟﻣﺷﻌﻌﺔ وﻏﯾر اﻟﻣﺷﻌﻌﺔ) ﺗم ﺗﻘدﯾرﻫﺎ ﺑﺈﺳﺗﺧدام اﻟﺗرﻛﯾز اﻟﺗﺛﺑﯾطﻰ اﻟﺟزﺋﻰ (FIC)، ﺣﯾث أﻋطت ﻛل
اﻟﻣﺧﺎﻟﯾط ﺗﻘرﯾﺑﺎ ﺗﺄﺛﯾر ﻏﯾر ﻣﺧﺗﻠف (Indifference) ﺑﯾﻧﻣﺎ اﻟﻣﺧﻠوط C0 X R0 أظﻬر ﺗﺄﺛﯾر ﺗﻌﺎوﻧﻰ إﯾﺟﺎﺑﻰ (Synergism) ﺗﺟﺎﻩ اﻟــــ B. cereus ﺣﯾث ﻛﺎﻧت ﻗﯾﻣﺔ اﻟـــ FIC 0.375 وﻛﺎﻧت أﯾﺿﺎ 0.375 ﺗﺟﺎﻩ اﻟـــــ E. coli . أﻣﺎ اﻟــــ FIC
ﻟﻣﺧﺎﻟﯾط C4 X R6 ،C2 X R6 ،R0 X T0 و R6 XT4 ﺗﺟﺎﻩ ﺑﻛﺗرﯾﺎ B. cereus ﻓﻘد أظﻬرت ﺗﺄﺛﯾر إﺿﺎﻓﺔ (Addition). 5
10. أظﻬرت ﻧﺗﺎﺋﺞ اﻟﺗﺟﺎرب اﻟﻣﺑدﺋﯾﺔ اﻟﺧﺎﺻﺔ ﺑﺗﺣدﯾد ﺣد اﻟﻘﺑول ﻣن اﻟزﯾت اﻟﻌطرى اﻟﻣﺿﺎف إﻟﻰ زﯾت دوار اﻟﺷﻣس أن إﺿﺎﻓﺔ زﯾت اﻟﻛﻣون ﻏﯾر اﻟﻣﺷﻌﻊ إﻟﻰ أدت إﻟﻰ ظﻬور اﻟراﺋﺣﺔ ﻋﻧد ﺗرﻛﯾز 0.2٪ واﻟﺗرﻛﯾزات اﻷﻋﻠﻰ ﻛﺎﻧت ﻣرﻓوﺿﺔ إﺣﺻﺎﺋﯾﺎ وﻟﻛﻧﻬﺎ ﻣﻘﺑوﻟﺔ ﺣﺳﯾﺎ ﺣﺗﻰ اﻟﺗرﻛﯾز 0.4٪. ﻋﻠﻰ اﻟوﺟﺔ اﻷﺧر، أظﻬرت ﻧﺗﺎﺋﺞ إﺿﺎﻓﺔ زﯾت ﺣﺻﺎﻟﺑﺎن واﻟزﻋﺗر إﻟﻰ زﯾت دوار اﻟﺷﻣس ظﻬور اﻟراﺋﺣﺔ ﻋﻧد اﻟﺗرﻛﯾز ﺑﻌد 0.1٪ وﻟﻛن اﻟﺗرﻛﯾزان 0.2٪ و 0.1٪ ﻓﻰ ﺣﺻﺎﻟﺑﺎن واﻟزﻋﺗر ﻋﻠﻰ اﻟﺗواﻟﻰ ﻛﺎﻧﺎ ﻋﺎﻟﯾﺎن وﻟﻛن ﻣﻘﺑوﻻن. 11. أظﻬرت ﻧﺗﺎﺋﺞ ﺗﻘدﯾر اﻟﺧواص اﻟﻣﺿﺎدة اﻟﺗﻰ ﻗدرت ﺑﺈﺳﺗﺧدام طرق اﻟــــ Rancimat واﻟـــــ DPPH-Radical Scavenging أﻧﻪ: اﻟﺛﺑﺎت اﻷﻛﺳﯾدى ﻟزﯾت دوار اﻟﺷﻣس اﻟﻣﺿﺎف إﻟﯾﻪ BHT ٪0.02 ﻛﺎن 162.5٪ ﺑﯾﻧﻣﺎ ﻛﺎن أﻋﻠﻰ ﺛﺑﺎت أﻛﺳﯾدى 107.3٪ ﻋﻧد إﺿﺎﻓﺔ 0.2٪ ﻣن زﯾت ﺣﺻﺎﻟﺑﺎن اﻟﻣﺷﻌﻊ ﺑﺟرﻋﺎت 4 و 6 ﻛﺟراى. أﻋﻠﻰ ﺛﺑﺎت ﻛﺎن ﻓﻰ ﺣﺎﻟﺔ إﺿﺎﻓﺔ 0.1٪ ﻣن زﯾت اﻟزﻋﺗر اﻟﻣﺷﻌﻊ ﺑﺟرﻋﺔ 4 ﻛﺟراى وﻛﺎن 122.7٪. ﺑﯾﻧﻣﺎ إﺿﺎﻓﺔ زﯾت اﻟﻛﻣون اﻟﻣﺷﻌﻊ ﺑﺟرﻋﺔ 4 ﻛﺟراى ﺑﺗرﻛﯾز 0.4٪ أدى ﻹﺣداث ﺛﺑﺎت أﻛﺳﯾدى ﺑﻧﺳﺑﺔ ٪114.1. 12. أظﻬرت ﻧﺗﺎﺋﺞ إﺧﺗﺑﺎر اﻟــ DPPH أن اﻟﻧﺷﺎط اﻟﻣﺿﺎد ﻟﻸﻛﺳدة ﻟﻠــــ BHT ﺑﺎﻟﺗرﻛﯾزات 200، 300، 400 و 500 ﺟزء ﻓﻰ اﻟﻣﻠﯾون ﻛﺎن 95.14، 95.86، 96.86 و 97.70٪، ﻋﻠﻰ اﻟﺗواﻟﻰ. وﻛﺎن أﻋﻠﻰ ﻧﺷﺎط ﻣﺿﺎد ﻟﻸﻛﺳدة ﻓﻰ زﯾت اﻟﻛﻣون ﻋﻧد ﺗرﻛﯾز 500 ﺟزء ﻓﻰ اﻟﻣﻠﯾون ﻓﻰ ﺣﺎﻟﺔ زﯾت اﻟﻛﻣون اﻟﻣﺷﻌﻊ ﺑﺟرﻋﺔ 4 ﻛﺟراى. ﻓﻰ ﺣﺎﻟﺔ زﯾت ﺣﺻﺎﻟﺑﺎن اﻟﻣﺷﻌﻊ ﺑﺟرﻋﺔ 6 ﻛﺟراى ﻋﻧد اﻟﺗرﻛﯾز 500 ﺟزء ﻓﻰ اﻟﻣﻠﯾون ﻛﺎن اﻟﻧﺷﺎط اﻟﻣﺿﺎد ﻟﻸﻛﺳدة ﺑﺈﺳﺗﺧدام اﻟـــ DPPH 97.71٪ وﻫذا اﻟﻧﺷﺎط ﻗرﯾب اﻟﻘﯾﻣﺔ ﺟدا ﻣن اﻟـــ BHT ﺑﺈﺳﺗﺧدام ﻧﻔس اﻟﺗرﻛﯾز. وأظﻬرت اﻟدراﺳﺔ أﻧﻪ ﻣن اﻟﻧﺗﺎﺋﺞ اﻟواﻋدة اﻟﺗﻰ ﯾﻣﻛن ﻣﻼﺣظﺗﻬﺎ ﻫو إﺳﺗﺧدام زﯾت اﻟزﻋﺗر اﻟﻣﺷﻌﻊ ﺑﺟرﻋﺔ 4 ﻛﺟراى ﺣﯾث أﻋطﻰ ﻗﯾﻣﺔ أﻛﺑر ﻣن ﻗﯾﻣﺔ اﻟﻧﺷﺎط اﻟﻣﺿﺎد ﻟﻸﻛﺳدة ﻟﻠــــ .BHT 6
أظﻬرت ﻧﺗﺎﺋﺞ ﻣﺧﺎﻟﯾط اﻟزﯾوت اﻟﻌطرﯾﺔ اﻟﻣﻛوﻧﺔ ﻣن زﯾوت ﻋطرﯾﺔ ﻏﯾر ﻣﺷﻌﻌﺔ أن
ﻣﺧﻠوط اﻟـــ C0 X T0 أﻋطﻰ ﻧﺷﺎط ﻣﺿﺎد ﻟﻸﻛﺳدة ﺑﺈﺧﺗﺑﺎر اﻟـــ DPPH ﺑﻣﻘدار 93.51٪ ﻋﻧد اﻟﺗرﻛﯾز 500 ﺟزء ﻓﻰ اﻟﻣﻠﯾون، وﻛﺎﻧت ﻫذﻩ اﻟﻘﯾﻣﺔ أﻋﻠﻰ ﻣن ﻗﯾﻣﺔ زﯾت اﻟﻛﻣون اﻟﻌطرى وﺣدﻩ وأﻗل ﻣن زﯾت اﻟزﻋﺗر وﺣدﻩ. ﻋﻠﻰ اﻟوﺟﺔ اﻷﺧر، ﻣﺧﺎﻟﯾط اﻟزﯾوت اﻟﻌطرﯾﺔ اﻟﻣﻛوﻧﺔ ﻣن زﯾوت ﻣﺷﻌﻌﺔ أﻋطت ﻗﯾم ﻧﺷﺎط ﺿد أوﻛﺳﯾدى أﻗل ﻣن ﻣﺛﯾﻠﺗﻬﺎ ﻏﯾر اﻟﻣﺷﻌﻌﺔ. 13. أظﻬرت اﻟﻧﺗﺎﺋﺞ اﻟﺳﺎﺑﻘﺔ اﻟﺧﺎﺻﺔ ﺑﺎﻟﻧﺷﺎط اﻟﻣﺿﺎد ﻟﻠﻣﯾﻛروﯾﺎت وﻛذﻟك اﻟﻣﺿﺎد ﻟﻸﻛﺳدة أن اﻟﺟرﻋﺔ اﻹﺷﻌﺎﻋﯾﺔ اﻟﻣﺛﻠﻰ ﻓﻰ ﻫذﻩ اﻟدراﺳﺔ ﻛﺎﻧت 4 ﻛﺟراى ﻓﻰ ﻛﻼ ﻣن زﯾت اﻟﻛﻣون واﻟزﻋﺗر اﻟﻌطرى ﺑﯾﻧﻣﺎ ﻛﺎﻧت 6 ﻛﺟراى ﻓﻰ زﯾت ﺣﺻﺎﻟﺑﺎن اﻟﻌطرى. وﻣن ﺛم أﻋطﻰ اﻟﺗﺣﻠﯾل اﻟﻛروﻣﺎﺗوﺟراﻓﻰ ﻟﺗﻠك اﻟزﯾوت اﻟﻣﺷﻌﻌﺔ ﻣﺎﯾﻠﻰ: إﻧﺧﻔض اﻟﻣﺣﺗوى ﻣن اﻟــــ β-pinene ﻣن 17.44٪ ﻓﻰ اﻟزﯾت ﻏﯾر اﻟﻣﺷﻌﻊ إﻟﻰ ٪3.82 ﻓﻰ اﻟزﯾت اﻟﻣﺷﻌﻊ ﻛذﻟك إﻧﺧﻔض اﻟﻣﺣﺗوى ﻣن اﻟـــ γ-terpinene ﻣن ٪19.49 إﻟﻰ 12.97٪. ﺑﯾﻧﻣﺎ زاد اﻟﻣﺣﺗوى ﻣن اﻟﻣرﻛب اﻟﻔﻌﺎل ﻓﻰ زﯾت اﻟﻛﻣون اﻟــــ cuminaldehyde ﻣن 24.93٪ إﻟﻰ 33.65٪. وزادت ﻗﯾﻣﺔ اﻟﻣرﻛﺑﺎت اﻷﻛﺳﯾﺟﯾﻧﯾﺔ ﻣن 52.82٪ ﻓﻰ اﻟزﯾت ﻏﯾر اﻟﻣﺷﻌﻊ إﻟﻰ 73.73٪ ﻓﻰ اﻟزﯾت اﻟﻣﺷﻌﻊ. أظﻬرت ﻧﺗﺎﺋﺞ اﻟﺗﺷﻌﯾﻊ اﻟﺟﺎﻣﻰ ﺑﺟرﻋﺔ 6 ﻛﺟراى ﻟزﯾت ﺣﺻﺎﻟﺑﺎن أن ﻣﺣﺗوى اﻟـــ α-pinene إﻧﺧﻔض ﻣن 17.1٪ ﻓﻰ اﻟزﯾت ﻏﯾر اﻟﻣﺷﻌﻊ إﻟﻰ 13.33٪ ﻓﻰ اﻟزﯾت اﻟﻣﺷﻌﻊ ﻛذﻟك إﻧﺧﻔض ﻣﺣﺗوى اﻟــــ cineol-1,8 ﻣن 33.2٪ إﻟﻰ 22.3٪. ﺑﯾﻧﻣﺎ زاد ﻣﺣﺗوى اﻟــ limonene ﻣن 5.93٪ إﻟﻰ 15.37٪ ﻛذﻟك ﻣﺣﺗوى اﻟـــ camphor ﻣن 14.5٪ إﻟﻰ 29.74٪. وﻣن ﺛم إﻧﺧﻔض ﻣﺣﺗوى اﻟزﯾت ﻣن اﻟﻣرﻛﺑﺎت اﻷﻛﺳﯾﺟﯾﻧﯾﺔ ﻣن 58.83٪ إﻟﻰ ٪25.77. أظﻬرت ﻧﺗﺎﺋﺞ زﯾت اﻟزﻋﺗر اﻟﻌطرى اﻟﻣﺷﻌﻊ ﺑﺟرﻋﺔ 4 ﻛﺟراى إﻧﺧﻔﺎض ﻣﺣﺗوى ﻛل ﻣن، ρ-cymene ،α-pinene ،α-thugene و γ-terpinene ﻧﺗﯾﺟﺔ اﻟﺗﺷﻌﯾﻊ اﻟﺟﺎﻣﻰ ﺑﯾﻧﻣﺎ إرﺗﻔﻊ ﻣﺣﺗوى ﻛل ﻣن اﻟـــ borneol ،linalool و thymol وﺑذﻟك إرﺗﻔﻊ ﻣﺣﺗوى اﻟﻣرﻛﺑﺎت اﻷﻛﺳﯾﺟﯾﻧﯾﺔ ﻣن 38.5٪ إﻟﻰ ٪70.71. 7
14. أظﻬرت ﻧﺗﺎﺋﺞ اﻟﺗطﺑﯾق اﻟﻐذاﺋﻰ أن اﻟﻣﺎﯾوﻧﯾز ﻣﺣﻠﻰ اﻟﺻﻧﻊ ﺗم ﺗﻘﯾﻣﻪ ﺣﺳﯾﺎ ﻣن ﺣﯾث اﻟﻣذاق واﻟراﺋﺣﺔ وذﻟك ﻟﺑﯾﺎن أﻗل ﺗرﻛﯾز ﻣن اﻟزﯾوت اﻟﻌطرﯾﺔ ﻣﺣل اﻟدراﺳﺔ ﯾﻣﻛن إﺳﺗﺧداﻣﺔ، ﺗم إﺿﺎﻓﺔ ﺗرﻛﯾزات 0.5، 0.7 و 0.9٪ ﻣن زﯾت اﻟﻛﻣون إﻟﻰ زﯾت دوار اﻟﺷﻣس، 0.4، 0.6 و 0.8٪ ﻣن زﯾت ﺣﺻﺎﻟﺑﺎن اﻟﻌطرى إﻟﻰ زﯾت دوار اﻟﺷﻣس و 0.3، 0.5 و 0.7٪ ﻣن زﯾت اﻟزﻋﺗر. وﻛﺎﻧت اﻟﻧﺗﺎﺋﺞ اﻟﻣﺗﺣﺻل ﻋﻠﯾﻬﺎ ظﻬور إﺧﺗﻼف ﻣﻌﻧوﯨﺎ ﺑﯾن زﯾت دوار اﻟﺷﻣس اﻟﻣﺿﺎف إﻟﯾﻪ اﻟزﯾوت اﻟﻌطرﯨﺔ ﻣﺣل اﻟدراﺳﺔ (ﻛل ﻋﻠﻰ ﺣد) وﺑﯾن اﻟزﯾوت ﻏﯾر اﻟﻣﺿﺎف إﻟﯾﻬﺎ زﯾت ﻋطري. 15. أظﻬرت ﻧﺗﺎﺋﺞ اﻟدراﺳﺔ أن اﻟﺣﻣل اﻟﻣﯾﻛروﺑﻰ ﻟﻠﻣﺎﯾوﻧﯾز ﻏﯾر اﻟﻣﻌﺎﻣل (اﻟﻣﻘﺎرﻧﺔ) ﻛﺎنx 5.6 410 وﺣدة ﺗﻛوﯾن ﻣﺳﺗﻌﻣرة / ﺟرام ﺑﯾﻧﻣﺎ ﻛﺎﻧت ﻋﻧد إﺿﺎﻓﺔ ﻣﺧﻠوط اﻟــــ 1 1 x 42 C0 X R0 10 وﺣدة ﺗﻛوﯾن ﻣﺳﺗﻌﻣرة / ﺟرام ، x 18 10 وﺣدة ﺗﻛوﯾن
ﻣﺳﺗﻌﻣرة/ﺟرام ﻓﻰ اﻟﻣﺧﻠوط C0 X T0. وﻋﻠﻰ اﻟﺟﺎﻧب اﻷﺧر ﻋﻧد إﺿﺎﻓﺔ ﻣﺧﺎﻟﯾط اﻟزﯾوت اﻟﻌطرﯾﺔ ﺑﺗرﻛﯾز 0.2٪ إﻟﻰ اﻟﻣﺎﯾوﻧﯾز اﻟﻣﻠﻘﺢ ﺑﻣﯾﻛروب اﻟﺳﺎﻟﻣوﻧﯾﻼ، أظﻬرت اﻟﻧﺗﺎﺋﺞ أن ﺟﻣﯾﻊ اﻟﻣﺧﺎﻟﯾط اﻟﻣﺳﺗﺧدﻣﺔ أدت إﻟﻰ ﺣدوث ﺗﺛﺑﯾط ﻛﺎﻣل ﻟﻠﺳﺎﻟﻣوﻧﯾﻼ ﻣن
أول ﺗﺧﻔﯾف وﺣﺗﻰ اﻟﺗﺧﻔﯾف اﻟﺧﺎﻣس ﻋدا اﻟﻣﺧﻠوطﯾن T4 X R6 و C4 X T4. وﻣن ﺛم ﺗﻘﺗرح اﻟدراﺳﺔ أن اﻟﻣﺧﺎﻟﯾط اﻟﻣﺳﺗﺧدﻣﺔ ﻣن اﻟزﯾوت اﻟﻌطرﯾﺔ ﺳواء
اﻟﻣﺷﻌﻌﺔ أو ﻏﯾر اﻟﻣﺷﻌﻌﺔ ﺧﺻوﺻﺎ اﻟﻣﺧﻠوط C0 X R0 ﯾﻣﻛن إﺳﺗﺧداﻣﻬﺎ ﻟﺗﻘﻠﯾل اﻟﺗرﻛﯾزات اﻟﻣﺳﺗﺧدﻣﺔ ﻣن اﻟزﯾوت اﻟﻌطرﯾﺔ واﻟﺗﻰ ﻛﺎﻧت ﺗﻌطﻰ ﺗﺄﺛﯾرا ﺣﺳﯾﺎ ﻏﯾر ﻣﻘﺑول ﻓﻰ اﻟﻐذاء.
ﺟﺎﻣﻌﺔ ﻋﯾــن ﺷﻣس ﻛﻠﯾﺔ اﻟـــــــــــزراﻋـــــــــﺔ
رﺳﺎﻟﺔ دﻛﺗـــــــــــــــــــوراﻩ
إﺳم اﻟطـــــــــــﺎﻟب : ﻣﺣﻣــد ﻋﺑد اﻟــرازق ﻋﺑد اﻟﻌﻠــﯾم ﺣﻧﻔﻰ ﻋﻧوان اﻟرﺳﺎﻟﺔ : دراﺳﺎت ﻋﻠﻰ اﻟﺗﺄﺛﯾر اﻟﺗﻌﺎوﻧﻰ ﻟﺑﻌض اﻟزﯾوت اﻟﻌطرﯾﺔ اﻟﻣﺷﻌﻌﺔ ﻓﻰ ﺑﻌض اﻟﻣﻧﺗﺟﺎت اﻟﻐذاﺋﯾﺔ إﺳم اﻟدرﺟـــــــــﺔ : دﻛﺗور ﻓﻠﺳﻔﺔ ﻓﻰ اﻟﻌﻠوم اﻟزراﻋﯾﺔ (ﻋﻠوم وﺗﻛﻧوﻟوﺟﯾﺎ اﻷﻏذﯾﺔ)
ﻟﺟﻧﺔ اﻹﺷراف د. اﺣﻣد ﯾوﺳف ﺟﺑرﯾل أﺳﺗﺎذ ﻋﻠوم وﺗﻛﻧوﻟوﺟﯾﺎ اﻷﻏذﯾﺔ اﻟﻣﺗﻔرغ ، ﻗﺳم ﻋﻠوم اﻷﻏذﯾﺔ ، ﻛﻠﯾﺔ اﻟزراﻋﺔ ، ﺟﺎﻣﻌﺔ ﻋﯾن ﺷﻣس
د. ﻋﻠﻰ ﺣﺳن ﻣﺣﻣد راﺿﻰ أﺳﺗﺎذ اﻟﺻﻧﺎﻋﺎت اﻟﻐذاﺋﯾﺔ اﻟﻣﺗﻔرغ ، ﻗﺳم اﻟﺑﺣوث اﻟﻧﺑﺎﺗﯾﻪ ، ﻣرﻛز اﻟﺑﺣوث اﻟﻧووﯾﺔ ، ﻫﯾﺋﺔ اﻟطﺎﻗﺔ اﻟذرﯾﺔ
د. ﺣﻧﺎن ﻣﺣﻣد ﻋﺑدﻩ اﻟﺳﯾد أﺳﺗﺎذ ﻋﻠوم وﺗﻛﻧوﻟوﺟﯾﺎ اﻷﻏذﯾﺔ اﻟﻣﺳﺎﻋد ، ﻗﺳم ﻋﻠوم اﻷﻏذﯾﺔ، ﻛﻠﯾﺔ اﻟزراﻋﺔ ، ﺟﺎﻣﻌﺔ ﻋﯾن ﺷﻣس
ﺗﺎرﯾﺦ اﻟﺗﺳﺟﯾل 2010/2/1
اﻟدراﺳﺎت اﻟﻌﻠﯾﺎ ﺧﺗم اﻹﺟ ﺎزة أﺟﯾزت اﻟرﺳﺎﻟﺔ ﺑﺗﺎرﯾﺦ 2013/ 5 / 29 ﻣواﻓﻘﺔ ﻣﺟﻠس اﻟﻛﻠﯾﺔ ﻣواﻓﻘﺔ ﻣﺟﻠس اﻟﺟﺎﻣﻌﺔ 2013/ / 2013/ /
ﺻﻔﺣـــﺔ اﻟﻣواﻓﻘــﺔ ﻋﻠﻰ اﻟرﺳﺎﻟـــﺔ
دراﺳﺎت ﻋﻠﻰ اﻟﺗﺄﺛﯾر اﻟﺗﻌﺎوﻧﻰ ﻟﺑﻌض اﻟزﯾوت اﻟﻌطرﯾﺔ اﻟﻣﺷﻌﻌﺔ ﻓﻰ ﺑﻌض اﻟﻣﻧﺗﺟﺎت اﻟﻐذاﺋﯾﺔ
رﺳﺎﻟﺔ ﻣﻘدﻣﺔ ﻣن ﻣﺣﻣــد ﻋﺑـد اﻟــرازق ﻋﺑـد اﻟﻌﻠــﯾم ﺣﻧﻔﻰ ﺑﻛﺎﻟورﯾوس ﻋﻠوم زراﻋﯾﺔ (ﻋﻠوم وﺗﻛﻧوﻟوﺟﯾﺎ اﻷﻏذﯾﺔ) ، ﺟﺎﻣﻌﺔ ﻋﯾن ﺷﻣس ، 1998 ﻣﺎﺟﺳﺗﯾر ﻋﻠوم زراﻋﯾﺔ (ﺻﻧﺎﻋﺎت ﻏذاﺋﯾﺔ) ، ﺟﺎﻣﻌﺔ اﻟﻘﺎﻫرة ، 2008
ﻟﻠﺣﺻول ﻋﻠﻰ درﺟﺔ دﻛﺗور ﻓﻠﺳﻔﺔ ﻓﻰ اﻟﻌﻠــوم اﻟزراﻋﯾــﺔ (ﻋﻠوم وﺗﻛﻧوﻟوﺟﯾﺎ اﻷﻏذﯾﺔ)
وﻗد ﺗﻣت ﻣﻧﺎﻗﺷﺔ اﻟرﺳﺎﻟﺔ واﻟﻣواﻓﻘﺔ ﻋﻠﯾﻬﺎ اﻟﻠﺟﻧــــــــــــــــــــــــــــــــــــــــــــــــﺔ د. ﺳﺎﻣﻰ إﺑراﻫﯾم اﻟﺻﯾﺎد...... أﺳﺗﺎذ اﻟﺻﻧﺎﻋﺎت اﻟﻐذاﺋﯾﺔ ، ﻛﻠﯾﺔ اﻟزراﻋﺔ ، ﺟﺎﻣﻌﺔ أﺳﯾوط د. ﺣﻣدى ﻣﺻطﻔﻰ ﻋﺑﯾد...... أﺳﺗﺎذ ﻋﻠوم وﺗﻛﻧوﻟوﺟﯾﺎ اﻻﻏذﯾﺔ ، ﻛﻠﯾﺔ اﻟزراﻋﺔ ، ﺟﺎﻣﻌﺔ ﻋﯾن ﺷﻣس د. اﺣﻣد ﯾوﺳف ﺟﺑرﯾل...... أﺳﺗﺎذ ﻋﻠوم وﺗﻛﻧوﻟوﺟﯾﺎ اﻻﻏذﯾﺔ اﻟﻣﺗﻔرغ ، ﻛﻠﯾﺔ اﻟزراﻋﺔ ، ﺟﺎﻣﻌﺔ ﻋﯾن ﺷﻣس د. ﺣﻧﺎن ﻣﺣﻣد ﻋﺑدﻩ اﻟﺳﯾد...... أﺳﺗﺎذ ﻋﻠوم وﺗﻛﻧوﻟوﺟﯾﺎ اﻷﻏذﯾﺔ اﻟﻣﺳﺎﻋد ، ﻛﻠﯾﺔ اﻟزراﻋﺔ ، ﺟﺎﻣﻌﺔ ﻋﯾن ﺷﻣس
ﺗﺎرﯾﺦ اﻟﻣﻧﺎﻗﺷﺔ 29/5/2013
دراﺳﺎت ﻋﻠﻰ اﻟﺗﺄﺛﯾر اﻟﺗﻌﺎوﻧﻰ ﻟﺑﻌض اﻟزﯾوت اﻟﻌطرﯾﺔ اﻟﻣﺷﻌﻌﺔ ﻓﻰ ﺑﻌض اﻟﻣﻧﺗﺟﺎت اﻟﻐذاﺋﯾﺔ
رﺳﺎﻟﺔ ﻣﻘدﻣﺔ ﻣن
ﻣﺣﻣــد ﻋﺑد اﻟــرازق ﻋﺑد اﻟﻌﻠــﯾم ﺣﻧﻔﻰ
ﺑﻛﺎﻟورﯾوس ﻋﻠوم زراﻋﯾﺔ(ﻋﻠوم وﺗﻛﻧوﻟوﺟﯾﺎ اﻷﻏذﯾﺔ) ، ﺟﺎﻣﻌﺔ ﻋﯾن ﺷﻣس ، 1998 ﻣﺎﺟﺳﺗﯾر ﻋﻠوم زراﻋﯾﺔ (ﺻﻧﺎﻋﺎت ﻏذاﺋﯾﺔ) ، ﺟﺎﻣﻌﺔ اﻟﻘﺎﻫرة ، 2008
ﻟﻠﺣﺻول ﻋﻠﻰ
درﺟﺔ دﻛﺗور ﻓﻠﺳﻔﺔ ﻓﻰ اﻟﻌﻠـوم اﻟزراﻋﯾــﺔ (ﻋﻠوم وﺗﻛﻧوﻟوﺟﯾﺎ اﻷﻏذﯾﺔ)
ﻗﺳم ﻋﻠوم اﻷﻏـذﯾﺔ ﻛﻠﯾــﺔ اﻟزراﻋــﺔ ﺟﺎﻣﻌﺔ ﻋﯾن ﺷﻣس
2013