Chemical composition antioxidant and antimicrobial activities of essential oils extracted from citrus varieties in vietnam

VIETNAM NATIONAL UNIVERSITY – HOCHIMINH CITY INTERNATIONAL UNIVERSITY CHEMICAL COMPOSITION, ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF ESSENTIAL OILS EXTRACTED FROM CITRUS VARIETIES IN VIETNAM A thesis submitted to the School of Biotechnology, International University in partial fulfillment of the requirements for the degree of MSc. in Biotechnology Student name: Phạm Thị Lan Chi – MBT04003 Supervisor: Phạm Văn Hùng, PhD. Nguyễn Thị Lan Phi, PhD. May/2013 i ABSTRACT Essential oils (EOs) are complex mixtures of biologically active substances used since a long time as flavoring agents and preservatives of a number of commercial products. The important characteristics of essential oils are their antioxidant and antimicrobial potential. In the present study, Vietnamese citrus essential oils from nine varieties were extracted by two methods, cold pressing and vacuum hydro-distillation. The essential oils were characterized for their chemical compositions, antioxidant and antimicrobial activities. GC analysis of the chemical compositions of the isolated essential oils revealed the presence of nine main compounds. The total concentrations of these compounds were from 63.06% to 99.27%, including α-pinene (0.04-1.98%), sabinene (0.15-3.89%), β-pinene (0.0221.89%), myrcene (0.96-41.95%), α-terpinene (0.02-11.41%), limonene (40.29- 95.78%), terpinolene (0.02-0.57%), ٧-terpinene (0.01-12.14%) and linalool (0.020.43%). Both cold pressed essential oils and vacuum hydro-distillated essential oils show strong antioxidant and antimicrobial activities. For antioxidant capacity, the lime showed the strongest, followed by pomelo and orange EOs. The lime also had the highest antimicrobial capacity as compared to other citrus essential oils. The minimal inhibition capacity (MIC) was a range of 0.66–42 mg/ml for lime EOs, 5.25- 42 mg/ml for orange EOs, 2.63-42 mg/ml for pomelo EOs. Although the yield of essential oils extracted by the vacuum hydro-distillation method was higher than that by the cold-pressing method, the antioxidant and antimicrobial capacities of hydro-distillated essential oils were lower than those of the cold pressed extracts for all citrus varieties. The results of this study show that the vacuum hydro-distillation method used in this study could be developed to be used in industrial application and the citrus essential oils could be widely used as flavoring and preservatives in food, cosmetic and pharmaceutical industries. ii ACKNOWLEDGEMENTS Firstly, I would like to express my love to my parents and my younger brother for their unconditional love, help and support for my study at International University. Secondly, I am so grateful to my supervisors, Dr Pham Van Hung and Dr Nguyen Thi Lan Phi for their help and guidance throughout my research. Without their enthusiastic help and advices, this thesis would not be completed. Moreover, I would like to give my thanks to all staffs in the laboratory rooms for pleasure provide me with all chemicals and equipments needed. Finally, I also would like to thank all my friends who are always there to give me support and provide their special knowledge and talent in this research. I am very grateful for meeting you and for our relationship. Your encouragement and help is endless. iii TABLE OF CONTENTS ABSTRACT ............................................................................................................................ i ACKNOWLEDGEMENTS........................................................................................................ iii TABLE OF CONTENTS .......................................................................................................... iv LIST OF FIGURES................................................................................................................ vi LIST OF TABLES................................................................................................................. vii ABBREVIATION ................................................................................................................ viii Chapter 1: INTRODUCTION ......................................................................................... 9 Chapter 2: LITERATURE REVIEW .............................................................................. 11 2.1 Essential oils .......................................................................................................... 11 2.2 Citrus essential oils ................................................................................................. 11 2.3 Extraction methods ................................................................................................. 13 2.3.1 Hydrodistillation method ................................................................................... 13 2.3.2 Solvent extraction ............................................................................................ 14 2.3.3 Supercritical carbon dioxide method ................................................................... 15 2.3.4 Cold pressing method ....................................................................................... 15 2.4 Gas chromatography ............................................................................................... 16 2.5 Food poisoned microorganisms ................................................................................. 18 2.5.1 Staphylococcus aureus ..................................................................................... 18 2.5.2 Bacillus cereus ................................................................................................. 19 2.5.3 Salmonella typhi .............................................................................................. 20 2.5.4 Pseudomonas aeruginosa .................................................................................. 21 2.5.5 Aspergillus flavus ............................................................................................. 22 2.5.6 Fusarium solani ............................................................................................... 23 2.6 Antimicrobial activity of essential oils ........................................................................ 24 2.7 Antioxidant activity of essential oils........................................................................... 26 Chapter 3: Materials and methods ............................................................................ 28 3.1 Experimental design ................................................................................................ 28 3.2 Materials collection and preparation .......................................................................... 28 3.3 Essential oils extraction ........................................................................................... 31 3.3.1 Cold pressing method ....................................................................................... 31 3.3.2 Vacuum distillation method ............................................................................... 31 iv 3.4 Chemical composition analysis ................................................................................. 32 3.5 Antimicrobial activities of citrus essential oils ............................................................. 33 3.5.1 Microbial strains............................................................................................... 33 3.5.2 Microorganisms counting .................................................................................. 33 3.5.3 Diffusion technique .......................................................................................... 34 3.5.4 Dilution technique ............................................................................................ 34 3.6 Antioxidant activities of citrus essential oils ................................................................ 34 3.6.1 DPPH assay ..................................................................................................... 34 3.6.2 Ferric thiocyanate (FTC) assay ........................................................................... 35 3.7 Data analysis ......................................................................................................... 36 Chapter 4: Results and Discussion ............................................................................ 37 4.1 Optimization conditions for vacuum distillation extraction ............................................ 37 4.1.1 Temperature optimization ................................................................................. 37 4.1.2 Time optimization ............................................................................................ 38 4.2 Yield of citrus essential oils ...................................................................................... 39 4.3 Chemical compositions of citrus essential oils ............................................................. 40 4.3.1 Chemical compositions of lime essential oils ........................................................ 40 4.3.2 Chemical compositions of orange essential oils .................................................... 43 4.3.3 Chemical compositions of pomelo essential oils .................................................... 45 4.4 Antioxidant activities of citrus essential oils ................................................................ 47 4.4.1 DPPH assay ..................................................................................................... 47 4.4.2 FTC assay ....................................................................................................... 49 4.5 Antimicrobial activities............................................................................................. 51 4.5.1 S. aureus ........................................................................................................ 51 4.5.2 B. cereus ........................................................................................................ 54 4.5.3 S. typhi .......................................................................................................... 56 4.5.4 P. aeruginosa .................................................................................................. 58 4.5.5 A. flavus ......................................................................................................... 59 4.5.6 F. solani .......................................................................................................... 62 Chapter 5: CONCLUSION ........................................................................................... 64 REFERENCES...................................................................................................................... 65 APPENDIX ......................................................................................................................... 75 v LIST OF FIGURES Figure 2 ‎ .1 Diagram of Gas chromatography ............................................................... 16 Figure 2 ‎ .2 Principle of Gas Chromatography ............................................................... 17 Figure 2 ‎ .3 Scanning electron micrograph of S. aureus. ................................................ 19 Figure 2 ‎ .4 Rod-shaped Bacillus cereus. ..................................................................... 20 Figure 2 ‎ .5 Flagella stain of Salmonella typhi. ............................................................. 21 Figure 2 ‎ .6 Pseudomonas aeruginosa ......................................................................... 22 Figure 2 ‎ .7 Aspergillus flavus colony surface ............................................................... 23 Figure 2 ‎ .8 Fusarium solani colony surface .................................................................. 24 Figure 3 ‎ .1 Flow chart of experimental process ............................................................ 28 Figure 3 ‎ .2 Model system used in extraction citrus essential oils in vacuum distillation process. ................................................................................................................ 32 Figure 4 ‎ .1 Effect of extraction time on yield (%) of essential oil ................................... 38 Figure 4 ‎ .2 Extraction yield of citrus peel extracts using different extracting methods. ..... 39 Figure 4 ‎ .3 IC50 values of investigated citrus oils by DPPH method. ............................... 47 Figure 4 ‎ .4 IC50 values of investigated citrus oils by FTC method. ................................. 49 vi LIST OF TABLES Table 2 ‎ .1: Scientific classification ............................................................................. 11 Table 2 ‎ .2: The main volatile components (%w/w) of several citrus essential oils ............ 12 Table 3 ‎ .1 Citrus samples in the study........................................................................ 29 Table 4 ‎ .1 Volatile compositions (%w/w) of Tan Trieu peel EOs extracted at 70 oC and 80oC by vacuum distillation method and cold pressing method. ............................................ 37 Table 4 ‎ .2 Volatile compositions (%w/w) of Vietnamese lime peel EOs extracted by cold pressing and vacuum distillation method ................................................................... 42 Table 4 ‎ .3 Volatile compositions (%w/w) of Vietnamese orange peel EOs extracted by cold pressing and vacuum distillation method ................................................................... 44 Table 4 ‎ .4 Volatile compositions (%w/w) of Vietnamese pomelo peel EOs extracted by cold pressing and vacuum distillation method ................................................................... 46 Table 4 ‎ .5 Zone of inhibition (mm) of citrus essential oils against S. aureus .................... 51 Table 4 ‎ .6 MIC values (mg/ml) of citrus essential oils for S. aureus ............................... 53 Table 4 ‎ .7 Zone of inhibition (mm) of citrus essential oils against B. cereus .................... 54 Table 4 ‎ .8 MIC values (mg/ml) of citrus essential oils for B. cereus ................................ 55 Table 4 ‎ .9 Zone of inhibition (mm) of citrus essential oils against S. typhi ...................... 56 Table 4 ‎ .10 MIC values (mg/ml) of citrus essential oils for S. typhi ................................ 57 Table 4 ‎ .11 Zone of inhibition (mm) of citrus essential oils against P. aeruginosa ............ 58 Table 4 ‎ .12 MIC values (mg/ml) of citrus essential oils for P. aeruginosa ........................ 59 Table 4 ‎ .13 Zone of inhibition (mm) of citrus essential oils against A.flavus .................... 60 Table 4 ‎ .14 MIC values (mg/ml) of citrus essential oils for A. flavus ............................... 61 Table 4 ‎ .15 Zone of inhibition (mm) of citrus essential oils against F.solani ..................... 62 Table 4 ‎ .16 MIC values (mg/ml) of citrus essential oils for F. solani ............................... 63 vii ABBREVIATION LLA-CP: Long An lime essential oil extracted by cold pressing method LLA-VA: Long An lime essential oil extracted by vacuum distillation method LDL-CP: Da Lat lime essential oil extracted by cold pressing method LDL-VA: Da Lat lime essential oil extracted by vacuum distillation method LD-CP: Dao lime essential oil extracted by cold pressing method LD-VA: Dao lime essential oil extracted by vacuum distillation method OX-CP: Xoan orange essential oil extracted by cold pressing method OX-VA: Xoan orange essential oil extracted by vacuum distillation method OPT-CP: Phu Tho orange essential oil extracted by cold pressing method OPT-VA: Phu Tho orange essential oil extracted by vacuum distillation method OV-CP: Vinh orange essential oil extracted by cold pressing method OV-VA: Vinh orange essential oil extracted by vacuum distillation method PTT-CP: Tan Trieu pomelo essential oil extracted by cold pressing method PTT-VA: Tan Trieu pomelo essential oil extracted by vacuum distillation method PTTR-CP: Thanh Tra pomelo essential oil extracted by cold pressing method PTTR-VA: Thanh Tra pomelo essential oil extracted by vacuum distillation method PDH-CP: Doan Hung pomelo essential oil extracted by cold pressing method PDH-VA: Doan Hung pomelo essential oil extracted by vacuum distillation method EOs: Essential oils CP: Cold pressing VA: Vacuum distillation TSB: Tryptone Soybean Broth TSA: Tryptone Soybean Agar PDA: Potato Dextrose Agar CFU: Colony forming unit MIC: Minimum inhibition concentration S. aureus: Staphylococcus aureus B. cereus: Bacilus cereus S. typhi: Salmonella typhi P. aeruginosa: Pseudomonas aeruginosa A. flavus: Aspergillus flavus F. solani: Fusarium solani viii 1 Chapter 1: INTRODUCTION The use of synthetic agents with antimicrobial and antioxidant activity is the technique for extending the shelf-life of foods. However, over the past two decades, there is a great public concern about safety and side effects of synthetic agents in food preservation besides health implications. These agents are known to have toxic and carcinogenic effects on human and food systems. Therefore, recent studies interest in developing safer compounds based on natural sources, as alternatives, to prevent from the deterioration of foods (Ayoola et al., 2008). Plants and plant extracts are recognized as potential sources of natural compounds to improve the shelf life and the safety of food. Especially, essential oils extracted from citrus species are the best candidates to replace synthetic additives in food preservation. Having functional properties such as antimicrobial and antioxidant activities, citrus essential oils can lengthen the food shelf life and avoid health-related problems, off odors, unpleasant tastes or changes in color. Citrus is a common term and genus of flowering plants that belongs to the rue family, Rutaceae, originating and growing extensively in tropical and subtropical southern regions of Asia. Citrus oils which obtained from citrus fruits like oranges, lime, and pomelo called argument oils that are considered generally recognized as safe (GRAS) (Kabara, 1991). The flavedo of citrus fruits is the main section containing essential oils. Citrus essential oils are a mixture of volatile compound and mainly consisted of monoterpenes hydrocarbons (Baik et al., 2008). In addition, these oils comprise over a hundred other constituents that can be divided into two fractions: sequiterpene hydrocarbons and oxygenated compounds (Sana et al., 2009, Baik et al., 2008). On the account of the fact that the yield, chemical composition and biological properties of the essential oils are affected by geographical regions and extraction methods (Njoroge et al., 2006), that is the reason why people put a high attention in the part of selecting locations and extracting methods for expected quality. There are two common methods used to extract citrus essential oils. Citrus essential oils are usually extracted by cold pressing method. In this technique, the smell of cold pressed oil is natural, chemical composition is conservative. Nevertheless, the yield of essential oils extracted using this process is low (Fils, 2000). Also, it is important to note that the oils extracted by this method have a relatively short shelf-life (Lan-Phi et al., 2006). The other method for citrus essential oils extraction is hydro-distillation. It is a method which has wide acceptance for large scale production because it produces high yield of essential oils. However, the drawback of this process is that the hydrolysable compounds such as ester, as well as thermally labile components, may be decomposed during the distillation process (Houghton and Raman, 1998). Furthermore, some chemical changes are related to antimicrobial and antioxidant activities of essential oils (Chemat, 2010). 9 It is well known that essential oils from citrus species possess antimicrobial activity against both bacteria and fungi (Lanciotti et al., 2004). Dilution and diffusion methods are basic techniques for the assessment of both antibacterial and antifungal activities of essential oils. Diffusion method is recommended as a pre-screening method for a large number of essential oils, so as the most active ones may be selected for further analysis by means of more sophisticated methods. On the other hand, the aim of dilution method is to determine the lowest concentration of the assayed antimicrobial agent (minimal inhibitory concentration, MIC) that, under defined test conditions, inhibits the visible growth of the bacterium, fungi being investigated. Numerous researchers demonstrated that citrus essential oils affected on the elimination of food-borne pathogens (Sana et al., 2009) and were manufactured as meat and dairy products preservatives (Fernández-López et al., 2007). Chaisawadi (2005) reported that citrus oils including Citrus hystrix DC. and Citrus aurantifolia inhibited growth of Staphylococcus aureus and Salmonella typhi. Citrus essential oils could represent as good candidates to improve the shelf life and the safety of minimally processed fruits (Lanciotti et al., 2004), skim milk and low-fat milk (Dabbah et al., 1970). The other functional property of citrus essential oils is antioxidant activity. Antioxidant activity is action against linoleic acid oxidation and 2,2-diphenyl-1- picrylhydrazyl radical scavenging. Some recent publications showed antioxidant activities of these essential oils (Baik et al., 2008). Other study observed the effects of essential oils of Citrus obovoides and Citrus natsudadai on DPPH radical scavenging, superoxide anion radical scavenging and nitric oxide radical scavenging activity (Kim et al., 2008). In that study, Citrus obovoides and Citrus natsudadai exhibited only superoxide anion radical scavenging activity. Malhotra (2009) also reported Citrus karna essential oils showed a significant inhibition for the oxidation of linoleic acid in the beta-carotene-linoleic acid system. Citrus essential oils bring a lot of benefits, many studies on citrus essential oils including extraction methods, chemical compositions and properties of essential oils from different varieties and different location have been done over the world. However, there is little information regarding the detailed evaluation of antioxidant and antimicrobial activities of citrus essential oils in Vietnam which is a country with a huge production of citrus fruits. In addition, two conventional methods for extraction possess disadvantages that affect on the yield and quality of citrus essential oils. Therefore, the objective of this study is to determine chemical composition, antimicrobial and antioxidant activities of Vietnamese citrus essential oils with emphasis on the possible future application of essential oils as alternative synthetic agents in food preservation. Additionally, vacuum distillation which is modified from hydro-distillation method to avoid altering volatile compounds and produce high yield is employed to extract the citrus essential oils. 10 2 2.1 Chapter 2: LITERATURE REVIEW Essential oils An essential oil is a concentrated hydrophobic liquid containing volatile aroma compounds from plants. It is less soluble in water. Essential oil is extracted from plant so it carries a distinctive scent, or essence, of the plant and is therefore applied in food flavoring and perfumery (Gunther, 1952). The occurrence of essential oils is restricted to over 2000 plant varieties from about 60 different families, however only about 100 varieties are the basis for the economically important production of essential oils in the world (Van de Braak and Leijten, 1999). The ability of plants to accumulate essential oils is quite high in both Gymnosperms and Angiosperms, although the most commercially important essential oil plant sources are related to the Angiosperms (Burt, 2004; Hussain et al., 2008; Anwar et al., 2009a). Essential oils are isolated from various parts of the plant, such as leaves (basil, patchouli, cedar), fruits (citrus) , bark (cinnamon), root (ginger), grass (citronella), gum (myrrh and balsam oils), berries (pimenta), seed (caraway), flowers (rose and jasmine), twigs (clove stem), wood (amyris), heartwood (cedar), and saw dust (cedar oil) (Dang et al., 2001; Burt, 2004; Sood et al., 2006; Cava et al., 2007; Hussain et al., 2008). 2.2 Citrus essential oils Citrus is a common term and genus of flowering plants that belongs to the rue family, Rutaceae, originating and growing extensively in tropical and subtropical southern regions of Asia including Vietnam. Table 2.1: Scientific classification (Dugo and Giacoma, 2002; Manner et al., 2006) Domain Eukarya Kingdom Plantae Class Magnoliopsida Family Rutaceae Subfamily Aurantioideae Genus Citrus L. The plants belong to the genus Citrus are eukaryotic organisms. The cells have a true nucleus, possess membrane-bond organelles, and the genetic material is DNA. In addition, they are multicellular and have cell walls made of cellulose, and participate in photosynthesis via chloroplasts. Moreover, they are flowering plant that uses a fruit body to protect its seeds and show characteristics of being a Dicotyledon such as secondary growth, non-parallel veins, and the presence of two cotyledons in their seeds (Dugo and Giacoma, 2002). The orders consist of woody trees, shrubs, and have strong scents. They 11 are generally edible and good sources of vitamin C. Hence, these organisms are characterized in Rutaceae family, Citrus genus (Dugo and Giacoma, 2002). In Vietnam, citrus fruits are grown in seven ecological regions, including three major subregions in the north of Vietnam (the mountainous midland region, the Red River Delta, and the northern central coast), in total accounting for 35-40% of citrus production in Vietnam. The Mekong River Delta in the south of Vietnam accounting for 55-60% of citrus production, and the rest concentrated in the central provinces (Agro, 2006). The citrusgrowing areas have increased year by year and citrus fruit production reached 700,000 tons in 2011 (Agro, 2011). The harvesting season that gives the highest production is normally September to December. The flavedo of citrus fruits is the main section containing essential oils. The main volatile compounds presenting in citrus essential oils are α-pinene, β-pinene, myrcene, limonene, citral, linalool, α-terpineol. The most abundant compound is limonene. Table 2.2 shows the main volatile component of citrus essential oils from several countries. Table 2.2: The main volatile components (%w/w) of several citrus essential oils No Compound Vietnamese Vietnamese Vietnamese India orange mandarin pomelo orange (Citrus sinensis) (Citrus reticulata (Citrus grandis (Citrus sinesis (Ref.71) Blanco) Osbeck) (L.) Osbeck) (Ref.71) (Ref.71) (Ref.105) 1. α-pinene 0.81 0.93 1.69 0.9 2. β-pinene 0.05 0.60 0.76 0.6 3. Myrcene 2.81 2.79 1.97 4.1 90.42 91.58 70.46 84.2 5. Citral 0.57 0.05 0.32 0.5 6. Linalool 0.73 0.24 0.12 4.4 7. α-terpineol 0.26 0.41 0.57 1.3 4. Limonene Ref.71: Lan-Phi et al., 2010. Ref.105: Sharma and Tripathi, 2008. The main abundant compounds presenting in orange (Citrus sinesis) oil cultivated in Vietnam was limonene (90.42%), followed by myrcene (2.81%) and α-pinene (0.81%). Linalool and α-terpineol are alcohols that have been reported to be the most important to orange flavor. The amount of linalool in the orange essential oil was 0.73% and α-terpineol was detected at the level of 0.26%. This compound, however, is a product of acidic and microbial degradation of limonene and also a contributor to off-flavor in stored orange juice (Shaw, 1979). 12 The major components found in mandarin (Citrus reticulata Blanco) essential oil were limonene (91.58%), followed by myrcene (2.79%), and α-pinene (0.93%). β-pinene was important to mandarin aroma and flavor. β-pinene presented at the level of 0.60% in this oil. Terpene compounds are the most reasons lead to antimicrobial activities of citrus essential oils. These components pass the cell membranes, penetrates into the interior of the cell and interact with critical intracellular sites (Cristani et al., 2007). The amount of linalool and α-terpineol was detected at low level of 0.24% and 0.41%, respectively. In case of pomelo (Citrus grandis Osbeck) oil, limonene was the most abundant compound, accounting for 70.46%. The other prominent compounds were myrcene (1.97%), α-pinene (1.69%) and β-pinene (0.76%). Although the proportion of α-terpineol in this oil remained at higher level than orange and mandarin oils, the amount of linalool was low (only 0.12%). Linalool, in previous studies, plays an important role in inhibition ability of peroxidation and caused essential oils possessing antioxidant activity (Hussain et al., 2008). For the volatile components of Indian Citrus sinesis (L.) Osbeck essential oil, limonene as the most abundant compound was identified at 84.2%, followed by linalool (4.4%), myrcene (4.1%) and α-terpineol (1.3%). β-pinene, α-pinene and citral remained at low levels of 0.9%, 0.6% and 0.5%, respectively. In comparison with the Vietnamese citrus essential oils, linalool represented higher concentration (up to 4.4%), whereas the percentage of this compound in Vietnamese oils only accounted for small amount (lower than 1%). Linalool, citral were compounds appreciated causing the antifungal capacities of citrus essential oils (Alma et al., 2004). Citral can form a charge transfer complex with an electron donor to fungal cells, which results in fungal death (Kurita et al., 1981). These results indicate that the different varieties and different growing location affect chemical composition of citrus essential oils resulting in different functional properties of these oils. 2.3 Extraction methods 2.3.1 Hydro-distillation method Hydro-distillation involves the use of water or steam to recover volatile principles from plant materials. The fundamental feature of hydro-distillation is that it enables a compound or mixture of compounds to be distilled and subsequently below that of the boiling point of the individual constituent. There have been three types of distillation: water distillation, water/steam distillation, and steam distillation. In water distillation, plant is soaked in a large chamber filled with water. Subsequently, the chamber is heated and essential oil is released from the plant by evaporation. The resulting steam from boiling water carries volatile oils with it and travels to a condenser, where the steam is cooled and is eventually turned back into water. 13 Eventually, the essential oil is equally returned to its former state and separated from water. In water/steam method, the plant material is placed on a grill above the hot water and steam passes through the plant material. The material must be carefully distributed on the grill to allow for steaming and extraction. In steam distillation, no water is placed inside the distillation tank. Instead, steam is directed into tank from an outside source. The essential oils are released from the plant material when the steam bursts the sacs containing the oil molecules. From this stage, the process of condensation and separation is standard (Chemat, 2010). This method has some important drawbacks. The elevated temperatures can cause modifications of the essential oil components and often a loss of the most volatile molecules (Chemat, 2010). In consequence, scent and quality of essential oils are deteriorated. However, hydro-distillation is one of the simplest methods for obtaining oils from plants. With high yield extraction and low cost requirement, it is also the most widely acceptable process for large scale of essential oils production. In present time, improving the hydro-distillation method has been conducted to produce essential oils with high quality. However, there is no public research that reports the new method producing high quality and high yield of essential oils, replacing for hydro-distillation method in large-scale production. Most of studies on extraction methods are only available in laboratory design. In addition, although the activities of the essential oils extracted by hydro-distillation are lower than natural oils, their quality is still ranged within acceptable limit. Therefore, this technique is used for essential oils production in industries. 2.3.2 Solvent extraction Another method is solvent extraction used to extract essential oils from delicate flowers and plant material which would be altered or damaged by hydro-distillation. First of all, the plant material is gradually mixed with a hydrocarbon solvent such as hexane, petroleum ether, benzene, toluene, ethanol, isopropanol, ethyl, acetone, etc. The solvent dissolves the plants constituents including essential oils, fatty acids and waxes. After the solvent is distilled off the remaining constituents make up the concrete. In addition, alcohol is used to extract the essential oil from the other constituents. Therefore, the fatty acids and waxes that are not alcohol soluble are left behind. Eventually, the alcohol is evaporated, leaving the absolute oil behind for harvesting (Rydberg et al., 2004). The solvent extraction is a simple method and does not require complex equipments. However, it possesses several disadvantages. The alcoholysis and evaporation may happen and could affect the quality and stability of oils. The important process of this method is the solvents eliminations from extracts, because of their harmfulness. 14 2.3.3 Supercritical carbon dioxide method When a gas is compressed to a sufficiently high pressure, it becomes liquid. If the gas is heated to a specific temperature, at the specific pressure, the hot gas will become supercritical fluid. This temperature is called the critical temperature and the corresponding vapor pressure is called the critical pressure. The values of the temperature and pressure are defined as critical point which is unique to a given substance. These states of the substances are called supercritical fluid when both the temperature and pressure exceed the critical point values. This fluid possesses both gas and liquid properties. It is suitable for extraction because of its characteristics such as favorable diffusivity, viscosity, surface tension and other physical properties. The diffuseness facilitates rapid mass transfer and faster completion of extraction than conventional liquid solvents. The low viscosity and surface tension enable it to easily penetrate the botanical materials from which the active components are extracted. The gas-like characteristics of supercritical fluid provide ideal conditions for extraction of solutes giving a high degree of recovery in a short period of time. Carbon dioxide is in its supercritical fluid state when both the temperature and pressure equal or exceed the critical point of 31°C and 73 atm. In its supercritical state, carbon dioxide has both gas-like and liquid-like qualities so it can fill any size of container, like a gas, and dissolve materials like a liquid. Carbon dioxide is prominent in comparison with other supercritical fluids because its critical temperature is remarkably low at only 31.1°C, so high temperatures are not necessary. This means supercritical carbon dioxide can be used as a solvent for materials that would be decomposed at higher temperatures. Hence, the essential oils produced from this method possess good quality. However, this method requires high-cost system and technical skills to perform. 2.3.4 Cold pressing method The cold pressing uses pressure to physically squeeze the oil from the plant tissue. It is used to obtain citrus fruit oils such as pomelo, mandarin, and orange oils. This is a simple method that uses machines that apply a centrifugal force for the purpose of separation of essential oil from other substances. In consequence, the essential oil is collected (Sawamura, 2010). The technique is a purely mechanical process while the hydro-distillation use steam from boiling water for carrying and extracting volatile oils. In comparison with hydro-distillation method, cold pressed extraction is carried out without applying heat to avoid the loss, chemical changes in the constituents, and formation of artifacts during hydro-distillation process of essential oil extraction. As a result, cold pressing method produces the essential 15 oil with native quality. On the other hand, this method produces low yield. Also, it is important to note that oil extracted using this method have a relatively short shelf life. There are reports in literature on the significance of extraction methods. Charles and Simon (1990) approved the hydro-distillation is a simpler and more rapid method for oil isolation. In comparison between hydro-distillation and ethyl acetate extraction, the study proved that the extraction yields of hydro-distillated oils and ethyl acetate extracts from fresh peels of citrus spp. widely varied depending on citrus cultivars. For each cultivar, the production yields of the hydro-distillated oils were much lower than that from extraction with ethyl acetate. In addition, several authors have compared the composition of essential oil obtained by hydro-distillation and the product obtained by super critical fluid extraction. They found that hydro-distillated oil contained higher percentages of terpene hydrocarbons. In contrast, the super critical extracted oil contained a higher percentage of oxygen compounds (Reverchon, 1997; Donelian et al., 2009). Khajeh et al. (2004) reported variation in the chemical composition of Carum copticum essential oil isolated by hydro-distillation and supercritical fluid extraction methods. 2.4 Gas chromatography Chromatography is the general name for separation technology whereby components in a mixture are separated through continuous repetition of concentration equilibration. When a gas is used as the mobile phase the technology is called gas chromatography (GC). The sample mixture is injected and instantaneously vaporized at the column inlet. The vaporized sample is then carried through the column by the carrier gas. While passing through the column, each component in the sample is adsorbed or is partitioned to the stationary phase according to its characteristic concentration ratio. As a result, the level of adsorption or partition for each component causes differences in the rate of movement for each component within the column. The components therefore elute separately from the column outlet (Sawamura, 2010). Figure 2.1 Diagram of Gas chromatography (Source: http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/gaschrm.htm) 16 The rate at which a compound travels through a particular GC system depends on the factors listed below:  Volatility of compound: Low boiling (volatile) components travel faster through the column than high boiling point components.  Polarity of compounds: Polar compounds move more slowly, especially if the column is polar.  Column temperature: Raising the column temperature speeds up all the compounds in a mixture.  Column packing polarity: Usually, all compounds move slower on polar columns, but polar compounds will show a larger effect.  Flow rate of the gas: Through the column, speeding up the carrier gas flow increases the speed with which all compounds move through the column.  Length of the column: The longer the column, the longer it will take all compounds to elute. Longer columns are employed to obtain better separation Figure 2.2 Principle of Gas Chromatography Gas chromatography–mass spectrometry (GC-MS) is another method based on the principle of GC. However, it combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. The gas chromatograph utilizes a capillary column which depends on the column's dimensions (length, diameter, film thickness) as well as the phase properties. The difference in the chemical properties between different molecules in a mixture will separate the molecules as the sample travels the length of the column. The molecules are retained by the column and then elute from the column at different times, and this allows the mass spectrometer downstream to capture, ionize, accelerate, deflect, and detect the ionized molecules separately. Today, GC and GC-MS are common instruments in most laboratories because they are sensitive, accurate, and convenient. 17 There is plenty of literature on determination of chemical composition of essential oils using gas chromatography (GC). Column and detection are two important elements in GC system. Capillary column with flame ionization detection (FID), are, in most cases, the method of choice for quantitative determinations because they enable more complex mixtures to be separated and resolved. Fisher and Phillips (2006) used GC system with capillary column and FID detector to analyze the composition of lemon, orange and bergamot essential oils. The results indicated three components, in which limonene was more abundant than citral or linalool in the oils tested. Also, Wungstintaweekul et al. (2010) selected capillary column and FID for the GC system to identify components of Citrus hystrix oil. Many researchers make use of mass spectrometers (MS), coupled with GC, to determine the identities of components. In a study, 67 components were identified in Citrus hystrix oils through GC-MS (Kirbaslar et al., 2009). Moreover, GC-MS analysis of Citrus sinensis (L.) Osbeck peel oil led to identification of 10 components (Sharma and Tripathi, 2008). Most of studies chose the capillary column with over 50 m in length to obtain better separation. Sokovic et al. (2007) analyzed 88 compounds included in Citrus limon and Citrus aurantium essential oils through GC-MS with capillary column (50m x 0.2mm i.d, 0.5 µm film thickness). Capillary columns selected, in most cases, are HP-5ms, DB-5 (cross-linked 5% diphenyl/95% dimethyl siloxane) or DB-1, also known as SE-30, (polydimethyl siloxane) stationary phases. These more non-polar stationary phases are often complimented by the use of a more polar stationary phase, such as polyethylene glycol (Cavaleiro et al., 2004). The composition of Citrus Turkish peel oils was analyzed by GC with DB-5 column (60m x 0.25mm i.d, 0.25µm film thickness) (Kirbaslar et al., 2009). 2.5 Food poisoned microorganisms 2.5.1 Staphylococcus aureus Staphylococcus aureus is facultative anaerobic gram-positive cocci which occur singly, in pairs, and irregular clusters. Typical colonies of S. aureus are usually large (6-8 mm in diameter), yellow to golden yellow in color, smooth, entire, slightly raised, often with hemolysis, when grown on blood agar plates. S. aureus is nonmotile, non-spore forming. The cell wall contains peptidoglycan and teichoic acid. The organisms are resistant to temperatures as high as 50°C, to high salt concentrations, and to drying. S. aureus usually affects on foods requiring hand preparation, such as potato salad, ham salad and sandwich spreads. It is frequently found as part of the normal skin flora on the skin and nasal passages. In normal, the bacteria do not cause disease. However, breached skin or other injury may allow the bacteria to overcome the natural protective mechanisms of the body, leading to infection such as pimples, impetigo, boils (furuncles), carbuncles, scalded skin syndrome and abscesses, bacteremia and septicemia. In infants, S. aureus 18 infection can cause a severe disease - staphylococcal scalded skin syndrome (SSSS) (John et al., 1980) In some researches, S. aureus was found to be the most susceptible bacterium for several essential oils. Upadhyay et al. (2010) reported that Citrus lemon and Azadirachta indica essential oils where inhibition zone of 23.10 mm and 23.23 mm, respectively, was recorded. Growth of this microorganism was completely inhibited by all of the citrus oils with 100% of reduction of inoculums (Dabbah et al., 1970). In addition, Turkish citrus peel oils also showed strong antimicrobial activity against S. aureus (Kirbaslar et al., 2009). Figure 2.3 Scanning electron micrograph of S. aureus. (Source: http://en.wikipedia.org/wiki/Staphylococcus_aureus) 2.5.2 Bacillus cereus Bacillus cereus is a Gram-positive, catalase, beta hemolytic bacterium that can be frequently isolated from soil and some food. B. cereus is aerobe, rod-shaped, and has the ability to form protective endospore, allowing the organism to tolerate extreme environmental conditions. Thus, B. cereus is more resistant to heat and chemical treatments than vegetative pathogens such as Salmonella, E. coli, Campylobacter, and Listeria monocytogenes (Jesen et al., 2003). Spores of B. cereus can be found widely in nature, including samples of dust, dirt, cereal crops, water, etc. Starchy foods such as rice, macaroni and potato dishes are the best environment for development of B. cereus. Bacillus food borne illnesses occur due to survival of the bacterial endospores when food is improperly cooked. Cooking temperatures less than or equal to 100 °C (212 °F) allows some B. cereus spores to survive (McKillip, 2000). Bacterial growth results in production of enterotoxins, one of which is highly resistant to heat and to pH between 2 and 11; ingestion leads to two types of illness: one type characterized by diarrhea and the other, called emetic toxin, by nausea and vomiting. Several reports studied on antibacterial activities of essential oils on B. cereus. Citrus lemon and Azadirachta indica essential oils showed high inhibition on this bacterium with 41.3mm and 45.63mm inhibition zone, respectively (Upadhyay et al., 2010). According to 19 the result of the other report, all of the citrus peel oils were more effective towards B. cereus (Kirbaslar et al., 2009). Chanthaphon et al. (2008) demonstrated that the extract from lime peel (Citrus aurantifolia Swingle) showed broad spectrum inhibitory against all Gram positive bacteria including B.cereus. The lime extract exhibited MIC value against B. cereus at 0.56 mg/ml. This result was correlated to the report of Chaisawadi et al. (2005) which cited Citrus aurantifolia displaying antibacterial activities on this microorganism. Figure 2.4 Rod-shaped Bacillus cereus. (Source: Courtesy of Frederick C. Michel, ASM MicrobeLibrary) 2.5.3 Salmonella typhi Salmonella typhi is gram-negative, motile, facultatively anaerobic bacterium, made up of nonspore-forming rods (Pelczar et al., 1993). It has a complex regulatory system, which mediates its response to the changes of external environment. In order to survive in the intestinal organs of its hosts where there are low levels of oxygen, Salmonella typhi has to be able to learn to use other sources other than oxygen as an electron acceptor. The electron acceptor of this strain is nitrogen such as nitrate, nitrite, fumarate, and dimethlysulphoxide. It is a food born pathogen and the most common source of infection is high protein foods such as meat, poultry, fish and eggs. S. typhi causes systemic infections, typhoid fever in humans (Doughari et al., 2007). It usually invades the surface of the intestine in humans, but have developed and adapted to grow into the deeper tissues of the spleen, liver, and the bone marrow. It is also able to inhibit the oxidative burst of leukocytes, making innate immune response ineffective. Symptoms most characterized by this disease often include a sudden onset of a high fever, a headache, and nausea. Other common symptoms include loss of appetite, diarrhea, and enlargement of the spleen (depending on where it is located) (Shah, 2012). The encounter of humans to S. typhi is made via fecal-oral route from infected individuals to healthy ones. Poor hygiene of patients shedding the organism can lead to secondary infection, as well as consumption of shellfish from polluted bodies of water. 20 There are many reports in literature regarding the antimicrobial activity of essential oils on S. typhi. Among the various essential oil treatments, the Citrus reticulata var. Tangarin exhibited the highest antibacterial activity on S. typhi (Ashok et al., 2011). Suganya et al. (2012) cited that the essential oil of Coriandrum sativam has the highest antibacterial activity against this bacterium with inhibition zone of 15mm in diameter. The essential oils distilled from Syzygium neesianum Arn, Elaeocarpus lanceifolius and Citrus sinesis also showed a significant inhibition on S. typhi (Maridass, 2010; Ashok et al., 2011). Figure 2.5 Flagella stain of Salmonella typhi. (approx. 1000 X) (Source: the Wistreich Collection, appearing exclusively on MicrobeWorld). 2.5.4 Pseudomonas aeruginosa Pseudomonas aeruginosa is a Gram-negative rod measuring 1-5 µm long and 0.5-1.0 µm wide. Almost all strains are motile by means of a single polar flagellum (Ryan and Ray, 2004). P. aeruginosa is an obligate respirer. It grows in the absence of oxygen and use nitrate as a respiratory electron acceptor. The pathogen is widespread in nature, inhabiting soil, water, plants, and animals (including humans). It is a common bacterium that can cause disease in animals, including humans (Iglewski, 1996). Vegetables, meats, milk and water are suitable environments for the infection of P. aeruginosa. Once infecting to human body, P. aeruginosa causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections and a variety of systemic infections, particularly in patients with burn and in cancer and AIDS patients who are immune-suppressed. P. aeruginosa infection is a serious problem in patients hospitalized with cancer, cystic fibrosis, and burns (Ryan and Ray, 2004). A number of publications demonstrated that various essential oils possessing antibacterial activities on P. aeruginosa. This microorganism was inhibited by the lemongrass (Cymbopogon citratus) oil with MIC value at 1% (v/v) whereas the lime (Citrus aurantifolia) oil showed the lower effect with MIC value at 2% (v/v) (Hammer et al., 1999). The essential oils Ammy visnaga L. exhibited strong inhibition effect of P. aeruginosa with 25 mm inhibition zone diameter (Khalfallah et al., 2011) 21 Figure 2.6 Pseudomonas aeruginosa (Source: http://fuckyeahmedicine.tumblr.com/post/455450061/pseudomonas-aeruginosainfections) 2.5.5 Aspergillus flavus Aspergillus flavus is a plant, animal, and human fungal pathogen. It grows by producing thread like branching filaments known as hyphae. Filamentous fungi such as A. flavus are sometimes called molds. Conidia are globose to subglobose (3-6 um in diameter), pale green and conspicuously echinulate. When young, the conidia of A. flavus appear yellow green in color. As the fungus ages, the spores turn a darker green. Conidial heads are typically radiate, mostly 300-400 µm in diameter, later splitting to form loose columns (Amaike and Keller, 2011). A network of its hyphae known as the mycelium secretes enzymes that break down complex food sources. Aspergillus flavus has a world-wide distribution and normally occurs in soil and on many kinds of decaying organic matter. The fungus infects seeds of corn, peanuts, cotton, and nut trees. It produces significant quantities of toxic compounds known as mycotoxins, commonly aflatoxin which is a toxic and carcinogenic compound. Aflatoxin is also the second leading cause of aspergillosis in humans (Agrios and George, 2005). Patients infected with A. flavus often have reduced or compromised immune systems (Amaike et al., 2011). Many plant oils showed inhibition abilities on A. flavus. The essential oils of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), orange (Citrus sinesis L.) and grapefruit (Citrus paradisi L.) possessed the capacity to reduce or inhibit the growth of the mold A. flavus. The total inhibition of growth was obtained with all citrus essential oils at the concentration of 0.94% (Martos et al., 2008). There was a highly marked inhibitory effect of all treatments EOs including marjoram, mint, basil, coriander, thyme, dill and rosemary on A. flavus strain growth. The highest growth inhibition rate of A. flavus was observed with the thyme Eos (Habib, 2012). 22 Figure 2.7 Aspergillus flavus colony surface (Source: William McDonald, 2011) 2.5.6 Fusarium solani Fusarium solani is a pathogenic fungus and is an important causal agent of several crop diseases, such as root and stem rot of pea, sudden death syndrome of soybean, foot rot of bean and dry rot of potato. It produces asexual spores which can be spread by air, equipment, and water (Cho et al., 2001). Colonies growing rapidly, 4.5 cm in 4 days, aerial mycelium white to cream, becoming bluish-brown when sporodochia are present. When young, the conidia of F. solani appear white to cream in color. Colonies grow rapidly, 4.5 cm in 4 days. As the fungus ages, the spores become a bluish-brown. The predominant hosts for Fusarium solani are potato, pea, bean, and members of the cucurbit family such as melon, cucumber, and pumpkin. It produces trichothecene mycotoxin that inhibits DNA and protein synthesis (Ueno, 1989; Thompson and Wannemacher, 1990). This mycotoxin also causes impairment of ribosome function and immune-suppression, allowing secondary and opportunistic bacterial infections and possibly delayed hypersensitivity (Ueno, 1989). There are numerous studies demonstrated that the the growth of F. solani and its potential pathogenic activity can be controlled. Origanum vulgare EOs showed strong antifungal activity against this mold (Laubach et al., 2012). Aziz et al. (2010) cited that the essential oil of Thymus serpyllum L. grown in the State of Jammu and Kashmir showed significant antifungal activity against Fusarium solani and moderate phytotoxic activity. Fungicidal activity against F. solani was observed at 5% concentration for essential oils from Pinus resinosa and Pinus strobus (Krauze-Baranowska et al., 2002) 23 Figure 2.8 Fusarium solani colony surface (Source: http://gahru-on.blogspot.com/) 2.6 Antimicrobial activity of essential oils Essential oils and other naturally occurring antimicrobials are attractive to the food industry for the following reasons: 1. It is highly unlikely that new synthetic compounds will be approved for use as food antimicrobials due to the expense of toxicological testing (Burt, 2004). 2. There exists a significant need for expanded antimicrobial activity both in terms of spectrum of activity and of broad food applications (Feng and Zheng, 2007). 3. Food processors are interested in producing “green” labels, i.e., ones without chemical names that apparently confuse consumers (Burt, 2004). 4. There are potential health benefits that come with the consumption of some naturally occurring antimicrobials (Ayoola et al., 2008). Recently, essential oils and extracts of certain plants have been shown to have antimicrobial effects, without effects on flavor of foods (Burt, 2004). Some citrus essential oils have shown promise as potential food safety interventions when added to minimally processed fruits (Lanciotti et al., 2003). There are many reports regarding the antimicrobial activity of essential oils (Kofidis et al., 2004; Singh et al., 2005). Martos et al. (2007) evaluated the antibacterial functions of the citrus essential oils from four varieties of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.), and orange (Citrus sinesis L.) and found that all of these essential oils displayed strong antibacterial activity against the strains tested. Testing and evaluation of antimicrobial activity of essential oils is difficult because of their characteristics such as volatility, water insolubility and complexity. Essential oils are hydrophobic and high viscosity compound. These properties may reduce the dilution ability 24 or cause unequal distribution of the oil in the medium even if a proper solvent agent is used. It has to be checked whether the applied concentrations of the emulsifier or solvent do not affect the growth and differentiation of tested microorganisms. Moreover, essential oils are very complex mixtures of volatile components. Consequently, long incubation periods may result in the evaporation or decomposition of some of the components during the testing period (Kalemba and Kunicka, 2003). For the antimicrobial assessment of essential oils, conventional methods of testing antibiotic abilities are usually applied. There are two basic techniques for the assessment of both antibacterial and antifungal activities of essential oils (Kalemba and Kunicka, 2003):  The agar diffusion method (paper disc or well): The diffusion method is the most widespread technique of antimicrobial activity assessment. According to this method, Petri dishes of 5-12 cm diameter (usually 9 cm) are filled with 10-20 ml of agar and inoculated with microorganisms. Two ways of essential oil incorporation are possible: on a paper disc (Simic et al., 2000; Omer et al., 1998) or into the well (hole) made in the agar medium. When a filter paper disc impregnated with essential oil is placed on agar or essential oil is added into the hole, the essential oil will diffuse from the disc or well into the agar. This diffusion will place the essential oil in the agar only around the disc or well. The solubility of essential oil and its molecular size will determine the size of the area of infiltration around the disc or well. If an organism is placed on the agar, it will not grow in the area around the disc or well because it is susceptible to the essential oil. This area of no growth around the disc or well is known as a “zone of inhibition” (Kalemba and Kunicka, 2003). This method is mostly used as a screening method when large numbers of essential oils and/or large numbers of bacterial isolates are to be screened (Deans et al., 1990; Dorman and Deans, 2000). In literature, both disc diffusion (Renzini et al., 1999; Senatore et al., 2000) and well diffusion (Dorman and Deans, 2000; Ruberto et al., 2000) assays are reported to evaluate the antimicrobial activity of essential oils.  The dilution method (agar or liquid broth): The serial dilution agar method is used for bacteria and fungi, but its modification with liquid broth is mostly applied for fungi. The broth micro dilution assay has become quite popular lately (Shapiro et al., 1994). The aim of broth and agar dilution methods is to determine the lowest concentration of the assayed antimicrobial agent (minimal inhibitory concentration, MIC) that, under defined test conditions, inhibits the visible growth of the bacterium, fungi being investigated. MIC values are used to determine susceptibilities of bacteria, fungi to drugs and also to evaluate the activity of new 25 antimicrobial agents. Agar dilution involves the incorporation of different concentrations of the antimicrobial substance into a nutrient agar medium followed by the application of a standardized concentration of organisms to the surface of the agar plate. For broth dilution, studies a number of different procedures exist for determining the MIC and MBC. The most common methods are that of measurement of optical density and the enumeration of colonies by viable count (Skandamis et al., 2001; Ultee and Smid, 2001). MIC was often determined in 96well microtiter plate format, bacteria are inoculated into a liquid growth medium in the presence of different concentrations of an antimicrobial agent. Growth is assessed after incubation for a defined period of time and the MIC value is read. 2.7 Antioxidant activity of essential oil From a biological point of view, antioxidants have been defined as substances that when present in concentrations lower than the oxidation substrate are capable of delaying or inhibiting oxidative processes (Choi, 2010). To extend the shelf-life of foods, phenolic compounds, such as butylated hydroxyanisole (BHA) butylated hydroxyl toluence (BHT), have been widely used as synthetic antioxidants in the food industry (Choi, 2010). However, over the past two decades, there is great public concern about the safety of the synthetic antioxidants in food preservation besides health implications. Some opinions concerned the safety and side effects of synthetic antioxidants as food additives. These synthetic antioxidants are known to have toxic and carcinogenic effects on human and food systems. Especially, they may cause liver swelling and influence liver system activities and cerebro-vascular diseases (Choi et al., 2007). Therefore, recent studies interest in developing safer antioxidants based on natural sources, as alternatives, to prevent the deterioration of foods. Essential oils and extracts from botanical materials are known to have varying degrees of antioxidant activities (Descalzo and Sancho 2008). Recent publications showed antioxidant activities of essential oils and extracts which have been reported to be more effective than some synthetic antioxidants (Hussain et al., 2008; Bendini et al., 2002). DPPH radical scavenging assay is the most popular method used for the determination of antioxidant activity of essential oils and plant extracts. The presence of an odd electron in the DPPH free radical gives a strong absorption maximum at 517 nm. As this electron becomes paired off in the presence of a hydrogen donor, the absorption strength is decreased, and the resulting decolonization is stoichiometric with respect to the number of electrons captured (Choi, 2010). Due to its simplicity and sensitivity, some authors only use DPPH method for evaluating the antioxidant activities of essential oils. Another method is to evaluate the antioxidant capacity of essential oils based on the reduction of Fe3+ to Fe2+. This method is known as Ferric Thiocyanate (FTC) method. In 26 this method, linoleic acid is added into mixture. Then, oxygen reacts with linoleic acid, which is unsaturated lipids (LH), through free radical initiation, propagation and termination processes (Frankel, 1980). Initiation takes place by loss of a hydrogen radical in the presence of trace metals, light or heat. The resulting lipid free radicals (L-) react with oxygen to form peroxy radicals (LOO-). In this propagation process, LOO- reacts with more LH to form lipid hydroperoxides (LOOH), which are the fundamental primary products of autoxidation (Frankel, 1984). LH  L-+HL-+O2  LOOLOO- + LH  LOOH + LAntioxidants (AH) can break this chain reaction by reacting with LOO- to form stable radicals (A-) which are either not too reactive or form non-radical products (Frankel, 1984). LOO-+AH  A- +LOOA-+LOO-  Non radical products A-+A- Non radical products In reaction of oxygen with linoleic acid, lipid hydroperoxides are formed, then oxidize Fe2+ to Fe3+. The latter ions form a complex with thiocyanate, and this complex has a maximum absorbance at 500 nm. Therefore, high absorbance indicates high linoleic acid oxidation. As a literature review, essential oils are extracted by four methods hydro-distillation, solvent extraction, supercritical carbon dioxide and cold pressing method. Their compositions are usually analyzed by GC or GC-MS instruments because they are sensitive, accurate, and convenient. Diffusion and dilution are two basic techniques for the assessment of both antibacterial and antifungal activities of essential oils. There are many methods used to define the antioxidant capacity of essential oils. However, DPPH radical scavenging and FTC method are two common assays. In this study, two extraction methods, cold pressing and vacuum distillation methods, will be employed to extract essential oils from the Vietnamese citrus peel and DPPH radical scavenging and FTC method will be used to determine antioxidant capacity of these EOs as well as diffusion and dilution will be used to check antimicrobial activities of these EOs. 27 3 3.1 Chapter 3: Materials and methods Experimental design Material collection and preparation Extraction essential oils by cold pressing method Evaluation of antimicrobial activities Evaluation of antimicrobial activities by dilution method Evaluation of antimicrobial activities by diffusion method Extraction essential oils by vacuum distillation method Evaluation of antioxidant activities Evaluation of antioxidant activities by DPPH assay Evaluation of antioxidant activities by lipid peroxidation assay Figure 3.1 Flow chart of experimental process 3.2 Materials collection and preparation: The time from flowering and fruiting to harvesting, which citrus fruits are in the major stage, is 8 months. It is the best time to collect materials for the study because the citrus fruits produce the highest amount of essential oils with the highest quality at this age. Therefore, citrus fruits were collected at mature stage (after fruiting 8 months) from farms in the provinces from North, Center and South region of Vietnam from September, 2012 to December, 2012, which is the main season of citrus fruits in Vietnam. The fruits were in earlier-ripe stage. After collecting, the samples were transferred to International University laboratory by airplane in same day. Therefore, the quality of fruits was not affected. All samples are shown in Table 3.1. 28 Table 3.1 Citrus samples in the study Common Scientific name Place collection Citrus sinensis Osbeck Lap Vo District, Dong Thap Image of whole fruit Image of cross section name Xoan orange Province Vinh orange Citrus sinensis Osbeck Quy Hop District, Nghe An Province Phu Tho Citrus hybrid - Citrus orange reticulata x Citrus Viet Tri City, Phu Tho Province maxima Tan Trieu Citrus grandis Osbeck pomelo Thanh Tra Vinh Cuu District, Dong Nai Province Citrus grandis Osbeck Thuy Bieu Ward, Hue City pomelo 29 Doan Hung Citrus grandis Osbeck Pomelo Long An lime Da Lat lime Doan Hung District, Phu Tho Province Citrus aurantifolia Ben Luc District, Long An Swingle Province Citrus limonia Osbeck Da Lat City, Lam Dong Province Dao lime Citrus hybrid – Citrus Thai Binh City, Thai Binh spp. Province. 30 The specimens were further identified and authenticated by Southern horticultural research institute (SOFRI) Vietnam. Subsequently, samples were primarily treated by washing with tap water to remove the surface adherents. Fruits were then sliced into 6 8 equal portions. The fruits albedo layers are peeled off carefully and discarded while the flavedo were kept for extracting the essential oils. 3.3 Essential oils extraction 3.3.1 Cold pressing method Peel oil were extracted by hand pressing of the flavedo layer with exposed oil sacs and collected in brine solution (saturated concentration: 40%) kept on ice, according to the method reported by Lan-Phi et al. (2006). The extract was centrifuged at 4000g for 15 minutes. The resulting supernatant was dried in anhydrous sodium sulphate at 5oC for 24 hours and then filtered. The oils were stored at -21oC until analyzed. The yield of extracted essential oil was calculated base on following formula (Njoroge and Sawamura, 2010): Yield (%)= x 100 3.3.2 Vacuum distillation method In order to determine the best conditions for citrus essential oils extraction. The boiling temperature and extraction duration were investigated and Tan Trieu pomelo was used to extract its essential oil at the appropriate conditions. 3.3.2.1 Temperature appropriate Samples of Tan Trieu pomelo peels were grinded in a blender to obtain small size (0.5 cm in diameter) for being extracted by a modified Clevenger-type apparatus (Figure 3.2). The temperature of boiling flask was controlled by a thermostatic bath (Daihan Scientific Co., Ltd; Wise Circu WCB-22), which replaced the chauffe ballon in the conventional hydro-distillation system to provide more efficient heat transfer to the flask and avoid localized overheating. In addition, the maintenance of the system pressure was made by a vacuum pump (KNF Neuberger, N811 KN.18). By fixing time extraction time for 3 hours and pressure of system at 0.7bar, 200g of grinded materials and 400ml distilled water were then submitted to the apparatus at 60oC, 70oC, 80oC, and 90oC. Subsequently, the distillates of essential oils were separated from water. Chemical compositions of essential oils extracted at different temperature conditions were further analyzed using gas chromatography and compared to the cold 31 pressed oil. The temperature at which the chemical compositions of extracted essential oils were not significant different from cold pressed oils was selected. Figure 3.2 Model system used in extraction citrus essential oils in vacuum distillation process. 3.3.2.2 Time appropriate The extraction procedures were repeated as described at the best temperature condition optimized previously for varying extraction times from 2.5 hours to 4 hours. After extraction, the yields were determined. The time at which the extraction yield reached the highest level was selected. 3.3.2.3 Extraction process After determination of the best conditions, these conditions were applied for extraction of the essential oils of all citrus samples as follow. 200g of citrus peels were grinded in a blender to obtain small size. The grinded materials and 400ml distilled water were then submitted to the apparatus for the optimal time, under 0.7bar and the optimal temperature. The distillates of essential oils were separated from water and stored at -21oC prior to analysis. 3.4 Chemical composition analysis The analysis of the essential oils was performed by Network GC system Agilent Technologies 6890N equipped with flame ionization detector (250oC FID) and a DB-1 column (30m x 0.25mm i.d, film thickness of 0.25µm). The column temperature was initially maintained at 70oC for 2 min, and gradually increased at the rate 2oC per min to 32 240oC, at which the temperature was held for 20 minutes. Nitrogen was used as carrier gas at flow rate of 0.7 ml/min. 3.5 Antimicrobial activities of citrus essential oils 3.5.1 Microbial strains In order to determine antimicrobial activities of citrus essential oils, four bacteria and two fungi were used. These microorganism included two gram-positive bacteria: Staphylococcus aureus with source from Institute of Drug Quality Control – Ho Chi Minh City and Bacillus cereus (VTCC-B-1005) obtained from Vietnam National University Institute of Microbiology and Biotechnology, two gram-negative bacteria: Salmonella typhi and Pseudomonas aeruginosa obtained from Institute of Drug Quality Control – Ho Chi Minh City and two fungi Aspergillus flavus (VTCC-F-824) and Fusarium solani (VTCCF-827) were obtained from the Vietnam National University Institute of Microbiology and Biotechnology. The concentration of bacteria tested in experiment was 106 colony forming unit (CFU)/ml while that of fungi was 105 CFU/ml. 3.5.2 Microorganisms counting A loop of microorganisms from a stock culture was placed into a 10mL tube of sterilized broth medium, and incubated for 24 h at 37oC. Then, 1mL aliquot was transferred into a fresh sterile 9mL of Tryptone Soybean Broth (TSB) to form a 1/10 or 10-1 dilution. The 1ml of 10-1 dilution was shake vigorously and transferred to the second 9ml TSB. This second dilution represented a 10 -2 dilution of the original sample. To be continued, a wide series of dilutions was performed (from 10-1 to 10-8). Subsequently, 100µl aliquot of microorganism suspension of each dilution was spread on plates then incubated at 37oC for 24 hours. Only the plates which contained between 30 and 300 colonies were selected (Breed, 1916). The number of colonies from plates of selected dilutions was used to determine the number of microorganisms in original sample based on the following formula (Benson, 2002): ρ Where N 1 VS D N: Number of colonies on plate (Colony forming unit: CFU) V S: Volume pipetted onto Petri plate (ml) D: Dilution factor for test tube plated out : Concentration of cells in original sample (CFU/ml) 33 3.5.3 Diffusion technique The antimicrobial activity of the selected essential oils was determined by the disc diffusion method (NCCLS, 1997). Briefly, 100µl suspension of bacteria and fungi were spread on petri dishes containing Tryptone Soybean Agar (TSA, Himedia, India) and Potato Dextrose Agar (PDA, Titan, India) medium, respectively. The agar plates were prepared in 90 mm petri dishes with 22mL of agar medium giving a final depth of 3 mm. The wells (9 mm in diameter) were punched in the culture media with essential oils and 100µl of extracts diluted in absolute ethanol to obtain a concentration 50% were added into the wells (Armando et al., 2009). Wells without samples were used as controls. The plates were incubated at 37 ºC for 24 hours for bacteria and at 28 °C for 48 hours for fungal strains. Antimicrobial activity was assessed by measuring the diameter of the growth-inhibition zone in millimeters (including well diameter of 9 mm) for the test organisms comparing to the controls. 3.5.4 Dilution technique: For minimum inhibitory concentration (MIC), a broth dilution susceptibility assay was used (Takhi et al., 2011). A serial twofold dilution of essential oils in absolute ethanol was prepared to obtain concentration of essential oils: 42mg/ml, 21mg/ml, 10.5mg/ml, 5.25mg/ml, 2.63mg/ml, 1.31mg/ml, and 0.66mg/ml. 500µl of diluted essential oils were transferred to the test tubes. Subsequently, a fixed volume 4ml of liquid culture medium was distributed into the test tubes and inoculated with 500µl of bacterial or fungal suspension (106CFU/ml for bacteria and 105CFU/ml for fungi) (Takhi et al., 2011). The experiment included three controls: the negative control tube containing culture medium and essential oils only, the positive control tube containing culture medium and microorganisms, and the solvent control tube containing ethanol, medium and microorganism. During the incubation period, the tubes were submitted to a manual agitation every hour. The test tubes were incubated for 24 hours at 37°C for bacteria and 48 to 25°C for fungi. After incubation, 100µl from each tube was spread on agar medium and incubated for 24 hours to determine MIC. The lowest concentration demonstrating no apparent growth was recorded as MIC. 3.6 Antioxidant activities of citrus essential oils 3.6.1 DPPH assay The antioxidant activity of the essential oils was assessed by measuring their ability to scavenging 2, 2‫׳‬-diphenyl-1-picrylhydrazyl stable radicals (DPPH) based on the method of Ghasemi et al. (2010). Depend on the activities, the essential oils were diluted in absolute methanol to obtain different concentrations: 5, 10, 15, 20, 25, 30, 40, 50, 60, 34 70, 80, 100, 120 and 140 mg/ml. The mixtures of essential oils were added, at an equal volume, to methanolic solution of DPPH (100 µM). After 15 minutes at room temperature, the absorbance of blank and resulting solutions was recorded. The control contained methanol and DPPH solution. The disappearance of DPPH was read spectrophotometrically at 517 nm using a spectrophotometer. DPPH inhibition (%) by essential oil was calculated in following way (Ghasemi et al., 2010): Inhibition (%) = x 100 Where A control is the absorbance of the negative control and A sample is the absorbance of the test compound. The concentration of essential oils causing 50% inhibition (IC50) was calculated from the graph-plotted scavenging percentage against essential oils concentration. 3.6.2 Ferric thiocyanate (FTC) assay The inhibitory capacity of extracts was tested against oxidation of linoleic acid by FTC method as previously reported by Hoa et al. (2007) and Sadaf et al. (2009). Essential oils were initially diluted into different concentrations depending on the activities of essential oils at 5 , 10 , 15 , 20, 25 , 30 ,40 , 50 , 60 , 70, 80 , 100 , 120, 140, 150, 200, 250, and 300mg/mL in absolute ethanol. Each concentration was added to a solution mixture of 4ml 2.51% linoleic acid, 8mL absolute ethanol and 8mL of 0.2 M sodium phosphate buffer (pH 7). All were tightly capped and kept at 40oC in the dark for 48 hours. During incubation, for every 24 hours, 0.1mL of each prepared samples above were dissolved in 9.7 mL of 75% ethanol, 0.1mL of 30% ammonium thiocyanate, 0.1ml of 20mM ferrous chloride in 3.5% HCl. Precisely 3 minutes after the addition of ferrous chloride to the reaction mixture, the absorbance of red color was measured at 500 nm. The investigation continued for every 24 hours until the absorbance of the control reached its maximum (48 hours). A mixture without a plant extract is used as a negative control. Synthetic antioxidant, BHT was used as positive control. The degree of linoleic acid peroxidation was calculated using the following formula (Pitchaon, 2011): Antioxidant activity = × 100 Where A control is the absorbance of the negative control after 48 hours and A sample is the absorbance of the test compound after 48 hours. The antioxidant activity was plotted against sample concentration in order to determine the concentration required to achieve a 50% inhibition of linoleic acid oxidation [AA50] (Pitchaon, 2011). 35 3.7 Data analysis Each parameter was tested in triplicate. Microsoft excel software is used to calculated means and standard deviations. Analysis of variance (ANOVA) was applied to the data to determine differences (p < 0.05). Statistical data analysis was undertaken using the Statistical Package for the Social Sciences (SPSS). 36 4 4.1 Chapter 4: Results and Discussion Optimization conditions for vacuum distillation extraction 4.1.1 Temperature optimization Table 4.1 lists the volatile compounds detected from various extraction methods. Detection and quantification of nine compounds were determined by GC. In order to optimize heating temperature for the vacuum distillation, a range of temperature from 60oC to 90oC with 10oC interval was employed. As a result, the heating temperature for vacuum distillation could not be lower than 70 oC or higher than 80oC because of low efficiency of essential oil extraction obtained. The power of vacuum pump in this study could only provide vacuum pressure of 0.7bar. At 0.7 bar of vacuum pressure, the boiling temperature of materials was only reduced up to 70 oC. At 60oC and 0.7 bar, the mixture of citrus peels and distilled water was not boiled to produce steam for extraction of essential oils. In case of 90oC, this temperature was high and closed to 100oC. The composition of citrus essential oils was affect seriously and most of compounds were degraded. Therefore, the compositions of essential oils extracted at 60oC and 90oC were not shown in Table 4.1. Table 4.1 Volatile compositions (%w/w) of Tan Trieu peel EOs extracted at 70oC and 80oC by vacuum distillation method and cold pressing method. No. Compounds Relative concentration (%) Cold pressing Vacuum distillation o Vacuum distillation at 70 C at 80oC 1 α-pinene 1.32±0.01 1.08±0.04 1.28±0.00 2 Sabinene 0.20±0.00 0.18±0.00 0.19±0.00 3 β-pinene 0.97±0.00 0.82±0.01 --- 4 myrcene 2.16±0.01 1.82±0.01 1.84±0.00 5 α-terpinene 0.33±0.01 0.25±0.00 0.27±0.00 6 limonene 80.55±0.06 80.08±0.46 81.89±0.01 7 terpinolene 0.03±0.00 0.04±0.00 0.05±0.00 8 ٧-terpinene 10.90±0.01 10.82±0.04 12.29±0.01 9 linalool Tr Tr --- Compositions of the Tan Trieu pomelo oil extracted by cold pressing method and the vacuum distillation at 70oC were mostly similar. There were 9 components detected in the oils extracted by these two methods. The most abundant compound in Tan Trieu pomelo essential oil was limonene. No significant differences were observed in the 37 concentration of limonene identified in the oils obtained by the cold pressing method (80.55%) and the vacuum distillation at 70oC (80.08%). In addition, the concentration of sabinene, β-pinene, α-terpinene and ٧-terpinene were 0.20%, 0.97%, 0.33% and 10.90%, respectively in cold pressed oil. Similar results were found in oils extracted by vacuum distillation at 70oC with sabinene (0.18%), β-pinene (0.82%), α-terpinene (0.27%) and ٧-terpinene (10.82%). The heating temperature of the vacuum distillation affected the composition of the essential oil. Although 9 main compounds were identified in the cold pressed oil and distillated oil at 70oC, two of them (β-pinene and linalool) were not detected in the essential oil extracted by vacuum distillation at 80 oC. These components were identified to strongly affect antioxidant and antimicrobial activities of essential oils (Belletti et al, 2009; Dorman and Deans, 2000). Linalool, in previous studies, plays an important role in inhibition ability of peroxidation and caused essential oils possessing antioxidant activity (Hussain et al., 2008; Malhotra et al., 2009). The lost of β-pinene and linalool might be due to high heating temperature. High temperature caused the missing of βpinene, linalool and moderate the amount of other compounds. Moreover, the concentration of the identified compounds was also apparently different. The oil extracted by distillation at 80oC contained higher percentage of limonene (81.89%) than did oil obtained by cold pressing method (80.55%). However, the concentrations of component of cold pressed oil such as ٧-terpinene (10.90%) and myrcene (2.16%) were significant different compared to those of vacuum distillated oil. In general, chemical compositions of extracted sample at 70oC were close to chemical compositions of cold pressed sample. As a result, the optimal temperature for vacuum distillation of citrus essential oil was 70oC. 4.1.2 Time optimization Figure 4.1 Effect of extraction time on yield (%) of essential oil 38 Figure 4.1 shows effect of extraction time on the yield of essential oil from peel of Tan Trieu pomelo. It was observed that by increasing the extraction time between 2.5 and 3.5 hours, the amount of oil extracted also increased from 0.19% to 0.29% whereby the maximum yield of extracted oil was achieved at 3.5 hours of process. Further increase in extraction process after 3.5 hours did not significantly increase in the oil yield. Thus, the optimal time for vacuum distillation of citrus essential oil was 3.5 hours. 4.2 Yield of citrus essential oils: The extraction yields of cold pressed and vacuum distillated essential oils from fresh peels of citrus spp. widely varied depending on citrus cultivars. For each cultivar, the production yields of the cold pressed essential oils were much lower than those of extraction by the vacuum distillation. The production yields of citrus essential oils extracted from 2 methods are given in Figure 4.2. Figure 4.2 Extraction yield of citrus peel extracts using different extracting methods. ( ) EOs extracted by the cold pressing method; ( ) EOs extracted by the vacuum distillation method. Vacuum distillation extraction of Long An lime, Da Lat lime, Dao lime, Xoan orange, Phu Tho orange, Vinh orange, Tan Trieu pomelo, Thanh Tra pomelo and Doan Hung pomelo peels provided the production yields of 0.14, 0.19, 0.16, 0.45, 0.59, 0.39, 0.30, 0.25 and 0.04%, whereas only 0.02, 0.04, 0.03, 0.24, 0.25, 0.14, 0.16, 0.08 and 0.03 % yield, respectively were obtained by the cold pressing method. Phu Tho orange peel yielded the highest amount of vacuum distillated and cold pressed essential oils 39 comparing to other citrus cultivars. The lowest yields of essential oils extracted by both methods were obtained from Doan Hung pomelo peel. Thus, the variety of EOs yield depends on several parameters including the locations where the plants grew and extraction method (Uribe-Hernandez et al., 1992). Viljoen et al. (2006) and Chalchat et al. (1995) reported variations in the yield of essential oils from Mentha longifolia (L.) and Tagetes minuta populations, collected from different geographical locations, respectively. The yield and of essential oils from Origanum vulgare ssp. hirtum essential oils from twenty three localities, scattered all over Greece were varied significantly (Vokou et al ., 1993). 4.3 Chemical compositions of citrus essential oils 4.3.1 Chemical compositions of lime essential oils The main components of lime oils extracted by cold pressing and vacuum methods were identified as presented in Table 4.2. The result indicated that nine components were detected in lime EOs extracted by cold pressing and vacuum distillation methods Limonene was the most abundant compound in lime essential oils. Among three different lime varieties, LLA oil showed the lowest content of limonene (40.29 and 50.64%). However, the LLA peel oils had the highest amounts of β-pinene (14.58 and 21.89%) and sabinene (2.54 and 3.89%). The high content of β-pinene was matched with various studies on key lime (Citrus aurantifolia) oils reported by Dugo et al. (2002). Besides, the portion of γ-terpinene was the lowest among 3 kinds of lime EOs (2.61 and 2.25%). Other components of LLA oils such as α-pinene (1.12 and 1.98 %), myrcene (2.54 and 3.89%), α-terpinene (0.08 and 0.02%), terpinolene (0.57 and 0.25%) and linalool (0.31 and 0.24%) also present significant distinctions as compared with LD and LDL oils. The differences in chemical compositions between 3 lime oils could be linked to the different geographical locations. Variable soil textures result in different chemical products (Hussain et al., 2008). Climatic factors such as heat and drought were also related to the essential oil profiles obtained (Uribe-Hermandez et al., 1992; Milos et al., 2001). Moreover, genetic make-up of the plant shows a greater influence on the chemical profile of the oil produced (Graven et al., 1990; Milos et al., 2001). For the volatile components of Da Lat EOs, nine compounds were also identified and quantified. The limonene was also found to be prominent in EOs extracted by cold pressing and vacuum distillation methods with 53.41% and 56.04%, respectively. The proportion of limonene was higher than that in the literature reported for Ben Tre lime (Citrus aurantifolia Swingle) oil (41.40%) (Lan-Phi, 2010). Among constituents, only γterpinene, which made up 12.14 and 2.25% in LDL-CP and LDL-VA, and α-terpinene, which represented 0.18 and 11.41%, were found to indicate considerably distinctions between these kinds of extracts. However, the total of 2 compounds in both LDL-CP and 40 LDL-VA was not considerably different. The distinction may not cause remarkable difference in activities of these 2 essential oils because the antioxidant and antimicrobial abilities of essential oils was stimulated by certain combination of the volatiles (Filtenborg et al., 1996). β-pinene (9.95 and 11.02%), α-pinene (1.74 and 1.91%), sabinene (1.76 and 1.85%), myrcene (1.50 and 1.36%), and terpinolene (0.21 and 0.17%) were also determined in LDL-CP and LDL-VA EOs. In addition, the only member of alcohol group, linalool, just occupied (0.30 and 0.26%) of the total content. In case of Dao lime EOs, both LD-CP and LD-VA had high content of monoterpene hydrocarbons with limonene (70.56% and 72.82%, respectively), γ-terpinene (10.40 % and 8.52%) and β-pinene (6.10% and 6.73%). In addition, the percentage of myrcene obtained in LD-CP and LD-VA accounted for 1.78% and 1.68%, followed by α-pinene (1.50 and 1.38%), sabinene (1.27 and 1.32%), terpinolene (0.30 and 0.26%), α- terpinene (0.19 and 0.06%) and linalool (0.17 and 0.13%). Most of contents in LD-CP EO were not significantly differently from LD-VA EO with exception of α-terpinene. The amount of α-terpinene obtained in LD-CP exceeded three times larger than in LD-VA EO. α-terpinene is one of components contributes to the antioxidant activities of essential oils (Rudback et al., 2012). Therefore, the low proportion of α-terpinene also affects on the quality of LD-VA. 41 Table 4.2 Volatile compositions (%w/w) of Vietnamese lime peel EOs extracted by cold pressing and vacuum distillation method No Compound Long An lime Da Lat lime Dao lime LLA-CP LLA-VA LDL-CP LDL-VA LD-CP LD-VA 1 α-pinene 1.12±0.02 1.98±0.01 1.74±0.02 1.91±0.02 1.50±0.02 1.38±0.02 2 Sabinene 2.54±0.02 3.89±0.03 1.76±0.02 1.85±0.04 1.27±0.00 1.32±0.01 3 β-pinene 14.58±0.31 21.89±0.26 6.73±0.07 11.02±0.06 6.10±0.02 6.73±0.03 4 myrcene 0.96±0.02 1.15±0.04 1.68±0.01 1.36±0.03 1.78±0.02 1.68±0.02 5 α-terpinene 0.08±0.00 0.02±0.00 0.18±0.00 11.41±0.13 0.19±0.00 0.06±0.00 6 limonene 40.29±0.38 50.64±0.44 53.41±0.29 56.04±0.28 70.56±0.15 72.82±0.17 7 terpinolene 0.57±0.00 0.25±0.01 0.21±0.00 0.17±0.00 0.30±0.00 0.26±0.00 8 ٧-terpinene 2.61±0.02 0.84±0.01 12.14±0.21 2.25±0.07 10.40±0.04 8.52±0.41 9 Linalool 0.31±0.00 0.24±0.01 0.30±0.00 0.26±0.00 0.17±0.00 0.13±0.00 Total 63.06±0.77 80.90±0.81 77.15±0.62 86.27±0.63 92.27±0.25 92.9±0.66 LLA-CP: Long An lime extracted by cold pressing method; LLA-VA: Long An lime extracted by vacuum distillation method. LDL-CP: Da Lat lime extracted by cold pressing method; LDL-VA: Da Lat lime extracted by vacuum distillation method. LD-CP: Dao lime extracted by cold pressing method; LD-VA: Dao lime extracted by vacuum distillation method 42 4.3.2 Chemical compositions of orange essential oils EOs of three different orange varieties were analyzed and their volatile components were identified. Chemical compositions of these EOs are displayed in Table 4.3. Orange EOs mainly consisted of limonene with more than 90 % in chemical profiles of these EOs. In case of Xoan orange EOs extracted by cold pressing and vacuum distillation methods (OX-CP and OX-VA), limonene (94.25 and 94.43%), myrcene (2.10 and 2.11 %), αpinene (0.61 and 0.52%), sabinene (0.32 and 0.28%) and linalool (0.30 and 0.18%) were the main constituents. Furthermore, α-terpinene (0.15 and 0.14 %) and β-pinene (0.02 and 0.02%) occupied small amount in OX-CP and OX-VA EOs. Terpinolene and ٧terpinene were also detected in both OX-CP and OX-VA EOs at trace amount. Alphapinene and linalool were identified to strongly inhibit growth of bacteria (Dorman and Deans, 2000; Fisher and Phillips, 2006). The difference in percentage of α-pinene and linalool between OX-CP and OX-VA EOs cause the distinction in antimicrobial capacities of these oils. Alcohols including linalool were reported to be the most important to orange flavor (Shaw, 1979). The amount of linalool in Vinh orange extracted by cold pressing and vacuum distillation methods (OV-CP and OV-VA) was 0.39% and 0.43%. This compound was identified at the higher level of 0.73% in Xa Doai orange (Citrus sinesis (L.) Osbeck) in previous study (Lan-Phi, 2010). β-pinene (0.02 and 0.02%), terpinolene (0.02 and 0.02%) and ٧-terpinene (0.02 and 0.01%) represented at low content in cold pressed and vacuum distillated oils. Only eight compounds were indentified in Phu Tho orange EOs extracted by cold pressing and vacuum distillation methods (OPT-CP and OPT-VA). This caused difference from other orange EOs. Limonene was the most abundant compound in both OPT-CP and OPT-VA EOs with 95.78 and 95.61%, respectively. In addition, myrcene (2.16 and 2.12 %), α-pinene (0.59 and 0.57%), linalool (0.43 and 0.02%), sabinene (0.15 and 0.17%) and β-pinene (0.14 and 0.03%) were obtained in both OPT-CP and OPT-VA EOs. The results show that linalool and β-pinene contents of OPT-CP were significantly higher than those of OPT-VA EOs. These components cause the inhibition of EOs on pathogens (Dorman and Deans, 2000; Fisher and Phillips, 2006). Also, terpinolene, which was found to be one of factors affecting on radical scavenging effect of EOs (Choi et al., 2010), made up small amount in cold pressed and vacuum distillated oils with the same percentage at 0.02%. All three kinds of orange EOs showed the high percentage of limonene. However, antioxidant and antimicrobial activities of these orange EOs were low because limonene was considered that it would not play principle role in determining antioxidant and antibacterial activities of essential oils (Choi, 2010; Sokovic et al., 2010). 43 Table 4.3 Volatile compositions (%w/w) of Vietnamese orange peel EOs extracted by cold pressing and vacuum distillation method No Compound Xoan orange Vinh orange Phu Tho orange OX-CP OX-VA OV-CP OV-VA OPT-CP OPT-VA 1 α-pinene 0.61±0.02 0.52±0.02 0.53±0.02 0.51±0.02 0.59±0.03 0.57±0.01 2 Sabinene 0.32±0.01 0.28±0.01 0.28±0.01 0.27±0.01 0.15±0.00 0.17±0.00 3 β-pinene 0.02±0.00 0.02±0.00 0.02±0.00 0.02±0.00 0.14±0.00 0.03±0.00 4 myrcene 2.10±0.10 2.03±0.08 2.67±0.08 2.11±0.05 2.16±0.05 2.12±0.08 5 α-terpinene 0.15±0.00 0.14±0.00 0.42±0.00 0.27±0.00 --- --- 6 limonene 94.25±1.37 94.43±0.18 94.35±1.76 95.04±1.21 95.78±1.69 95.61±1.35 7 terpinolene Tr Tr 0.02±0.00 0.02±0.00 0.02±0.00 0.02±0.00 8 ٧-terpinene Tr Tr 0.02±0.00 0.01±0.00 Tr Tr 9 Linalool 0.30±0.00 0.18±0.00 0.39±0.39 0.43±0.00 0.43±0.02 0.02±0.00 Total 97.75±1.50 97.60±0.29 98.70±2.26 98.68±1.29 99.27±1.79 98.54±1.44 Tr: Trace, weight percent quantified less than 0.01% OX-CP: Xoan orange extracted by cold pressing method; OX-VA: Xoan orange extracted by vacuum distillation method. OV-CP: Vinh orange extracted by cold pressing method; OV-VA: Vinh orange extracted by vacuum distillation method. OPT-CP: Phu Tho orange extracted by cold pressing method; OPT-VA: Phu Tho orange extracted by vacuum distillation method 44 4.3.3 Chemical compositions of pomelo essential oils Table 4.4 show the data of chemical compositions of the pomelo essential oils extracted by two methods. In case of Tan Trieu pomelo EOs extracted by cold pressing and vacuum distillation methods (PTT-CP and PTT-VA), the most abundant compound was limonene (80.55 and 80.08%), followed by ٧-terpinene (10.90 and 10.82%) and α-pinene (1.32 and 1.08%). The contents of myrcene (2.16 and 1.82%), sabinene (0.20 and 0.18%) and α-terpinene (0.33 and 0.25%) were in agreement with Nam Roi pomelo EOs in previous study (Lan-Phi, 2010). Linalool, which plays an important role in antioxidant and antimicrobial ability of EOs (Alma et al., 2004; Hussain et al., 2008), was detected in both PTT-CP and PTT-VA EOs at trace amount. A total of nine main compounds were found in Thanh Tra pomelo extracted by cold pressing and vacuum distillated methods (PTTR-CP and PTTR-VA). Limonene (58.44 and 64.06%), myrcene (26.43 and 24.29%) and ٧-terpinene (7.63 and 6.57%) were the major components. Subsequently, α-pinene (0.93 and 0.78%), β-pinene (0.85 and 0.77%) and α-terpinene (0.17 and 0.07%) were also obtained in PTTR-CP and PTTR-VA EOs. The contents of α-pinene, β-pinene and α-terpinene of cold pressed oil were significantly higher than those of vacuum distillated oil. High amount of these compounds in EOs were corresponding to the high antioxidant and antimicrobial activities (Dorman and Deans, 2000; Choi, 2010; Rudback et al., 2012). Only 7-8 compounds were detected in Doan Hung pomelo EOs extracted by cold pressing and vacuum distillation methods (PDH-CP and PDH-VA). Limonene (42.93 and 47.26%) was the most abundant compound, followed by myrcene (41.95 and 38.22%), α-pinene (0.33 and 0.04%) and β-pinene (0.29 and 0.46%). The contents of ٧-terpinene (0.10 and 0.08%) in Doan Hung EOs were lower than those in Tan Trieu and Thanh Tra EOS. α-terpinene was not detected in both PDH-CP and PDH-VA EOs. Generally, chemical compositions of cold pressed EOs presented better quality compared with vacuum-distillation EOs. The differences in volatile compositions of these pomelo peels EOs were due to differences in varieties and extraction methods (Njoroge et al., 2006; Lota et al., 2000). Cold pressed EOs offered better quality because this technique did not affect by heat, pressure, or solvent. On the contrary, 70oC from vacuum hydro-distillation apparatus still had effect on EOs. At this temperature, chemical transformation may happen during distillation. 45 Table 4.4 Volatile compositions (%w/w) of Vietnamese pomelo peel EOs extracted by cold pressing and vacuum distillation method No Compound Tan Trieu pomelo Thanh Tra pomelo Doan Hung pomelo PTT-CP PTT-VA PTTR-CP PTTR-VA PDH-CP PDH-VA 1 α-pinene 1.32±0.01 1.08±0.04 0.93±0.00 0.78±0.00 0.33±0.00 0.04±0.00 2 Sabinene 0.20±0.00 0.18±0.00 0.20±0.00 0.19±0.00 0.16±0.00 0.17±0.00 3 β-pinene 0.97±0.00 0.82±0.01 0.85±0.00 0.77±0.00 0.29±0.00 0.46±0.00 4 myrcene 2.16±0.01 1.82±0.01 26.43±0.01 24.29±0.01 41.95±0.02 38.22±0.00 5 α-terpinene 0.33±0.01 0.25±0.00 0.17±0.01 0.07±0.00 --- --- 6 limonene 80.55±0.06 80.08±0.46 58.44±0.02 64.06±0.30 42.93±0.04 47.26±0.23 7 terpinolene 0.03±0.00 0.04±0.00 0.20±0.00 0.17±0.00 0.07±0.00 0.01±0.00 8 ٧-terpinene 10.90±0.01 10.82±0.04 7.63±0.00 6.57±0.18 0.10±0.00 0.08±0.00 9 Linalool Tr Tr 0.11±0.00 0.01±0.00 0.04±0.00 --- Total 96.46±0.10 95.09±0.56 94.96±0.04 96.91±0.49 85.87±0.06 86.24±0.23 Tr: Trace, weight percent quantified less than 0.01%. PTT-CP: Tan Trieu pomelo extracted by cold pressing method; PTT-VA: Tan Trieu pomelo extracted by vacuum distillation method. PTTR-CP: Thanh Tra pomelo extracted by cold pressing method; PTTR-VA: Thanh Tra pomelo extracted by vacuum distillation method. PDH-CP: Doan Hung pomelo extracted by cold pressing method; PDH-VA: Doan Hung pomelo extracted by vacuum distillation method. 46 4.4 Antioxidant activities of citrus essential oils: 4.4.1 DPPH assay Antioxidant activity of the essential oils of citrus fruits, as assessed by DPPH radical scavenging assay as well as expressed in terms of 50% inhibition concentration (IC50) is given in Figure 4.3. The use of the DPPH free radical is more beneficial in evaluating antioxidant activity because it is more stable than hydroxyl and super oxide (LayinaPathirana et al., 2006). DPPH is a stable free radical having maximum absorption at 517 nm that accepts an electron or hydrogen atom to become a stable diamagnetic molecule. In the presence of a substance capable of donating a hydrogen atom, its free radical nature is lost and hence the reduction in DPPH radical was determined by the decrease in its absorbance at 517nm. In this assay, the violet color of DPPH was reduced to a yellow color due to the abstraction of hydrogen atom from antioxidant compound. The more antioxidant occurred in the extract, the more DPPH reduction will occur. High reduction of DPPH is related to the high scavenging activity performed by particular sample (Blois, 1958). IC50 values denote the concentration of sample, which is required to scavenge 50% of DPPH free radicals. The lower IC50 value is, the higher antioxidant activity is. Figure 4.3 IC50 values of investigated citrus oils by DPPH method. ( ) EOs extracted from cold pressing method; ( ) EOs extracted from vacuum distillation. 47 As can be clearly seen from the figure, all citrus EOs tested possed radical scavenging activities. Also, the radical scavenging capacity varied significantly (p0.05) according to Tukey’s Values followed by the same according to Tukey’s test. zone (mm) including well diameter of 9 mm) are mean ± standard small letter within the same column are not significant different test. letter within the same line are not significant different (p>0.05) In case of EOs extracted by cold pressing method, Long An lime EO, Doan Hung pomelo and Tan Trieu pomelo EOs showed the highest effects with the inhibition zone diameter of 27.67, 27.17 and 26.83 mm, respectively. From the results, other citrus EOs were found highly susceptible to S. aureus as growth inhibition zone diameters were obtained in a range from 20.00 to 24.00 mm. Three orange EOs (Xoan, Vinh and Phu Tho orange) possessed the least effects on this bacterium. The results were in agreement with previous reports. Sharma and Tripathi (2006) cited that orange oils did not showed powerful activity against bacteria. In a report, Thailand pomelo EO obtained from cold 51 pressing method had inhibition zone with 9.50 mm in diameter against S. aureus (Mungdee et al., 2012), whereas 3 kinds of pomelo essential oils in this study possessed larger zone ranging from 21.58 to 27.17 mm. This indicated that Vietnamese pomelo EOs in this assay had higher effect on this strain than Thailand pomelo EO. Among vacuum distillation samples, Long An lime, Da Lat lime, Dao lime and Doan Hung pomelo EOs also exhibited appreciable antimicrobial activities with inhibition zones (IZ) of 25.17, 21.08, 18.33 and 18.17 mm against this strain, respectively. Two kinds of pomelo, Tan Trieu and Thanh Tra pomelo essential oils showed the lowest antibacterial activities against S. aureus with zone diameters 14.42 and 15.08 mm, respectively. Three orange EOs presented moderate effects. The MIC values of different citrus oils are presented in Table 4.6. Most of cold pressed oils showed high susceptibility against S. aureus, as MIC values obtained were very low. The lowest MIC values among cold pressed extracts were Long An lime, Tan Trieu pomelo and Doan Hung pomelo with the same value at 2.63 mg/ml, followed by Dao lime, Da Lat lime and Thanh Tra pomelo EOs with 5.25, 10.5, 10.5 mg/ml in MIC values, respectively. Three kinds of orange essential oils exhibited the highest MIC values (21 mg/ml). In other word, these orange extracts possessed the lowest antimicrobial activity on this bacterium. These results were suitable with the above data from zone of inhibition (mm) of citrus essential oils. As regards essential oils extracted by the vacuum distillation, most of essential oils showed low antimicrobial capacity on this strain with MIC values were higher than 42 mg/ml except for Long An and Da Lat lime EOs. The reason may be due to the degradation of some active compounds of essential oils extracted by vacuum distillation. As a literature review, peel extracts of Citrus aurantifolia and Citrus hystrix DC exhibited antibacterial activities on S. aureus with the inhibition zone of 15.5 and 16 mm, respectively (Chaisawadi et al., 2005). Some authors proved that ethyl acetate extracts of pomelo peel from Khao-nahm-peung and Khao-paen varieties exhibited better inhibition activities against this bacterium. At concentration 200 mg/ml, the inhibition zones of these oils affecting on S. aureus were 10 and 7.5 mm (Suklampoo et al., 2012). Additionally, Citrus sinesis peel of aqueous extracts showed a very good antimicrobial capacity when compared to Citrus limon. The inhibition zone of Citrus sinesis was 9mm, whereas Citrus limon did not showed antibacterial activity on this strain (Ashok et al., 2011). Upadhyay et al. (2010) reported that Citrus lemon and Azadirachta indica essential oils had inhibition zones of 23.10 mm and 23.23 mm, respectively, were recorded. In one research, S. aureus was found to be the most susceptible bacterium for Citrus limon and Citrus aurantium essential oils with MIC values of 6.0 µg/ml for both (Sokovic et al., 2017). Growth of this microorganism was completely inhibited by all of the citrus oils with 100% of reduction of inoculums (Dabbah et al., 1970). In addition, 52 Turkish citrus peel oils also showed strong antimicrobial activity against S. aureus (Kirbaslar et al., 2009). The growth of S. aureus was affected by the appearance of α-pinene in the composition of essential oils (Dorman and Deans, 2000). In addition, linalool was also significantly effective inhibitory on this bacterium (Fisher and Phillips, 2006). Possible action mechanisms by which microbial growth may be reduced or totally inhibited have been suggested. It is commonly accepted that it is the toxic effects of the EO components on the functionality and structure of the cell membrane that is responsible for the antimicrobial activity. Uribe et al. (1985) related the respiration and alter the permeability of the microbe cell membrane would be induced by low EO concentrations with changes in the cell structure, while high concentrations would prompt severe damage to the membrane and the loss of homeostasis, resulting in cell death (Carson et al., 2002 ). Connerand and Beuchat (1984) recommended that interactions caused by EOs in the enzymatic systems related with energy production and in the synthesis of structural components of the microbial cells produces the antimicrobial capacity of EOs. On the other hand, Omidbeygi et al. (2007) proposed that components of the essential oils cross the cell membrane, interacting with the enzymes and proteins of the membrane, so producing a flux of protons towards the cell exterior which creates changes in the cells and, ultimately, their death. Cristani et al. (2007) reported that the antimicrobial activity is related to ability of terpenes to affect not only permeability but also other functions of cell membranes, these compounds might cross the cell membranes, thus penetrating into the interior of the cell and interacting with critical intracellular sites. Table 4.6 MIC values (mg/ml) of citrus essential oils for S. aureus Methods Cold pressing Vacuum distillation Long An lime 2.63 5.25 Da Lat lime 10.5 10.5 Dao lime 5.25 42 Xoan orange 21 >42 Vinh orange 21 >42 Phu Tho orange 21 42 Tan Trieu pomelo 2.63 >42 Thanh Tra pomelo 10.5 >42 Doan Hung pomelo 2.63 42 Essential oils 53 4.5.2 B. cereus Antimicrobial activities against pathogenic B. cereus of the cold pressed extracts and the vacuum distillated extracts were investigated and compared. According to the results of table 4.7, all EOs tested showed inhibition activity on B. cereus. For cold pressed extracts, most of citrus EOs exhibited good effects against this baterium. Only Xoan orange and Phu Tho orange EOs showed the least inhibition capacity on B. cereus with the inhibition zones of 17.17 and 18.42 mm, respectively. The highest antibacterial ability of cold pressed oils belonged to Long An lime EO (IZ=28.17mm). Da Lat lime, Dao lime, Vinh orange, Thanh Tra pomelo and Doan Hung pomelo EOs displayed potential effects with the inhibition zones of 22.42, 26.67, 24.17, 24.83, 26.17 mm, in the order given. Table 4.7 Zone of inhibition (mm) of citrus essential oils against B. cereus Methods Cold pressing Vacuum distillation Long An lime 28.17±0.29gB 23.83±0.14gA Da Lat lime 22.42±0.38dA 22.08±0.14fA Dao lime 26.67±0.29fB 20.33±0.29eA Xoan orange 17.17±0.29aB 12.67±0.29aA Vinh orange 24.17±0.29eB 17.67±0.14dA Phu Tho orange 18.42±0.14bB 13.83±0.14bA Tan Trieu pomelo 20.42±0.14cB 16.00±0.00cA Thanh Tra pomelo 24.83±0.14eB 22.25±0.25fA Doan Hung pomelo 26.17±0.29fB 22.17±0.14fA Essential oils Values (Diameter of inhibition deviation. Values followed by the same (p>0.05) according to Tukey’s Values followed by the same according to Tukey’s test. zone (mm) including well diameter of 9 mm) are mean ± standard small letter within the same column are not significant different test. letter within the same line are not significant different (p>0.05) With regard to EOs extracted by vacuum distillation method, the strongest inhibitory effect was Long An lime EO with IZ of 23.83 mm, whereas the least effect was recorded in Xoan orange (IZ=12.67 mm). Other citrus EOs showed antibacterial abilities against this strain with their inhibition zones ranging from 13.83 to 22.25 mm in diameters. These results indicated that different EOs had diverse antibacterial activities. The difference may be due to the various chemical compositions of EOs due to geographical regions (Njoroge et al., 2006). Additionally, most of cold pressed EOs were more effective than vacuum distillated EOs. Only Da Lat Lime EO was special case. A report cited the antibacterial ability of citrus 54 essential oils on B. cereus caused by the presence of linalool in EOs (Fisher and Phillips, 2006). The percentages of linalool component in cold pressed oils were higher than those in vacuum distillated oils. This reason may explain for the higher inhibition capacities of oils extracted by cold pressing method. MIC of the investigated citrus essential oils obtained by dilution method are shown in Table 4.8. Among cold pressed oils, Long An lime EO showed the best effect on B. cereus with MIC value at 1.31mg/ml. The lowest effect were recored in Xoan orange and Phu Tho orange EOs (MIC at 42 mg/ml). Three kinds of pomelo EOs possessed MIC values which were larger than 2.63 mg/ml. The results were matched with the conclusion of the study of Chanthaphon et al. (2008) who revealed that MIC value of pomelo essential oil on B. cereus was larger than 2.25 mg/ml. As regards citrus oils extracted by vacuum distillation method, Long An lime exhibited the lowest MIC (5.25 mg/ml). Therefore, it maintained the highest effect against B. cereus. Xoan orange, Phu Tho orange and Tan Trieu pomelo EOs possessed the less antimicrobial activities with MIC values more than 42 mg/ml. The results of MIC values was corelated with the data of inhibition zone. Table 4.8 MIC values (mg/ml) of citrus essential oils for B. cereus Methods Cold pressing Vacuum distillation Long An lime 1.31 5.25 Da Lat lime 10.5 10.5 Dao lime 2.63 21 Xoan orange 42 >42 Vinh orange 5.25 42 Phu Tho orange 42 >42 Tan Trieu pomelo 21 >42 Thanh Tra pomelo 5.25 10.5 Doan Hung pomelo 2.63 10.5 Essential oils Several researches studied on antibacterial activities of essential oils on B. cereus. Citrus lemon and Azadirachta indica essential oils showed high inhibition on this bacterium with 41.3mm and 45.63mm inhibition zone, respectively (Upadhyay et al., 2010). According to the result of the other report, all of the citrus peel oils were more effective towards B. cereus (Kirbaslar et al., 2009). Mungdee et al. (2012) reported that only extracts from pomelo peel indicated inhibition activities against B. cereus (IZ=8.17 mm) while those from pomelo albedo and seed showed no inhibition activities. Chaisawadi et al. (2005) cited EOs of Citrus aurantiifolia Swing and Citrus hystrix DC showed antimicrobial effect against this strain. Chanthaphon et al. (2008) demonstrated that the extract from lime 55 peel (Citrus aurantifolia Swingle) showed broad spectrum inhibitory against all Gram positive bacteria including B. cereus. The lime extract exhibited MIC value against B. cereus at 0.56 mg/ml. This result was correlated to the report of Chaisawadi et al. (2005) which cited Citrus aurantifolia displaying antibacterial activities on this microorganism. 4.5.3 S. typhi Screening of antibacterial activities using well diffusion method of peel extracts from citrus varieties performed various degree of growth inhibition against S. typhi. The results are shown in Table 4.9. The cold pressed EOs showed stronger antimicrobial activities than the ones obtained from vacuum distillation technique. The variance in chemical compositions of EOs due to extraction methods may be the main cause in difference in antibacterial activities (Njoroge et al., 2006). Table 4.9 Zone of inhibition (mm) of citrus essential oils against S. typhi Methods Cold pressing Vacuum distillation Long An lime 26.33±0.29eB 24.17±0.14gA Da Lat lime 23.17±0.14dA 23.00±0.00fA Dao lime 28.83±0.29fB 18.33±0.29cA Xoan orange 17.08±0.14aB 16.17±0.29aA Vinh orange 23.00±0.00cdB 20.08±0.14dA Phu Tho orange 20.33±0.29bB 18.17±0.29cA Tan Trieu pomelo 20.42±0.14bB 17.17±0.29bA Thanh Tra pomelo 22.42±0.14cB 20.50±0.25deA Doan Hung pomelo 22.58±0.38cdB 21.08±0.14eA Essential oils Values (Diameter of inhibition deviation. Values followed by the same (p>0.05) according to Tukey’s Values followed by the same according to Tukey’s test. zone (mm) including well diameter of 9 mm) are mean ± standard small letter within the same column are not significant different test. letter within the same line are not significant different (p>0.05) The highest antibacterial ability of cold pressed oils belonged to Dao lime EO (IZ=26.17mm). The other citrus EOs also possessed high potential against this bacterium with inhibition zone in a range between 17.08 and 26.33 mm. There were not significantly different in inhibtion zones among Da Lat lime, Vinh orange and Doan Hung pomelo with zone diameters of 23.17, 23.00 and 22.58 mm, respectively. In addition, Phu Tho orange and Tan Trieu pomelo EOs displayed the same effects on S. typhi. 56 For samples from vacuum distillation method, most of EOs exhibited low ability against this strain with inhibition zone lower than 20.50 mm. The highest effect was replaced by Long An lime EO with zone diameter of 24.17 mm. The result of MIC values of citrus essential oils are presented in Table 4.10. Among cold pressed oils, Long An lime essential oil displayed the highest capacity in inhibition on this pathogen (MIC value at 1.31 mg/ml). The lowest effect belonged to Xoan orange with MIC at 42 mg/ml. The MIC values of cold pressed oils were in wide range from 1.31 to 42 mg/ml, while those of vacuum distillated extracts between 5.25 and more than 42 mg/ml. These data demonstrated that the inhibition abilities of citrus extracted by cold pressing method were higher than those extracted by vacuum distillation method on S. typhi. However, Da Lat lime EO was exception. The LDL-CP and LDL-VA had the same effect on this strain with the MIC value at 5.25 mg/ml. Table 4.10 MIC values (mg/ml) of citrus essential oils for S. typhi Methods Cold pressing Vacuum distillation Long An lime 2.63 5.25 Da Lat lime 5.25 5.25 Dao lime 1.31 42 Xoan orange 42 >42 Vinh orange 5.25 21 Phu Tho orange 21 42 Tan Trieu pomelo 21 42 Thanh Tra pomelo 10.5 21 Doan Hung pomelo 10.5 10.5 Essential oils There are many reports regarding the antimicrobial activity of essential oils on S. typhi. Among the various essential oil treatments, the Citrus reticulata var. Tangarin exhibited the highest antibacterial activity on S. typhi (Ashok et al., 2011). The essential oils distilled from Syzygium neesianum Arn, Elaeocarpus lanceifolius and Citrus sinesis showed a significant inhibition on S. typhi (Maridass, 2010; Ashok et al., 2011). Citrus sisnesis (Linn.) also possessed potential activities against S. typhi with inhibition zone of 10 mm and MIC at a concentration of 0.25 mg/ml (Lawal et al., 2013). This strain was also found to be more susceptible to the Citrus aurantifolia and Citrus hystrix DC essential oils (Chaisawadi et al., 2005). 57 4.5.4 P. aeruginosa The efficiency of two extraction methods for antimicrobial activities of various citrus peels on P. aeruginosa was investigated. The results are shown in Table 4.11. Overall, citrus extracts from the cold pressing method exhibited the stronger inhibitory effects than those from the vacuum distillation method on P. aeruginosa. However, LDL-CP and LDL-VA had not significant difference in inhibition zone on P. aeruginosa. For cold pressed EOs, the antibacterial activity of Dao lime was predominant against P. aeruginosa with inhibition zone of 28.67 mm in diameter. This bacterium was also estimated as more sensitive to Long An lime, Da Lat lime, Vinh orange and Doan Hung pomelo with zone diameters of 23.33, 22.33, 23.58, 22.33 mm. The lowest effect was seen at Xoan orange EO (IZ=19.00 mm). Among the vacuum distillation samples, most citrus essential oils tested exhibited low effect with the inhibition zones in a range from 18.08 to 20.58 mm. The highest activities against P. aeruginosa were demonstrated at Dao lime EO (IZ=23.08 mm) and Da Lat lime EO (IZ=22.08 mm). Table 4.11 Zone of inhibition (mm) of citrus essential oils against P. aeruginosa Methods Cold pressing Vacuum distillation Long An lime 23.33±0.29dB 20.58±0.14cA Da Lat lime 22.33±0.38cA 22.08±0.14dA Dao lime 28.67±0.29eB 23.08±0.14eA Xoan orange 19.00±0.25aB 18.08±0.14aA Vinh orange 23.58±0.14dB 20.33±0.29cA Phu Tho orange 20.58±0.14bB 17.42±0.38aA Tan Trieu pomelo 20.08±0.14bB 17.33±0.29aA Thanh Tra pomelo 21.83±0.14cB 19.33±0.29bA Doan Hung pomelo 22.33±0.29cB 19.83±0.14cA Essential oils Values (Diameter of inhibition deviation. Values followed by the same (p>0.05) according to Tukey’s Values followed by the same according to Tukey’s test. zone (mm) including well diameter of 9 mm) are mean ± standard small letter within the same column are not significant different test. letter within the same line are not significant different (p>0.05) As shown in Table 4.12, cold pressed oils displayed higher MIC than oils extracted by vacuum distillation method. In case of cold pressed oils, Dao lime EO exhibited the strongest effect on P. aeruginosa with MIC value of 1.31 mg/ml, whereas Xoan orange, Phu Tho orange and Tan Trieu pomelo were recorded as the weakest with the same MIC values of 21 mg/ml. 58 Turning to extracts from the vacuum distillation method, Dao lime EO had the lowest MIC (5.25 mg/ml). In addition, 3 kinds of lime EOs showed very high susceptibility against this pathogen, as MIC values obtained were very low. The rest of citrus EOs possessed low inhibition capacities on P. aeruginosa because of high MIC values A number of publications demonstrated that various essential oils possessing antibacterial activities on P. aeruginosa. Antimicrobial activity of the peel oils was found to be directly concerning with the components that they contained. The studies showed β-pinene, α-terpinene and geraniol were effective toward P. aeruginosa (Dorman and Deans, 2000). Several reports investigated the antibacterial activities of essential oils on P. aeruginosa. The Turkish citrus peel oils exhibited strong inhibition effect of P. aeruginosa with 10-12 mm inhibition zone diameter (Kirbaslar et al., 2009). In another research, cold water extract of orange peel performed a high antimicrobial effect against bacteria tested, especially P. aeruginosa with zone diameter of 13 mm (Jwanny et al., 2012). The essential oils Ammy visnaga L. exhibited strong inhibition effect of P. aeruginosa with 25 mm inhibition zone diameter (Khalfallah et al., 2011). This microorganism was inhibited by the lemongrass (Cymbopogon citratus) oil with MIC value at 1% (v/v) whereas the lime (Citrus aurantifolia) oil showed the lower effect with MIC value at 2% (v/v) (Hammer et al., 1999). Table 4.12 MIC values (mg/ml) of citrus essential oils for P. aeruginosa Methods Cold pressing Vacuum distillation Long An lime 5.25 21 Da Lat lime 10.5 10.5 Dao lime 1.31 5.25 Xoan orange 21 42 Vinh orange 5.25 21 Phu Tho orange 21 42 Tan Trieu pomelo 21 42 Thanh Tra pomelo 10.5 21 Doan Hung pomelo 10.5 21 Essential oils 4.5.5 A. flavus The antimicrobial activity of citrus extracts against A. flavus employed and its activity potentials were assessed by the presence or absence of inhibition zones. The data obtained from well diffusion method indicated that citrus EOs displayed a variable degree of antimicrobial activity on this fungus. 59 As regards EOs extracted by cold pressing method, Dao lime EO showed the strongest activity against A. flavus (IZ=25.42 mm), while Xoan orange and Tan Trieu pomelo extracts possessed the least antifungal capacities with zone diameters of 17.17 mm for both EOs. Long An lime, Da Lat lime, Thanh Tra pomelo and Doan Hung pomelo samples exhibited zone diameters of 24.25, 23.67, 22.33 and 23.08 mm, respectively. Among vacuum distillated EOs, the Xoan orange and Phu Tho orange EOs exhibited the lowest effects on A. flavus. This strain was recorded to be resistant to these oils with no inhibition zone, while the zone diameters of other EOs were between 14.17 and 23.42 mm. The strongest inhibitory effect belonged to Dao lime EO (IZ=24.17 mm). Moreover, all cold pressed oils exhibited larger inhibition zones than vacuum distillated oils. Limonene showed the most abundant content in citrus oils. This component in vacuum distillated oils was predominant that in cold pressed oils. However, it had showed the lowest effect against microorganisms (Fisher and Phillips (2006). α-pinene and linalool which are found in appreciable amounts in cold pressed oils showed considerable antifungal activity (Chutia et al., 2009). Table 4.13 Zone of inhibition (mm) of citrus essential oils against A. flavus Methods Cold pressing Vacuum distillation Long An lime 24.25±0.25fB 23.42±0.29eA Da Lat lime 23.67±0.14eA 23.17±0.14eA Dao lime 25.42±0.14gB 24.17±0.14fA Xoan orange 17.17±0.14aB 9.00±0.00aA Vinh orange 18.33±0.14bB 15.08±0.14cA Phu Tho orange 17.67±0.14aB 9.00±0.00aA Tan Trieu pomelo 17.17±0.29aB 14.17±0.29bA Thanh Tra pomelo 22.33±0.29cB 15.17±0.29cA Doan Hung pomelo 23.08±0.14dB 17.08±0.14dA Essential oils Values (Diameter of inhibition deviation. Values followed by the same (p>0.05) according to Tukey’s Values followed by the same according to Tukey’s test. zone (mm) including well diameter of 9 mm) are mean ± standard small letter within the same column are not significant different test. letter within the same line are not significant different (p>0.05) The results of dilution method are presented in Table 4.14. Most cold pressed oils exhibited low MIC value from 0.66 to 10.5 mg/ml. Also, Dao lime EO showed the lowest MIC value (0.66 mg/ml), followed by Long An lime, Dao lime and Doan Hung pomelo with the same MIC of 1.31 mg/ml. 3 kinds of orange and Tan Trieu pomelo possessed the highest MIC (10.5 mg/ml). 60 With regard to vacuum distillated oils, three kinds of lime displayed strong antimicrobial activities on A. flavus, as MIC values obtained were very low (only 1.31 mg/ml). In addition, Xoan orange and Phu Tho orange did not show effects against this fungus. Few studied have examined the antifungal activity of citrus essential oils. Martos et al. (2008) examined the antifungal activities of citrus EOs such as Citrus lemon L., Citrus reticulata L., Citrus paradisi L. and Citrus sinesis L. against A. flavus. They reported that Citrus reticulata L. EO was the most effective at reducing the growth of this strain. In another study, the Nigerian lime (Citrus aurantifolia) and orange (Citrus sinesis) EOs exhibited antifungal activities against A. flavus. The MIC values of these EOs were recorded at 50 mg/ml (Jeff-Agboola et al., 2012). Table 4.14 MIC values (mg/ml) of citrus essential oils for A. flavus Methods Cold pressing Vacuum distillation Long An lime 1.31 1.31 Da Lat lime 1.31 1.31 Dao lime 0.66 1.31 Xoan orange 10.5 NI Vinh orange 10.5 21 Phu Tho orange 10.5 NI Tan Trieu pomelo 10.5 42 Thanh Tra pomelo 2.63 21 Doan Hung pomelo 1.31 10.5 Essential oils NI: No inhibition Citrus essential oils are a complex mixture of volatile compounds that show, among other properties, antifungal activity by reducing or totally inhibiting fungal growth in a dose response manner (Sharma and Tripathi, 2006b). Several authors have cited the antifungal ability of citrus essential oils to the presence of components such as linalool (Alma et al., 2004). Caccioni et al. (1998) suggested that the antimicrobial activities of EOs could be the results of the synergic effect of various compounds. French (1985) proposed that the various components of any oils may act sinergically while several compounds may have a stimulating action on fungal spore germination. Although, some volatile compounds show significantly distinctive activities according to their abundance, the synergic and additive effects of volatile compounds in citrus oils may prevail over contrasting effects of each single compound (Caccioni et al., 1998). Daferera et al. (2000) reported that the antimicrobial activity of EOs may have been due to formation of 61 hydrogen bonds between the hydroxyl group of oil phenolics and active sites of target enzymes. 4.5.6 F. solani The results of antifungal activities of EOs on F. solani tested are presented in Table 4.15. Generally, EOs extracted from cold pressing method showed larger growth inhibition zone diameters in comparison to those extracted by vacuum distillation technique with the exception of Da Lat lime EO. There was no significant difference in zone diameters between LDL-CP EO and LDL-VA EO. For cold pressing samples, various EOs exhibited excellent antifungal activities against this fungus with the inhibition zone more than 21.17 mm, which was the zone diameter of Tan Trieu pomelo EO. The strongest antifungal ability was recorded in Dao lime EO with zone diameter of 27.58 mm, followed by Long An lime, Da Lat lime, Doan Hung pomelo, Vinh orange, Thanh Tra pomelo extracts with inhibition zones of 26.17, 25.33, 24.83, 24.58, 24.08 mm, respectively. Furthermore, there was no significant difference in inhibition zone between Xoan orange (IZ=23.17 mm) and Phu Tho orange EOs (IZ=23.08 mm). Table 4.15 Zone of inhibition (mm) of citrus essential oils against F. solani Methods Cold pressing Vacuum distillation Long An lime 26.17±0.29fB 24.33±0.29eA Da Lat lime 25.33±0.14eA 25.00±0.00fA Dao lime 27.58±0.29gB 25.33±0.14eA Xoan orange 23.17±0.14bB 13.17±0.14aA Vinh orange 24.58±0.14cdB 14.67±0.14bA Phu Tho orange 23.08±0.14bB 13.33±0.29aA Tan Trieu pomelo 21.17±0.29aB 15.17±0.29bA Thanh Tra pomelo 24.08±0.14cB 17.17±0.14cA 24.83±0.14deB 18.17±0.29dA Essential oils Doan Hung pomelo Values (Diameter of inhibition deviation. Values followed by the same (p>0.05) according to Tukey’s Values followed by the same according to Tukey’s test. zone (mm) including well diameter of 9 mm) are mean ± standard small letter within the same column are not significant different test. letter within the same line are not significant different (p>0.05) The results of MIC values are shown in Table 4.16. Among cold pressed oils, the lowest MIC value was recorded in Dao lime EO (1.31mg/ml), followed by Long An lime and Dao lime with MIC values at 2.63 mg/ml. Tan Trieu pomelo showed the highest MIC (10.5 mg/ml). Most of citrus EOs extracted by vacuum distillation method possessed higher 62 MIC than those extracted by cold pressing method. In case of EOs extracted by vacuum distillation method, the lowest MIC were recorded in Da Lat lime and Dao lime EOs with the same MIC at 2.63 mg/ml. Table 4.16 MIC values (mg/ml) of citrus essential oils for F. solani Methods Cold pressing Vacuum distillation Long An lime 2.63 5.25 Da Lat lime 2.63 2.63 Dao lime 1.31 2.63 Xoan orange 5.25 >42 Vinh orange 5.25 >42 Phu Tho orange 5.25 >42 Tan Trieu pomelo 10.5 >42 Thanh Tra pomelo 5.25 42 Doan Hung pomelo 5.25 42 Essential oils Some author mentioned that the presence of phenolic compounds leads to the antifungal activity of EOs (Veldhuizen et al., 2006). In fact, the hydrophilic part of the molecule interacts with the polar part of the membrane, while the hydrophobic benzene ring and the aliphatic side chains are buried in the hydrophobic inner part of the bacterial membrane (Cristani et al., 2007). Furthermore, the involvement of the hydroxyl group in the formation of hydrogen bonds and the acidity of these phenolic compounds may have other possible explanations (Cristani et al., 2007). Lucini et al., (2006) indicated that the monoterpenes presenting in essential oils causes mycelial growth inhibition. The increase of quantity of lipid peroxides such as hydroxyl, alkoxyl and alkoperoxyl radicals is induced by monoterpenes. Thus, it brings about cell death. For Sharma and Tripathi (2006b), the EOs would act on the hyphae of the mycelium, stimulating the exit of components from the cytoplasm, the loss of rigidity and integrity of the hyphae cell wall, resulting in its collapse and death of the mycelium. A positive correlation between monoterpenes other than limonene and sequiterpene content of the oils and the pathogenic fungi inhibition was observed and reported (Caccioni et al., 1998). Although antifungal activities of EOs were found to be related their compositions. However, the variation of each component amount depends on several parameters including ripeness of fruits, vegetative stage of plant, storage condition, geographical region and extraction methods (Lota et al., 2000; Njoroge et al., 2006). 63 5 Chapter 5: CONCLUSION In the present study, the yields of EOs extracted by the vacuum distillation method were higher than those extracted by the cold pressing method. Three kinds of orange essential oils possessed the highest yields among citrus EOs investigated. Both cold pressed essential oils and vacuum hydro-distillated essential oils showed high antioxidant and antimicrobial activities. In addition, most of vacuum hydro-distillated essential oils of citrus varieties tested had less antioxidant and antimicrobial capacities than the cold pressed extracts. Antioxidant activities of lime essential oils were the highest. Moreover, these lime EOs showed the strongest activity against all microorganism tested. The lowest antioxidant and antimicrobial activities were recorded in orange EOs. This study has some limitations. The vacuum pump does not support high power enough to reduce temperature of boiling flask to 50oC for avoiding changes in chemical composition and functional properties of essential oils. If we can construct the best optimization for vacuum system, we can produce better quality of essential oils and better biological activities as natural oils but also high yields for applications in food industry. The result of study provides important baseline information for the use of vacuum hydrodistillation system in further research. 64 REFERENCES 1. Ago, 2011. Report of Agriculture and Rural development in 2011. http://www.agroviet.gov.vn 2. Agrios and George, N., 2005. Plant Pathology, fifth ed. Elsevier Academic Press. 3. Agro, 2006. Actual situation and sustainable development strategy of edible fruit production up to 2015. http://www.agro.gov.vn. 4. Alma, M.H., Nitz, S., Kollmannsberger, H., Digrak, M., Efe, F.T., Yilmaz, N., 2004. Chemical composition and antimicrobial activity of the essential oils from the gum of Turkish Pistachio (Pistacia vera L.). Journal of Agriculture and Food Chemistry 52(12), 3911-3914. 5. Amaike, S., Keller, N.P., 2011. Aspergillus flavus. Annual Review of Phytopathology 49, 107-133. 6. Anwar, F., Ali, M., Hussain, A.I., Shahid, M., 2009a. Antioxidant and antimicrobial activities of essential oils and extracts of fennel (Foeniculum vulgare Mill.) seeds from Pakistan. Flavour and Fragrance Journal 24, 170-176. 7. Armando, C.C., Rahma H.Y., 2009. Evaluation of the yiled and the antimicrobial activity of the essential oils from: Eucalyptus globulus, Cymbopogon citratus and Rosmarinus officinalis in Mbarara District (Uganda). Revista Colombiana de Ciencia Animal 1(2), 240-249. 8. Ashok, K.K., Narayani, M., Subanthini, A., Jayakumar, M., 2011. Antimicrobial activity and phytochemical analysis of citrus fruit peels – Utilization of fruit waste. International Journal of Engineering Science and Technology 3(6), 5414-5421. 9. Ayoola, G.A., Johnsonl, O.O., Adelowotan, T., Aibinu, I.E., Adenipekun, E., Adepoju-Bellol, A.A., Coker, H.A.B., Odugbemi, T.O., 2008. Evaluation of the chemical constituents and the antimicrobial activity of the volatile oil of Citrus reticulata fruit (Tangerine fruit peel) from South West Nigeria. African Journal of Biotechnology 7(13), 2227-2231. 10. Aziz, S., Rehman, H., Irshad, M., Asghar, S.F., Hussain, H., Ahmed, I., 2010. Phytotoxic and Antifungal Activities of Essential Oils of Thymus serpyllu Grown in the State of Jammu and Kashmir. Journal of essential oil-bearing plant, 224-229. 11. Baik, J.S., Kim, S.S., Lee, J.A., Oh, T.H., Kim ,J.Y., Lee, N.H., Hyun, C.G., 2008. Chemical composition and biological activities of essential oils extracted from Korean endemic citrus species. Journal of Microbiology and Biotechnology 18(1), 74-9. 12. Belletti, N., Kamdem, S.S., Tabanelli, G., Lanciotti, R., Gardin, F., 2009. Modeling of combined effects of citral, linalool and β -pinene used against Saccharomyces 65 cerevisiae in citrus-based beverages subjected to a mild heat treatment. International Journal of Food Microbiology 136 (2010), 283– 289. 13. Bendini, A., Gallina-Toschi, T., Lercker, G., 2002. Antioxidant activity of oregano (Origanum vulgare L.) leaves. Italian Journal of Food Science 14, 17–23. 14. Benson, H. J., 2002. Bacterial Population Counts, in: Microbiological Applications, eighth ed. McGraw Hill, New York. 15. Blois, M.S., 1958. Antioxidants determination by the use of a stable free radical. Nature 181,1199-1200. 16. Breed, R., Dotterrer, W.D., 1916. The Number of Colonies Allowable On Satisfactory Agar Plates. Journal of Bacteriology 1, 321-331. 17. Burt, S. 2004. Essential oils: their antimicrobial properties and potential application in foods-A review. International Jour nal of Food Microbiology. 94: 223-253. 18. Carccioni, D.R.L., Guizzardi, M., Biondi, D.M., Renda, A., Ruberto, G., 1998. Relationship between volatile components of citrus fruit essential oils and antimicrobial action on Penicillium digitatum and Penicillium italicum. International Journal of Food Microbiology 43(1/2), 73-79. 19. Carson, C.F., Me, B.J., Riley, T.V., 2002. Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy. Antimicrobial agents and chemotherapy 46, 1914-1920. 20. Cava, R., Nowak, E., Taboada, A., Mari n-Iniesta, F., 2007. Antimicrobial Activity of Clove and Cinnamon Essential Oils against Listeria monocytogenes in Pasteurized Milk. Journal of Food Protection 70, 2757-2763. 21. Cavaleiro, C., Pinto, E., Goncalves , M. J., Salgueiro.L., 2006. Antifungal activity of Juniperus essential oils against dermatophyte, Aspergillus and Candida strains. Journal of Applied Microbiology 100,1333–1338. 22. Chaisawadi, S., Thongbutr, D., Methawiriyasilp, W., Chaisawadi, A., Pitakworarat, N., Jaturonrasamee, K., Khemkhaw, J., Tanuthumchareon, W., 2005. Preliminary study of antimicrobial activities on medicinal herbs of Thai food’s ingredients. Acta Hort 675, 111-114 23. Chalchat, J. C., Garry, R.P., Muhayimana, A., 1995. Essential oil of Tagetes minuta from Rwanda and France: chemical composition according to harvesting location, growth stage and part of plant extract. Journal of Essential Oil Research 7, 375-386. 24. Chanthphon, S., Suphitchaya, C., Hongpattarakere, T., 2008. Antimicrobial activities of essential oils and crude extracts from tropical Citrus spp. against 66 food-related microorganisms. Songklanakarin Journal of Science and Technology 30, 125-131. 25. Charles, D. J., Simon, J.E., 1990. Comparison of extraction methods for the rapid determination of essential oil content and composition. Journal of the American Society for Horticultural Science 115, 458-462. 26. Chemat, F., 2010. Techniques for oil extraction, in: Sawamura, M., (Eds.), Citrus Essential Oils: Flavor and Fragrance. Wiley. 27. Cho, J. H., Rupe, J. C., Cummings, M. S., Gbur, E. E. J., 2001. Isolation and identification of Fusarium solani f. sp. glycines from soil on modified Nash and Snyder’s medium. Plant Disease 85, 256-260. 28. Choi, H.S., 2010. Functional Propertie, in: Sawamura, M., (Eds.), Citrus Essential oils - Flavor and Fragrance. Wiley. 29. Choi, Y., Jeong, H. S., Lee, J., 2007. Antioxidant activity of methanolic extracts from some grains consumed in Korea. Food Chemistry 103, 130-138. 30. Chutia, M., Bhuyan, P.D., Pathak, M.G., Sarna, T.C., Boruah, P., 2009. Antifungal activity and chemical composition of Citrus reticulata Blanco essential oil against phytopathogens from North East India. Food Science and Technology 42(2009), 777-780. 31. Conner, D. E., Beuchat, L. R., 1984. Effects of essential oils from plants on growth of food spoilage yeasts. Journal of Food Science 49(2), 429-434. 32. Cristani, M., D'Arrigo, M., Mandalari, G., Castelli, F., Sarpietro, M.G., Micieli, D., 2007. Interaction of four monoterpenes contained in essential oils with model membranes: Implications for their antibacterial activity. Journal of Agriculture and Food Chemistry 55(15), 6300-6308. 33. Dabbah, R., Edwards, V.M., Moats, W.A., 1970. Antimicrobial Action of Some Citrus Fruit Oils on Selected Food-Borne Bacteria. Apply and Environmental Microbiology 19, 27-31. 34. Daferera, D.J., Ziogas, B.N., Polissiou, M.G., 2000. GC-MS analysisof essential oils from some Greek aromatic plants and their fungitoxicity on Penicillium digitatum. Journal Agricultural and Food Chemistry 48, 2576-2581. 35. Dang, M. N., Takacsova, M., Nguyen, D.V., Kristianova, K., 2001. Antioxidant activity of essential oils from various spices. Nahrung/Food 45, 64–66. 36. Deans, S. G, Svoboda, K.P., 1990. The antimicrobial proprieties of marjoram (Origanum majorana L.) volatile oil. Flavour and Fragrance Journal 5, 187–190. 37. Descalzo, A. M., Sancho, A. M., 2008. A review of natural antioxidants and their effects on oxidative status, odor and quality of fresh beef produced in Argentine. Meat Science 79, 423-436. 67 38. Donelian, A., Carlson, L. H. C., Lopes, T. J., and Machado, R. A. F., 2009. Comparison of extraction of patchouli (Pogostemon cablin) essential oil with supercritical CO2 and by steam distillation. The Journal of Supercritical Fluids 48, 15-20. 39. Dorman, H. J. D., Deans, S.G., 2000. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. Journal of Applied Microbiology 88, 308–316. 40. Doughari, J.H., Pukuma, M.S., De, N., 2007. Antibacterial effects of Balanites aegyptiaca L. Drel. and Moringa oleifera Lam. on Salmonella typhi. Biotechnology 6, 2212-2215. 41. Dugo, G. and Giacoma, A.D., 2002. Citrus: the genus Citrus, Taylor & Francis, London, England, UK. 42. Feng, W., and Zheng, X., 2007. Essential oils to control Alternaria alternatain vitro and in vivo. Food Control 18, 1126–1130. 43. Fernandez-Lopez, J., Viuda-Martos, M., Sendara, E., Sayas-Barbera, E., Navarro, C., Perez-Alvarez, J.A., 2007. Orange fibre as potential functional ingredient for dry-cured sausages. European Food Research and Technology 226, 1-6. 44. Fils, J.M., 2000. The Production of Oils, in: Hamm, W., Hamilton, R.J. (Eds.), Edible Oil Processing. Sheffield: Sheffield Academy Press, U.K. 45. Fisher, K., Phillips, C.A., 2006. The effect of lemon, orange and bergamot essential oils and their components on the survival of Campylobacter jejuni, Escherichia coli O157, Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus in vitro and in food systems. Journal of Applied Microbiology 101 (2006), 1232-1240. 46. Frankel, E.N., 1980. Lipid oxidation. Progress in Lipid Research 19, 1-22. 47. Frankel, E.N., 1984. Lipid oxidation Mechanisms, Products and Biological Significance. Journal of the American Oil Chemists’ Society 61(12), 1908-1917. 48. French, R.C., 1985. The bio-regulatory action of flavor compounds on fungal spores and other propagules. Annual Review Phytopatology 23, 173-199. 49. Ghasemi, K., Ghasemi, Y., Ebrahimzadeth, M.A., 2009. Antioxidant activity, phenol and flavonoid contents of 13 Citrus species peels and tissues. Journal of Pharmaceutical Sciences 22(3), 277 – 281. 50. Graven, E.H., Webber, L., Venter, M., Gardner, J.B., 1990. The development of Artemisia afra (Jacq.) as a new essential oil crop. Journal of Essential Oil Research 2, 215-220. 51. Gunther, E., 1952. The essential Oils. D. Van Nostrand Company, New York, London. 68 52. Habib, R.E., 2012. Antifungal activity of some essential oils on Aspergillus flavus growth and aflatoxin production. Journal of Food, Agriculture and Environment 10(40), 274-279. 53. Hammer, K.A., Carson, C.F., Riley, T.V., 1999. Antimicrobial activity of essential oils and other plant extracts. The society for applied microbiology 86, 985-990. 54. Hoa, T.T.V., Thao, T.T.P., Tam, V.T.T., 2007. Chemical compositions and antioxidative properties of Curcuma zedoaria Berg. from Vietnam. Journal of Science and Technology Development 10(4), 37-48. 55. Houghton, P. J., Raman, A., 1998. Laboratory handbook for the fractionation of natural extracts, first ed. London: Chapman and Hall. 56. Hussain, A. I., Anwar, F., Sherazi, S. T. H., Przybylski, R., 2008. Chemical composition. Antioxidant and antimicrobial activities of basil (Ocimum basilicum) essential oils depends on seasonal variations. Food Chemistry 108, 986-995. 57. Iglewski, B.H., 1996. Pseudomonas, in: Baton, S., (Eds.), Baron's Medical Microbiology. 58. Jeff-Agboola, Y.A., Onifade, A.K., Akinyele, B.J., Osho, I.B., 2012. In vitro antifungal activities of essential oil from Nigerian medicinal plants against toxigenic Aspergillus flavus. Journal of Medicinal Plants Research 6(23). 40484056. 59. Jesen, G.B., Hansen, B.M., Eilenberg, J., Mahillon, J., 2003. The hidden lifestyles of Bacillus cereus and relatives. Environmental microbiology 5, 631-640. 60. John P., M.D., Curran, Farouk, L., Al-Salihi, M.D., 1980. Neonatal staphylococcal scalded skin syndrome: massive outbreak due to an unusual phage type. Pediatrics 66(2), 285-90. 61. Jwanny, E.W., El-Sayed, S.T., Salem, A.M., Mabrouk, N.A., Shehata, A.N., 2012. Fraction, identification and biological activities of Egyptian Citrus peel extracts. Astralian Journal of Basic and Applied Sciences 6(4), 34-40. 62. Kabara, J.J., 1991. Phenols and chelators. Food preservatives, 200-214. 63. Kalemba, D., Kunicka A., 2003. Antibacterial and Antifungal Properties of Essential Oils. Current Medicinal Chemistry 10, 813-829. 64. Khajeh, M., Yamini, Y., Sefidkon, F., Bahramifar, N., 2004. Comparison of essential oil composition of Carum copticum obtained by supercritical carbon dioxide extraction and hydro-distillation methods. Food chemistry 86, 587-591 65. Khalfalliah, A., Label, A., Semra, Z., Kaki, B.A., Kabouche, A., Touzani, R., and Kabouche, Z., 2011. Antibacterial activity and chemical composition of the essential oil of Ammi visnaga L. (Apiaceae) from Constantine, Algeria. International Journal of Medicinal and Aromatic Plants 1(3), 302-305. 69 66. Kim, S.S., Baik, J.S., Oh, T.H., Yoon, W.J., Lee, N.H., Hyun, C.G., 2008. Biological activities of Korean Citrus obovoides and Citrus natsudaidai essential oils against acne-inducing bacteria. Bioscience, Biotechnoly and Biochemistry 72(10), 2507-13. 67. Kirbaslar, F.G., Tavman, A., Dulger, B., Turker, G., 2009. Antimicrobial activity of Turkish citrus peel oils. Parkistan Journal of botany 41(6), 3207-3212. 68. Kofidis, G., Bosabalidis, A., Kokkini, S., 2006. Seasonal variations of essential oils in a linalool-rich chemotype of Mentha spicata grown wild in Greece. Journal of Essential Oil Research 16, 469-472. 69. Krauze-Baranowska, M., Mardarowicz, M., Wiwart, M., Pobłocka, L., Dynowska, M., 2002. Antifungal Activity of the Essential Oils from Some Species of the Genus Pinus. Verlag der Zeitschrift für Naturforschung, 479-482. 70. Kurita, N., Miyaji, M., Kurane, R., Takahara, Y., 1981. Antifungal activity of components of essential oils. Agriculture and Biology Chemistry 45, 945–952. 71. Lanciotti, R., Fianotti, A., Patrignani, F., Belletti, N., Guerzoni, M.E., Gardini, F., 2004. Use of natural aroma compounds to improve shelf-life and safety of minimally processed fruits. Trends in Food Science and Technology 15, 201-208 72. Lan-Phi, N.T., 2010. Compositional Analysis, in: Sawamura, M., (Eds.), Citrus Essential Oils: Flavor and Fragrance. Wiley. 73. Lan-Phi, N.T., Nishiyama, C., Choi, H.S, Sawamura, M., 2006. Evaluation of Characteristic aroma compounds of Citrus natsudadai Hayata (Natsudadai) ColdPressed peel oil. Bioscience, Biotechnology and Biochemistry 70(8), 1832-1838. 74. Lawal, D., Bala, J.A., Aliyu, S.Y., Huhuma, M.A., 2013. Phytochemical screening and in vitro anti-bacterial studies of the ethanolic extract of Citrus sinesis (Linn) peel against some clinical bacterial isolates. International Journal of Innovation and Applied Studies 2(2), 138-145. 75. Layina-Pathirana, C.M., Shahidi, F., Alasalvar, C., 2006. Antioxidant activity of cherry laurel fruit (Laurocerasus officinalis Roem.) and its concentrated juice. Food Chemistry 99, 121-128. 76. Lota, M., Laure, S.D., De Rocca, T.F., Casanova, J., 2000. Chemical variability of peel and leaf essential oils of mandarins from Citrus reticulata Blanco. Journal Biochemistry Systematics and Ecology 28, 61-78. 77. Lucini, E.I., Zunino, M.P., Lopez, M.L., Zygadlo, J.A., 2006. Effect of monoterpenes on lipid composition and sclerotial development of Sclerotium cepivorum Berk. Journal of Phytopathology 154, 441-446. 78. Malhotra ,S., Suri, S., Tuli, R., 2009. Antioxidant activity of Citrus cultivars and chemical composition of Citrus karna essential oil. Planta Medica 75, 62-64. 70 79. Manner, H.I., Bulker, R.S., Smith, V.E., Ward, D., and Elevitch, C.R., 2006. Citrus (citrus) and Fortunella (kumquat), in: Elvitch, C.R. (Eds.). Species Profiles for Pacific Island Agroforestry. Hawaii. 80. Maridass, M., 2010. Antibacteri al activity of essential oi ls of Aromatic plants from South India. International Journal of Advances in Pharmaceutical Sciences 1, 176-180. 81. Maridass, M., 2010. Antibacteri al activity of essential oils of aromatic plants from South India. International Journal of Advances in Pharmaceutical Sciences 1 (2010), 176-180. 82. Martos, M.V., Navajas, Y.R., Lopez, J.F., Alvarez, J.P., 2007. Antbacterial activity of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.) and orange (Citrus sinesis L.) essential oils. Journal of food safety 28, 567-576. 83. Martos, M.V., Navajas, Y.R., Lopez, J.F., Alvarez, J.P., 2008. Antifungal activity of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapfruit (Citrus paradisi L.) and orange (Citrus sinesis L.) essential oils. Food control 19, 1130-1138. 84. McKillip, J.L., 2000. Prevalence and expression of enterotoxins in Bacillus cereus and other Bacillus spp., a literature review. Antonie Van Leeuwenhoek 77(4), 9393. 85. Milos, M., Radonic, A., Bezic, N., Dunkic, V., 2001. Localities and seasonal variations in the chemical composition of essential oils of Satureja montana L. and S. cuneifolia Ten. Flavor and Fragrance Journal 16, 157-160. 86. Mungdee, S., Chaisawadi, S., Panapiyasakunchai, J., Keawbunrueng, S., 2012. Natural antibacterial activities extracts from Thai pomelo, in: 38 th Congress on Science and Technology of Thailand. 87. NCCLS (National Committee for Clinical Laboratory Standards). 1997. Performance standards for antimicrobial disk susceptibility test. 88. Njoroge, S.M., Mungal, H.N., Koaze, H., Phi, N.T.L, Sawamura, M., 2006. Volatile constituents of mandarin Citrus reticulata Blanco peel oil from Burundi. Journal of Essential Oil Research 18, 659-662. 89. Njoroge, S.M., Sawamura, M., 2010. Africa, in: Swamura, M., (Eds.), Citrus Essential Oils: Flavor and Fragrance. Wiley. 90. Omer E. A., Khattab, M. E., Ibrahim, M. E., 1998. First cultivation trial of Perilla frutescens L. in Egypt. Flavor and Fragrance Journal 13, 221. 91. Omidbeygi, M., Barzegar, M., Hamidi, Z., Nafhdibadi, H., 2007. Antifungal activity of thyme, summer savory and clove essential oils against Aspergillus flavus in liquid medium and tomato paste. Food Control 18(12), 1518-1523. 71 92. Pelczar, M.J., Chan, E.C.S., Krieg, N.R., 1993. Microbiology Concepts and Application, first ed. 93. Pitchaon, M., 2011. Antioxidant capacity of extracts and fractions from mango (Mangifera indica Linn.) seed kernels. International Food Research Journal 18, 523-528 94. Renzini, G., Scazzocchio, F., Lu, M., Mazzanti, G., Salvatore, G., 1999. Antibacterial and cytotoxic activity of Hyssopus officina-lis L. oils. Journal of Essential Oil Research 11, 649 – 654. 95. Reverchon, E., 1997. Supercritical fluid extraction and fractionation of essential oils and related products. Journal of Supercritical Fluids 10, 1-37. 96. Ruberto, G., Baratta, M.G., 2000. Antioxidant activity of selected essential oil components in two lipid model systems. Food Chemistry 69, 167–174. 97. Rudback, J., Bergstrom, M.A., Borje, A., Nilsson, U., Karlberg, A.T., 2012. α- Terpinene, an antioxidant in tea tree oil, autoxidizes rapidly to skin allergens on air exposure. Chemical Research in Toxicology 25(3), 21-713. 98. Ryan, K.J., Ray, C.G., 2004. Sherris Medical Microbiology, fourth ed. McGraw Hill. 99. Rydberg, J., Choppin, G.R., Musikas, C., Sekine, T., 2004. Solvent Extraction Equilibra, in: Rydberg, J., Cox, M., Musikas, C. (Eds.), Solvent Extraction Principles and Practice. Marcel Dekker, New York. 100. Sadaf, S., Bhatti, H.N., Iqbal, Z., Shahid, M., 2009. Chemical composition, antioxidant and antimicrobial activities of Citrus Jambhiri Lush and Citrus Reticulata Blanco essential oils. Journal of the Chemical Society of Pakistan 31(5), 838-844. 101. Sawamura, M., 2010. Citrus essential oils: Flavor and fragance. Wiley. 102. Senatore, F., Napolitano, F., Ozcan, M., 2000. Composition and antibacterial activity of the essential oil from Crithmum maritimum L. (Apiaceae) growing wild in Turkey. Flavor and Fragrance Journal 15, 186– 189. 103. Shah M., 2012. Foodborne and Waterborne Bacterial Pathogens: Epidemiology, Evolution and Molecular Biology. Caister Academic Press. 104. Shapiro, S., Meier, A., Guggenheim, B., 1994. The antimicrobial activity of essential oils and essential oil components towards oral bacteria. Molecular Oral Microbiology 9, 202. 105. Sharma, N., Tripathi, A., 2006b. Effects of Citrus sinesis (L.) Osbeck epecarp essential oil on growth and morphogenesis of Aspergillus niger (L.) Van Tieghem. Microbiological Research, Available online. 106. Sharma, N., Tripathi, A., 2008. Effects of Citrus sinesis (L.) Osbeck epicarp essential oil on growth and morphogenesis of Aspergillus niger (L.) Van Tieghem. Microbiology Research 163, 337-344. 72 107. Shaw, P.E., 1979. A review of quantitative analysis of citrus essential oils. Journals of Agricultural and Food chemistry 27, 246-257. 108. Simic, N.P.R., Vajs, V., Milosavljevic, S., Djokovic, D., 2000. Composition and antibacterial activity of Achillea chrysocoma essential oil. Journal of Essential Oil Research 12, 784. 109. Singh, A. K., Raina, V.K., Naqvi, A.A., Patra, N.K., Kumar, B., Ram, P., Khanuja, S.P.S., 2005. Essential oil composition and chemo arrays of menthol mint (Mentha arvensis L. f. Piperascens Malinvaud ex. Hoimes) cultivars. Flavor and Fragrance Journal 20, 302-305. 110. Skandamis, P., Koutsoumanis, K., Fasseas, K., Nychas, G.J.E., 2001. Inhibition of oregano essential oil and EDTA on Escherichia coli O157:H7. Italian Journal of Food Science 13 (1), 65 – 75. 111. Sokovic, M., Marin, P.D., Brkic, D., Griensven, L.J.L.D., 2007. Chemical composition and antibacterial activity of essential oils of ten aromatic plants against human pathogenic bacteria. Global Science Books Journal 1(1). 112. Sood, S., Dhiraj, V., Nagar, P.K., 2006. Physiological and biochemical studies during flower development in two rose species. Scientia Hoticulturae 108, 390396 113. Suganya, S., Bharathidasan, Panneerselvam, A., 2012. Coriandrum sativam R., Senthilkumar, G., Madhanraj, P. and Antibacterial activity of essential oil extracted from (L.) and GC-MS analysis. Journal of Chemical and Pharmaceutical Research 4(3), 1846-1850. 114. Suklampoo, L., Thawai, C., Weethong, R., Champathong, W., Wongwongsee, W., 2012. Antimicrobial activities of crude extracts from pomelo peel of Khao-nahmpeung and Khao-paen varieties. King Mongkut's Institute of Technology Ladkrabang Science and Technology Journal 12(1), 55-61. 115. Takhi, D., Ouinten M., Yousfi, M., 2011. Study of Antimicrobial Activity of Secondary Metabolites Extracted from Spontaneous Plants from the Area of Laghouat, Algeria. Advances in Environmental Biology 5(2), 469-476. 116. Thompson, W.L., Wannemacher, R.W.Jr., 1990. In vivo effects of T-2 mycotoxin on synthesis of proteins and DNA in rat tissues. Toxicology and Applied Pharmacology 105(3), 482–491. 117. Ueno, Y., 1989.Trichothecene mycotoxins: Mycology, chemistry, and toxicology. Advances in Food & Nutrition Research 3, 301–353. 118. Ultee, A., Smid, E.J., 2001. Influence of carvacrol on growth and toxin production by Bacillus cereus. International Journal of Food Microbiology 64, 373–378. 73 119. Upadhyay, R.K., Dwivedi, P., and Ahmad, S., 2010. Screening of antibacterial activity of six plant essential oils against pathogenic bacterial strains. Asian Journal of medicinal sciences 2(3), 152-158. 120. Uribe, S., Ramirez, J., Pena, A., 1985. Effects of beta-pinene on yeast membrane functions. Journal of Bacteriology 161, 1195-1200. 121. Uribe-Hernandez, C. J., Hurtado-Ramos, J.B., Olmedo-Arcega, E.R., MartinezSosa, M.A., 1992. The essential oil of Lippia graveolens H.B.K. from Jalisco, Mexico. Journal of Essential Oil Research 4, 647-649. 122. Van de Braak, S. A. A. J., Leijten, G. C. J. J., 1999. Essential Oils and Oleoresins: A Survey in the Netherlands and other Major Markets in the European Union. CBI, Centre for the Promotion of Imports from Developing Countries, Rotterdam. 123. Veldhuizen, E.J., Tjeerdsma-van Bokhoven, J.L., Zeijtzer, C., Burt, S.A., Haagsman, H.P., 2006. Structural requirements for the animicrobial activity of carvacrol. Journal of Agricultural and Food Chemistry 54, 1874-1879. 124. Viljoen, A. M., Petkar, Van-Vuuren, S.F., Cristina Figueiredo, A., Pedro, L.G., Barroso, J.G., 2006. Chemo-Geographical Variation in Essential Oil Composition and the Antimicrobial Properties of "Wild Mint"- Mentha longifolia subsp. polyadena (Lamiaceae) in Southern Africa. Journal of Essential Oil Research 18, 60-65. 125. Vokou, D., Kokkini, S., Bessiere, J.M., 1993. Geographic variation of Greek oregano (Origanum vulgare ssp. hirtum) essential oils. Biochemical Systematic and Ecology 21, 287–295. 126. Wungstintaweekul, J., Sitthithaworn, W., Putalun, W., Pfeifhoffer, H.W., Brantner, A., 2010. Antimicrobial, antioxidant activities and chemical composition of selected Thai spices. Songklanakarin Journal of Science and Technology 32(6), 589-598. 74 APPENDIX Appendix 1. Chromatogram of LD-CP (on the top) and LD-VA EOs (on the bottom). 1: α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7: terpinolele, 8: ٧-terpinene, 9: Linalool 75 Appendix 2. Chromatogram of LDL-CP (on the top) and LDL-VA EOs (on the bottom). 1: α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7: terpinolele, 8: ٧-terpinene, 9: Linalool 76 Appendix 3. Chromatogram of LLA-CP (on the top) and LLA-VA EOs (on the bottom). 1: α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7: terpinolele, 8: ٧-terpinene, 9: Linalool 77 Appendix 4. Chromatogram of PTT-CP (on the top) and PTT-VA EOs at 70oC (on the middle) and 80oC (on the bottom). 1: α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7: terpinolele, 8: ٧-terpinene, 9: Linalool 78 Appendix 9. Chromatogram of PTTR-CP (on the top) and PTTR-VA EOs (on the bottom). 1: α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7: terpinolele, 8: ٧-terpinene, 9: Linalool 79 Appendix 10. Chromatogram of PDH-CP (on the top) and PDH-VA EOs (on the bottom). 1: α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7: terpinolele, 8: ٧-terpinene, 9: Linalool 80 Appendix 11. Chromatogram of OV-CP (on the top) and OV-VA EOs (on the bottom). 1: α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7: terpinolele, 8: ٧-terpinene, 9: Linalool 81 Appendix 12. Chromatogram of OX-CP (on the top) and OX-VA EO (on the bottom). 1: α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7: terpinolele, 8: ٧-terpinene, 9: Linalool 82 Appendix 13. Chromatogram of OPT-CP (on the top) and OPT-VA EOs (on the bottom). 1: α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7: terpinolele, 8: ٧-terpinene, 9: Linalool 83 B. cereus LD LDL LLA PTT PTTR PDH OV OX OPT Appendix 14. Illustration of control and Citrus EOs on the growth of B. cereus (first row: cold-pressed EO, the second row: vacuum distillated EO) S. Typhi LD LDL LLA PTT PTTR PDH OV OX OPT Appendix 15. Illustration of control and Citrus EOs on the growth of S. typhi (first row: cold-pressed EO, the second row: vacuum distillated EO) 84 P.aerugi nosa LD LDL LLA PTT PTTR PDH OV OX OPT Appendix 16. Illustration of control and Citrus EOs on the growth of P. aeruginosa (first row: cold-pressed EO, the second row: vacuum distillated EO) S. aureus LD LDL LLA PTT PTTR PDH OV OX OPT Appendix 17. Illustration of control and Citrus EOs on the growth of S. aureus (first row: cold-pressed EO, the second row: vacuum distillated EO) 85 A. Flavus LD LDL LLA PTT PTTR PDH OV OX OPT Appendix 18. Illustration of control and Citrus EOs on the growth of A. flavus (first row: cold-pressed EO, the second row: vacuum distillated EO) F. solani LD LDL LLA PTT PTTR PDH OV OX OPT Appendix 19. Illustration of control and Citrus EOs on the growth of F. solani (first row: cold-pressed EO, the second row: vacuum distillated EO) 86 Samples Culture tubes Controls TSB medium & Bacteria TSB medium & Ethanol & Bacteria Concentration (mg/ml) 0.655 1.31 2.63 5.25 10.5 21 42 PTT-CP PTT-VA PTTRCP PTTRVA PDH-CP 87 PDH-VA OX-CP OX-VA OV-CP OV- VA OPT-CP 88 OPT-VA LLA-CP LLA-VA LDL-CP LDL-VA LD-CP 89 LD-VA Appendix 20. Effect of Citrus EOs B. cereus at different essential oil concentrations. 90 Samples Culture tubes Controls TSB medium & Bacteria TSB medium & Ethanol & Bacteria Concentration (mg/ml) 0.655 1.31 2.63 5.25 10.5 21 42 PTT-CP PTT-VA PTTRCP PTTRVA PDH-CP 91 PDH-VA OX-CP OX-VA OV-CP OV- VA OPT-CP 92 OPT-VA LLA-CP LLA-VA LDL-CP LDL-VA LD-CP 93 LD-VA Appendix 21. Effect of Citrus EOs S.typhi at different essential oil concentrations. 94 Samples Culture tubes TSB Controls TSB medium medium & & Bacteria Ethanol 0.655 1.31 Concentration (mg/ml) 2.63 5.25 10.5 21 42 & Bacteria PTT-CP PTT-VA PTTR-CP PTTRVA 95 PDH-CP PDH-VA OX-CP OX-VA OV-CP OV-VA 96 OPT-CP OPT-VA LLA-CP LLA-VA LDL-CP LDL-VA 97 LD-CP LD-VA Appendix 22. Effect of Citrus EOs P. aeruginosa at different essential oil concentrations. 98 Samples Culture tubes TSB Controls TSB medium medium & & Bacteria Ethanol 0.655 1.31 Concentration (mg/ml) 2.63 5.25 10.5 21 42 & Bacteria PTT-CP PTT-VA PTTR-CP PTTRVA 99 PDH-CP PDH-VA OX-CP OX-VA OV-CP OV-VA 100 OPT-CP OPT-VA LLA-CP LLA-VA LDL-CP LDL-VA 101 LD-CP LD-VA Appendix 23. Effect of Citrus EOs S. aureus at different essential oil concentrations. 102 Samples Culture tubes TSB Controls TSB medium medium & Fungi & 0.655 1.31 Concentration (mg/ml) 2.63 5.25 10.5 21 42 Ethanol & Fungi PTT-CP PTT-VA PTTR-CP PTTRVA 103 PDH-CP PDH-VA OX-CP OX-VA NI OV-CP OV-VA 104 OPT-CP OPT-VA NI LLA-CP LLA-VA LDL-CP LDL-VA 105 LD-CP LD-VA Appendix 24. Effect of Citrus EOs A. flavus at different essential oil concentrations. 106 Samples Culture tubes TSB Controls TSB medium medium & Fungi & 0.655 1.31 Concentration (mg/ml) 2.63 5.25 10.5 21 42 Ethanol & Fungi PTT-CP PTT-VH PTTR-CP 107 PTTRVA PDH-CP PDH-VA OX-CP OX-VA OV-CP 108 OV-VA OPT-CP OPT-VA LLA-CP LLA-VA LDL-CP 109 LDL-VA LD-CP LD-VA Appendix 24. Effect of Citrus EOs F. solani at different essential oil concentrations 110 [...]... antibacterial functions of the citrus essential oils from four varieties of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.), and orange (Citrus sinesis L.) and found that all of these essential oils displayed strong antibacterial activity against the strains tested Testing and evaluation of antimicrobial activity of essential oils is difficult because of their characteristics... regions, including three major subregions in the north of Vietnam (the mountainous midland region, the Red River Delta, and the northern central coast), in total accounting for 35-40% of citrus production in Vietnam The Mekong River Delta in the south of Vietnam accounting for 55-60% of citrus production, and the rest concentrated in the central provinces (Agro, 2006) The citrusgrowing areas have increased... the proportion of α-terpineol in this oil remained at higher level than orange and mandarin oils, the amount of linalool was low (only 0.12%) Linalool, in previous studies, plays an important role in inhibition ability of peroxidation and caused essential oils possessing antioxidant activity (Hussain et al., 2008) For the volatile components of Indian Citrus sinesis (L.) Osbeck essential oil, limonene... 2.2 shows the main volatile component of citrus essential oils from several countries Table 2.2: The main volatile components (%w/w) of several citrus essential oils No Compound Vietnamese Vietnamese Vietnamese India orange mandarin pomelo orange (Citrus sinensis) (Citrus reticulata (Citrus grandis (Citrus sinesis (Ref.71) Blanco) Osbeck) (L.) Osbeck) (Ref.71) (Ref.71) (Ref.105) 1 α-pinene 0.81 0.93... collection and preparation Extraction essential oils by cold pressing method Evaluation of antimicrobial activities Evaluation of antimicrobial activities by dilution method Evaluation of antimicrobial activities by diffusion method Extraction essential oils by vacuum distillation method Evaluation of antioxidant activities Evaluation of antioxidant activities by DPPH assay Evaluation of antioxidant activities. .. in mandarin (Citrus reticulata Blanco) essential oil were limonene (91.58%), followed by myrcene (2.79%), and α-pinene (0.93%) β-pinene was important to mandarin aroma and flavor β-pinene presented at the level of 0.60% in this oil Terpene compounds are the most reasons lead to antimicrobial activities of citrus essential oils These components pass the cell membranes, penetrates into the interior of. .. growth results in production of enterotoxins, one of which is highly resistant to heat and to pH between 2 and 11; ingestion leads to two types of illness: one type characterized by diarrhea and the other, called emetic toxin, by nausea and vomiting Several reports studied on antibacterial activities of essential oils on B cereus Citrus lemon and Azadirachta indica essential oils showed high inhibition... bacteremia, bone and joint infections, gastrointestinal infections and a variety of systemic infections, particularly in patients with burn and in cancer and AIDS patients who are immune-suppressed P aeruginosa infection is a serious problem in patients hospitalized with cancer, cystic fibrosis, and burns (Ryan and Ray, 2004) A number of publications demonstrated that various essential oils possessing antibacterial... and a DB-1 column (30m x 0.25mm i.d, film thickness of 0.25µm) The column temperature was initially maintained at 70oC for 2 min, and gradually increased at the rate 2oC per min to 32 240oC, at which the temperature was held for 20 minutes Nitrogen was used as carrier gas at flow rate of 0.7 ml/min 3.5 Antimicrobial activities of citrus essential oils 3.5.1 Microbial strains In order to determine antimicrobial. .. REVIEW Essential oils An essential oil is a concentrated hydrophobic liquid containing volatile aroma compounds from plants It is less soluble in water Essential oil is extracted from plant so it carries a distinctive scent, or essence, of the plant and is therefore applied in food flavoring and perfumery (Gunther, 1952) The occurrence of essential oils is restricted to over 2000 plant varieties from ... of citrus essential oils 39 4.3 Chemical compositions of citrus essential oils 40 4.3.1 Chemical compositions of lime essential oils 40 4.3.2 Chemical compositions of. .. yield and quality of citrus essential oils Therefore, the objective of this study is to determine chemical composition, antimicrobial and antioxidant activities of Vietnamese citrus essential oils. .. of inhibition (mm) of citrus essential oils against S aureus 51 Table ‎ MIC values (mg/ml) of citrus essential oils for S aureus 53 Table ‎ Zone of inhibition (mm) of citrus essential oils