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

In the present study, Vietnamese citrus essential oils from nine varieties were extracted by two methods, cold pressing and vacuum hydro-distillation.. Although the yield of essential o

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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

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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 (0.02-21.89%), 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 (0.02-0.43%) 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

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

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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

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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

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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

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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 70oC 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

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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

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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)

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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 picrylhydrazyl radical scavenging Some recent publications showed antioxidant activities

2,2-diphenyl-1-of these essential oils (Baik et al., 2008) Other study observed the effects 2,2-diphenyl-1-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

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)

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

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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

sub-regions 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 citrus- growing 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 orange

(Citrus sinensis)

(Ref.71)

Vietnamese mandarin

(Citrus reticulata Blanco)

(Ref.71)

Vietnamese pomelo

(Citrus grandis Osbeck)

(Ref.71)

India orange

(Citrus sinesis (L.) Osbeck)

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)

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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

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

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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

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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 dioxidehas 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

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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)

Today, GC and GC-MS are common instruments in most laboratories because they are sensitive, accurate, and convenient

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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

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

20

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

21

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)

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)

23

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)

24

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

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

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

27

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

LOO-+AH  A- +LOO-

A-+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

28

3 Chapter 3: Materials and methods

3.1 Experimental design

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

Material collection and preparation

Extraction essential oils

DPPH assay

Evaluation of antioxidant activities by

lipid peroxidation

diffusion

method

29

Table 3.1 Citrus samples in the study

Common

name

Scientific name Place collection Image of whole fruit Image of cross section

Xoan orange Citrus sinensis Osbeck Lap Vo District, Dong Thap

Viet Tri City, Phu Tho Province

31

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):

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

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

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 F-824) and Fusarium solani

(VTCC-F-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):

Where

N: Number of colonies on plate (Colony forming unit: CFU)

VS: Volume pipetted onto Petri plate (ml)

D: Dilution factor for test tube plated out

: Concentration of cells in original sample (CFU/ml)

D

1 V

N ρS



34

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

35

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):

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):

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)

36

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)

37

4 Chapter 4: Results and Discussion

4.1 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 70oC 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 70oC 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

Cold pressing Vacuum distillation

Tr

1.08±0.04 0.18±0.00 0.82±0.01 1.82±0.01 0.25±0.00 80.08±0.46 0.04±0.00 10.82±0.04

Tr

1.28±0.00 0.19±0.00 - 1.84±0.00 0.27±0.00 81.89±0.01 0.05±0.00 12.29±0.01 -

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

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 80oC 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 β-pinene, 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

39

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

40

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

γ-terpinene, which made up 12.14 and 2.25% in LDL-CP and LDL-VA, and α-γ-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