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: />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: />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: />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: />
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