Thông tin tài liệu
VIETNAM NATIONAL UNIVERSITY – HOCHIMINH CITY
INTERNATIONAL UNIVERSITY
CHEMICAL COMPOSITION, ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES OF
ESSENTIAL OILS EXTRACTED FROM CITRUS VARIETIES IN VIETNAM
A thesis submitted to
the School of Biotechnology, International University
in partial fulfillment of the requirements for the degree of
MSc. in Biotechnology
Student name: Phạm Thị Lan Chi – MBT04003
Supervisor:
Phạm Văn Hùng, PhD.
Nguyễn Thị Lan Phi, PhD.
May/2013
i
ABSTRACT
Essential oils (EOs) are complex mixtures of biologically active substances used since a
long time as flavoring agents and preservatives of a number of commercial products. The
important characteristics of essential oils are their antioxidant and antimicrobial potential.
In the present study, Vietnamese citrus essential oils from nine varieties were extracted by
two methods, cold pressing and vacuum hydro-distillation. The essential oils were
characterized for their chemical compositions, antioxidant and antimicrobial activities. GC
analysis of the chemical compositions of the isolated essential oils revealed the presence of
nine main compounds. The total concentrations of these compounds were from 63.06% to
99.27%, including α-pinene (0.04-1.98%), sabinene (0.15-3.89%), β-pinene (0.0221.89%),
myrcene
(0.96-41.95%),
α-terpinene
(0.02-11.41%),
limonene
(40.29-
95.78%), terpinolene (0.02-0.57%), ٧-terpinene (0.01-12.14%) and linalool (0.020.43%). Both cold pressed essential oils and vacuum hydro-distillated essential oils show
strong antioxidant and antimicrobial activities. For antioxidant capacity, the lime showed
the strongest, followed by pomelo and orange EOs. The lime also had the highest
antimicrobial capacity as compared to other citrus essential oils. The minimal inhibition
capacity (MIC) was a range of 0.66–42 mg/ml for lime EOs, 5.25- 42 mg/ml for orange
EOs, 2.63-42 mg/ml for pomelo EOs. Although the yield of essential oils extracted by the
vacuum hydro-distillation method was higher than that by the cold-pressing method, the
antioxidant and antimicrobial capacities of hydro-distillated essential oils were lower than
those of the cold pressed extracts for all citrus varieties. The results of this study show
that the vacuum hydro-distillation method used in this study could be developed to be
used in industrial application and the citrus essential oils could be widely used as flavoring
and preservatives in food, cosmetic and pharmaceutical industries.
ii
ACKNOWLEDGEMENTS
Firstly, I would like to express my love to my parents and my younger brother for their
unconditional love, help and support for my study at International University.
Secondly, I am so grateful to my supervisors, Dr Pham Van Hung and Dr Nguyen Thi Lan
Phi for their help and guidance throughout my research. Without their enthusiastic help
and advices, this thesis would not be completed.
Moreover, I would like to give my thanks to all staffs in the laboratory rooms for pleasure
provide me with all chemicals and equipments needed.
Finally, I also would like to thank all my friends who are always there to give me support
and provide their special knowledge and talent in this research. I am very grateful for
meeting you and for our relationship. Your encouragement and help is endless.
iii
TABLE OF CONTENTS
ABSTRACT ............................................................................................................................ i
ACKNOWLEDGEMENTS........................................................................................................ iii
TABLE OF CONTENTS .......................................................................................................... iv
LIST OF FIGURES................................................................................................................ vi
LIST OF TABLES................................................................................................................. vii
ABBREVIATION ................................................................................................................ viii
Chapter 1: INTRODUCTION ......................................................................................... 9
Chapter 2: LITERATURE REVIEW .............................................................................. 11
2.1
Essential oils .......................................................................................................... 11
2.2
Citrus essential oils ................................................................................................. 11
2.3
Extraction methods ................................................................................................. 13
2.3.1
Hydrodistillation method ................................................................................... 13
2.3.2
Solvent extraction ............................................................................................ 14
2.3.3
Supercritical carbon dioxide method ................................................................... 15
2.3.4
Cold pressing method ....................................................................................... 15
2.4
Gas chromatography ............................................................................................... 16
2.5
Food poisoned microorganisms ................................................................................. 18
2.5.1
Staphylococcus aureus ..................................................................................... 18
2.5.2
Bacillus cereus ................................................................................................. 19
2.5.3
Salmonella typhi .............................................................................................. 20
2.5.4
Pseudomonas aeruginosa .................................................................................. 21
2.5.5
Aspergillus flavus ............................................................................................. 22
2.5.6
Fusarium solani ............................................................................................... 23
2.6
Antimicrobial activity of essential oils ........................................................................ 24
2.7
Antioxidant activity of essential oils........................................................................... 26
Chapter 3: Materials and methods ............................................................................ 28
3.1
Experimental design ................................................................................................ 28
3.2
Materials collection and preparation .......................................................................... 28
3.3
Essential oils extraction ........................................................................................... 31
3.3.1
Cold pressing method ....................................................................................... 31
3.3.2
Vacuum distillation method ............................................................................... 31
iv
3.4
Chemical composition analysis ................................................................................. 32
3.5
Antimicrobial activities of citrus essential oils ............................................................. 33
3.5.1
Microbial strains............................................................................................... 33
3.5.2
Microorganisms counting .................................................................................. 33
3.5.3
Diffusion technique .......................................................................................... 34
3.5.4
Dilution technique ............................................................................................ 34
3.6
Antioxidant activities of citrus essential oils ................................................................ 34
3.6.1
DPPH assay ..................................................................................................... 34
3.6.2
Ferric thiocyanate (FTC) assay ........................................................................... 35
3.7
Data analysis ......................................................................................................... 36
Chapter 4: Results and Discussion ............................................................................ 37
4.1
Optimization conditions for vacuum distillation extraction ............................................ 37
4.1.1
Temperature optimization ................................................................................. 37
4.1.2
Time optimization ............................................................................................ 38
4.2
Yield of citrus essential oils ...................................................................................... 39
4.3
Chemical compositions of citrus essential oils ............................................................. 40
4.3.1
Chemical compositions of lime essential oils ........................................................ 40
4.3.2
Chemical compositions of orange essential oils .................................................... 43
4.3.3
Chemical compositions of pomelo essential oils .................................................... 45
4.4
Antioxidant activities of citrus essential oils ................................................................ 47
4.4.1
DPPH assay ..................................................................................................... 47
4.4.2
FTC assay ....................................................................................................... 49
4.5
Antimicrobial activities............................................................................................. 51
4.5.1
S. aureus ........................................................................................................ 51
4.5.2
B. cereus ........................................................................................................ 54
4.5.3
S. typhi .......................................................................................................... 56
4.5.4
P. aeruginosa .................................................................................................. 58
4.5.5
A. flavus ......................................................................................................... 59
4.5.6
F. solani .......................................................................................................... 62
Chapter 5: CONCLUSION ........................................................................................... 64
REFERENCES...................................................................................................................... 65
APPENDIX ......................................................................................................................... 75
v
LIST OF FIGURES
Figure 2
.1 Diagram of Gas chromatography ............................................................... 16
Figure 2
.2 Principle of Gas Chromatography ............................................................... 17
Figure 2
.3 Scanning electron micrograph of S. aureus. ................................................ 19
Figure 2
.4 Rod-shaped Bacillus cereus. ..................................................................... 20
Figure 2
.5 Flagella stain of Salmonella typhi. ............................................................. 21
Figure 2
.6 Pseudomonas aeruginosa ......................................................................... 22
Figure 2
.7 Aspergillus flavus colony surface ............................................................... 23
Figure 2
.8 Fusarium solani colony surface .................................................................. 24
Figure 3
.1 Flow chart of experimental process ............................................................ 28
Figure 3
.2 Model system used in extraction citrus essential oils in vacuum distillation
process. ................................................................................................................ 32
Figure 4
.1 Effect of extraction time on yield (%) of essential oil ................................... 38
Figure 4
.2 Extraction yield of citrus peel extracts using different extracting methods. ..... 39
Figure 4
.3 IC50 values of investigated citrus oils by DPPH method. ............................... 47
Figure 4
.4 IC50 values of investigated citrus oils by FTC method. ................................. 49
vi
LIST OF TABLES
Table 2
.1: Scientific classification ............................................................................. 11
Table 2
.2: The main volatile components (%w/w) of several citrus essential oils ............ 12
Table 3
.1 Citrus samples in the study........................................................................ 29
Table 4
.1 Volatile compositions (%w/w) of Tan Trieu peel EOs extracted at 70 oC and 80oC
by vacuum distillation method and cold pressing method. ............................................ 37
Table 4
.2 Volatile compositions (%w/w) of Vietnamese lime peel EOs extracted by cold
pressing and vacuum distillation method ................................................................... 42
Table 4
.3 Volatile compositions (%w/w) of Vietnamese orange peel EOs extracted by cold
pressing and vacuum distillation method ................................................................... 44
Table 4
.4 Volatile compositions (%w/w) of Vietnamese pomelo peel EOs extracted by cold
pressing and vacuum distillation method ................................................................... 46
Table 4
.5 Zone of inhibition (mm) of citrus essential oils against S. aureus .................... 51
Table 4
.6 MIC values (mg/ml) of citrus essential oils for S. aureus ............................... 53
Table 4
.7 Zone of inhibition (mm) of citrus essential oils against B. cereus .................... 54
Table 4
.8 MIC values (mg/ml) of citrus essential oils for B. cereus ................................ 55
Table 4
.9 Zone of inhibition (mm) of citrus essential oils against S. typhi ...................... 56
Table 4
.10 MIC values (mg/ml) of citrus essential oils for S. typhi ................................ 57
Table 4
.11 Zone of inhibition (mm) of citrus essential oils against P. aeruginosa ............ 58
Table 4
.12 MIC values (mg/ml) of citrus essential oils for P. aeruginosa ........................ 59
Table 4
.13 Zone of inhibition (mm) of citrus essential oils against A.flavus .................... 60
Table 4
.14 MIC values (mg/ml) of citrus essential oils for A. flavus ............................... 61
Table 4
.15 Zone of inhibition (mm) of citrus essential oils against F.solani ..................... 62
Table 4
.16 MIC values (mg/ml) of citrus essential oils for F. solani ............................... 63
vii
ABBREVIATION
LLA-CP: Long An lime essential oil extracted by cold pressing method
LLA-VA: Long An lime essential oil extracted by vacuum distillation method
LDL-CP: Da Lat lime essential oil extracted by cold pressing method
LDL-VA: Da Lat lime essential oil extracted by vacuum distillation method
LD-CP: Dao lime essential oil extracted by cold pressing method
LD-VA: Dao lime essential oil extracted by vacuum distillation method
OX-CP: Xoan orange essential oil extracted by cold pressing method
OX-VA: Xoan orange essential oil extracted by vacuum distillation method
OPT-CP: Phu Tho orange essential oil extracted by cold pressing method
OPT-VA: Phu Tho orange essential oil extracted by vacuum distillation method
OV-CP: Vinh orange essential oil extracted by cold pressing method
OV-VA: Vinh orange essential oil extracted by vacuum distillation method
PTT-CP: Tan Trieu pomelo essential oil extracted by cold pressing method
PTT-VA: Tan Trieu pomelo essential oil extracted by vacuum distillation method
PTTR-CP: Thanh Tra pomelo essential oil extracted by cold pressing method
PTTR-VA: Thanh Tra pomelo essential oil extracted by vacuum distillation method
PDH-CP: Doan Hung pomelo essential oil extracted by cold pressing method
PDH-VA: Doan Hung pomelo essential oil extracted by vacuum distillation method
EOs: Essential oils
CP: Cold pressing
VA: Vacuum distillation
TSB: Tryptone Soybean Broth
TSA: Tryptone Soybean Agar
PDA: Potato Dextrose Agar
CFU: Colony forming unit
MIC: Minimum inhibition concentration
S. aureus: Staphylococcus aureus
B. cereus: Bacilus cereus
S. typhi: Salmonella typhi
P. aeruginosa: Pseudomonas aeruginosa
A. flavus: Aspergillus flavus
F. solani: Fusarium solani
viii
1
Chapter 1: INTRODUCTION
The use of synthetic agents with antimicrobial and antioxidant activity is the technique for
extending the shelf-life of foods. However, over the past two decades, there is a great
public concern about safety and side effects of synthetic agents in food preservation
besides health implications. These agents are known to have toxic and carcinogenic effects
on human and food systems. Therefore, recent studies interest in developing safer
compounds based on natural sources, as alternatives, to prevent from the deterioration of
foods (Ayoola et al., 2008). Plants and plant extracts are recognized as potential sources
of natural compounds to improve the shelf life and the safety of food. Especially, essential
oils extracted from citrus species are the best candidates to replace synthetic additives in
food preservation. Having functional properties such as antimicrobial and antioxidant
activities, citrus essential oils can lengthen the food shelf life and avoid health-related
problems, off odors, unpleasant tastes or changes in color.
Citrus is a common term and genus of flowering plants that belongs to the rue family,
Rutaceae, originating and growing extensively in tropical and subtropical southern regions
of Asia. Citrus oils which obtained from citrus fruits like oranges, lime, and pomelo called
argument oils that are considered generally recognized as safe (GRAS) (Kabara, 1991).
The flavedo of citrus fruits is the main section containing essential oils. Citrus essential oils
are a mixture of volatile compound and mainly consisted of monoterpenes hydrocarbons
(Baik et al., 2008). In addition, these oils comprise over a hundred other constituents that
can be divided into two fractions: sequiterpene hydrocarbons and oxygenated compounds
(Sana et al., 2009, Baik et al., 2008). On the account of the fact that the yield, chemical
composition and biological properties of the essential oils are affected by geographical
regions and extraction methods (Njoroge et al., 2006), that is the reason why people put a
high attention in the part of selecting locations and extracting methods for expected
quality.
There are two common methods used to extract citrus essential oils. Citrus essential oils
are usually extracted by cold pressing method. In this technique, the smell of cold pressed
oil is natural, chemical composition is conservative. Nevertheless, the yield of essential oils
extracted using this process is low (Fils, 2000). Also, it is important to note that the oils
extracted by this method have a relatively short shelf-life (Lan-Phi et al., 2006). The other
method for citrus essential oils extraction is hydro-distillation. It is a method which has
wide acceptance for large scale production because it produces high yield of essential oils.
However, the drawback of this process is that the hydrolysable compounds such as ester,
as well as thermally labile components, may be decomposed during the distillation process
(Houghton and Raman, 1998). Furthermore, some chemical changes are related to
antimicrobial and antioxidant activities of essential oils (Chemat, 2010).
9
It is well known that essential oils from citrus species possess antimicrobial activity against
both bacteria and fungi (Lanciotti et al., 2004). Dilution and diffusion methods are basic
techniques for the assessment of both antibacterial and antifungal activities of essential
oils. Diffusion method is recommended as a pre-screening method for a large number of
essential oils, so as the most active ones may be selected for further analysis by means of
more sophisticated methods. On the other hand, the aim of dilution method is to
determine the lowest concentration of the assayed antimicrobial agent (minimal inhibitory
concentration, MIC) that, under defined test conditions, inhibits the visible growth of the
bacterium, fungi being investigated. Numerous researchers demonstrated that citrus
essential oils affected on the elimination of food-borne pathogens (Sana et al., 2009) and
were manufactured as meat and dairy products preservatives (Fernández-López et al.,
2007). Chaisawadi (2005) reported that citrus oils including Citrus hystrix DC. and Citrus
aurantifolia inhibited growth of Staphylococcus aureus and Salmonella typhi. Citrus
essential oils could represent as good candidates to improve the shelf life and the safety of
minimally processed fruits (Lanciotti et al., 2004), skim milk and low-fat milk (Dabbah et
al., 1970). The other functional property of citrus essential oils is antioxidant activity.
Antioxidant
activity
is
action
against
linoleic
acid
oxidation
and
2,2-diphenyl-1-
picrylhydrazyl radical scavenging. Some recent publications showed antioxidant activities
of these essential oils (Baik et al., 2008). Other study observed the effects of essential oils
of Citrus obovoides and Citrus natsudadai on DPPH radical scavenging, superoxide anion
radical scavenging and nitric oxide radical scavenging activity (Kim et al., 2008). In that
study, Citrus obovoides and Citrus natsudadai exhibited only superoxide anion radical
scavenging activity. Malhotra (2009) also reported Citrus karna essential oils showed a
significant inhibition for the oxidation of linoleic acid in the beta-carotene-linoleic acid
system.
Citrus essential oils bring a lot of benefits, many studies on citrus essential oils including
extraction methods, chemical compositions and properties of essential oils from different
varieties and different location have been done over the world. However, there is little
information regarding the detailed evaluation of antioxidant and antimicrobial activities of
citrus essential oils in Vietnam which is a country with a huge production of citrus fruits. In
addition, two conventional methods for extraction possess disadvantages that affect on the
yield and quality of citrus essential oils. Therefore, the objective of this study is to
determine chemical composition, antimicrobial and antioxidant activities of Vietnamese
citrus essential oils with emphasis on the possible future application of essential oils as
alternative synthetic agents in food preservation. Additionally, vacuum distillation which is
modified from hydro-distillation method to avoid altering volatile compounds and produce
high yield is employed to extract the citrus essential oils.
10
2
2.1
Chapter 2: LITERATURE REVIEW
Essential oils
An essential oil is a concentrated hydrophobic liquid containing volatile aroma compounds
from plants. It is less soluble in water. Essential oil is extracted from plant so it carries a
distinctive scent, or essence, of the plant and is therefore applied in food flavoring and
perfumery (Gunther, 1952).
The occurrence of essential oils is restricted to over 2000 plant varieties from about 60
different families, however only about 100 varieties are the basis for the economically
important production of essential oils in the world (Van de Braak and Leijten, 1999). The
ability of plants to accumulate essential oils is quite high in both Gymnosperms and
Angiosperms, although the most commercially important essential oil plant sources are
related to the Angiosperms (Burt, 2004; Hussain et al., 2008; Anwar et al., 2009a).
Essential oils are isolated from various parts of the plant, such as leaves (basil, patchouli,
cedar), fruits (citrus) , bark (cinnamon), root (ginger), grass (citronella), gum (myrrh and
balsam oils), berries (pimenta), seed (caraway), flowers (rose and jasmine), twigs (clove
stem), wood (amyris), heartwood (cedar), and saw dust (cedar oil) (Dang et al., 2001;
Burt, 2004; Sood et al., 2006; Cava et al., 2007; Hussain et al., 2008).
2.2
Citrus essential oils
Citrus is a common term and genus of flowering plants that belongs to the rue family,
Rutaceae, originating and growing extensively in tropical and subtropical southern regions
of Asia including Vietnam.
Table 2.1: Scientific classification (Dugo and Giacoma, 2002; Manner et al., 2006)
Domain
Eukarya
Kingdom
Plantae
Class
Magnoliopsida
Family
Rutaceae
Subfamily
Aurantioideae
Genus
Citrus L.
The plants belong to the genus Citrus are eukaryotic organisms. The cells have a true
nucleus, possess membrane-bond organelles, and the genetic material is DNA. In addition,
they are multicellular and have cell walls made of cellulose, and participate in
photosynthesis via chloroplasts. Moreover, they are flowering plant that uses a fruit body
to protect its seeds and show characteristics of being a Dicotyledon such as secondary
growth, non-parallel veins, and the presence of two cotyledons in their seeds (Dugo and
Giacoma, 2002). The orders consist of woody trees, shrubs, and have strong scents. They
11
are generally edible and good sources of vitamin C. Hence, these organisms are
characterized in Rutaceae family, Citrus genus (Dugo and Giacoma, 2002).
In Vietnam, citrus fruits are grown in seven ecological regions, including three major subregions in the north of Vietnam (the mountainous midland region, the Red River Delta, and
the northern central coast), in total accounting for 35-40% of citrus production in Vietnam.
The Mekong River Delta in the south of Vietnam accounting for 55-60% of citrus
production, and the rest concentrated in the central provinces (Agro, 2006). The citrusgrowing areas have increased year by year and citrus fruit production reached 700,000
tons in 2011 (Agro, 2011). The harvesting season that gives the highest production is
normally September to December. The flavedo of citrus fruits is the main section
containing essential oils.
The main volatile compounds presenting in citrus essential oils are α-pinene, β-pinene,
myrcene, limonene, citral, linalool, α-terpineol. The most abundant compound is limonene.
Table 2.2 shows the main volatile component of citrus essential oils from several countries.
Table 2.2: The main volatile components (%w/w) of several citrus essential oils
No
Compound
Vietnamese
Vietnamese
Vietnamese
India
orange
mandarin
pomelo
orange
(Citrus sinensis)
(Citrus reticulata
(Citrus grandis
(Citrus sinesis
(Ref.71)
Blanco)
Osbeck)
(L.) Osbeck)
(Ref.71)
(Ref.71)
(Ref.105)
1. α-pinene
0.81
0.93
1.69
0.9
2. β-pinene
0.05
0.60
0.76
0.6
3. Myrcene
2.81
2.79
1.97
4.1
90.42
91.58
70.46
84.2
5. Citral
0.57
0.05
0.32
0.5
6. Linalool
0.73
0.24
0.12
4.4
7. α-terpineol
0.26
0.41
0.57
1.3
4. Limonene
Ref.71: Lan-Phi et al., 2010.
Ref.105: Sharma and Tripathi, 2008.
The main abundant compounds presenting in orange (Citrus sinesis) oil cultivated in
Vietnam was limonene (90.42%), followed by myrcene (2.81%) and α-pinene (0.81%).
Linalool and α-terpineol are alcohols that have been reported to be the most important to
orange flavor. The amount of linalool in the orange essential oil was 0.73% and α-terpineol
was detected at the level of 0.26%. This compound, however, is a product of acidic and
microbial degradation of limonene and also a contributor to off-flavor in stored orange
juice (Shaw, 1979).
12
The major components found in mandarin (Citrus reticulata Blanco) essential oil were
limonene (91.58%), followed by myrcene (2.79%), and α-pinene (0.93%). β-pinene was
important to mandarin aroma and flavor. β-pinene presented at the level of 0.60% in this
oil. Terpene compounds are the most reasons lead to antimicrobial activities of citrus
essential oils. These components pass the cell membranes, penetrates into the interior of
the cell and interact with critical intracellular sites (Cristani et al., 2007). The amount of
linalool and α-terpineol was detected at low level of 0.24% and 0.41%, respectively.
In case of pomelo (Citrus grandis Osbeck) oil, limonene was the most abundant compound,
accounting for 70.46%. The other prominent compounds were myrcene (1.97%), α-pinene
(1.69%) and β-pinene (0.76%). Although the proportion of α-terpineol in this oil remained
at higher level than orange and mandarin oils, the amount of linalool was low (only
0.12%). Linalool, in previous studies, plays an important role in inhibition ability of
peroxidation and caused essential oils possessing antioxidant activity (Hussain et al.,
2008).
For the volatile components of Indian Citrus sinesis (L.) Osbeck essential oil, limonene as
the most abundant compound was identified at 84.2%, followed by linalool (4.4%),
myrcene (4.1%) and α-terpineol (1.3%). β-pinene, α-pinene and citral remained at low
levels of 0.9%, 0.6% and 0.5%, respectively. In comparison with the Vietnamese citrus
essential oils, linalool represented higher concentration (up to 4.4%), whereas the
percentage of this compound in Vietnamese oils only accounted for small amount (lower
than 1%). Linalool, citral were compounds appreciated causing the antifungal capacities of
citrus essential oils (Alma et al., 2004). Citral can form a charge transfer complex with an
electron donor to fungal cells, which results in fungal death (Kurita et al., 1981).
These results indicate that the different varieties and different growing location affect
chemical composition of citrus essential oils resulting in different functional properties of
these oils.
2.3
Extraction methods
2.3.1 Hydro-distillation method
Hydro-distillation involves the use of water or steam to recover volatile principles from
plant materials.
The fundamental feature of hydro-distillation
is
that
it
enables
a
compound or mixture of compounds to be distilled and subsequently below that of
the boiling point of the individual constituent.
There have been three types of distillation: water distillation, water/steam distillation, and
steam distillation. In water distillation, plant is soaked in a large chamber filled with water.
Subsequently, the chamber is heated and essential oil is released from the plant by
evaporation. The resulting steam from boiling water carries volatile oils with it and travels
to a condenser, where the steam is cooled and is eventually turned back into water.
13
Eventually, the essential oil is equally returned to its former state and separated from
water. In water/steam method, the plant material is placed on a grill above the hot water
and steam passes through the plant material. The material must be carefully distributed on
the grill to allow for steaming and extraction. In steam distillation, no water is placed
inside the distillation tank. Instead, steam is directed into tank from an outside source. The
essential oils are released from the plant material when the steam bursts the sacs
containing the oil molecules. From this stage, the process of condensation and separation
is standard (Chemat, 2010).
This method has some important drawbacks. The elevated temperatures can cause
modifications of the essential oil components and often a loss of the most volatile
molecules (Chemat, 2010). In consequence, scent and quality of essential oils are
deteriorated. However, hydro-distillation is one of the simplest methods for obtaining oils
from plants. With high yield extraction and low cost requirement, it is also the most widely
acceptable process for large scale of essential oils production. In present time, improving
the hydro-distillation method has been conducted to produce essential oils with high
quality. However, there is no public research that reports the new method producing high
quality and high yield of essential oils, replacing for hydro-distillation method in large-scale
production. Most of studies on extraction methods are only available in laboratory design.
In addition, although the activities of the essential oils extracted by hydro-distillation are
lower than natural oils, their quality is still ranged within acceptable limit. Therefore, this
technique is used for essential oils production in industries.
2.3.2 Solvent extraction
Another method is solvent extraction used to extract essential oils from delicate flowers
and plant material which would be altered or damaged by hydro-distillation. First of all, the
plant material is gradually mixed with a hydrocarbon solvent such as hexane, petroleum
ether, benzene, toluene, ethanol, isopropanol, ethyl, acetone, etc. The solvent dissolves
the plants constituents including essential oils, fatty acids and waxes. After the solvent is
distilled off the remaining constituents make up the concrete. In addition, alcohol is used
to extract the essential oil from the other constituents. Therefore, the fatty acids and
waxes that are not alcohol soluble are left behind. Eventually, the alcohol is evaporated,
leaving the absolute oil behind for harvesting (Rydberg et al., 2004).
The solvent extraction is a simple method and does not require complex equipments.
However, it possesses several disadvantages. The alcoholysis and evaporation may happen
and could affect the quality and stability of oils. The important process of this method is
the solvents eliminations from extracts, because of their harmfulness.
14
2.3.3 Supercritical carbon dioxide method
When a gas is compressed to a sufficiently high pressure, it becomes liquid. If the gas is
heated to a specific temperature, at the specific pressure, the hot gas will become
supercritical
fluid.
This
temperature
is
called
the
critical
temperature
and
the
corresponding vapor pressure is called the critical pressure. The values of the temperature
and pressure are defined as critical point which is unique to a given substance. These
states of the substances are called supercritical fluid when both the temperature and
pressure exceed the critical point values. This fluid possesses both gas and liquid
properties. It is suitable for extraction because of its characteristics such as favorable
diffusivity, viscosity, surface tension and other physical properties. The diffuseness
facilitates rapid mass transfer and faster completion of extraction than conventional liquid
solvents. The low viscosity and surface tension enable it to easily penetrate the botanical
materials from which the active components are extracted. The gas-like characteristics of
supercritical fluid provide ideal conditions for extraction of solutes giving a high degree of
recovery in a short period of time.
Carbon dioxide is in its supercritical fluid state when both the temperature and pressure
equal or exceed the critical point of 31°C and 73 atm. In its supercritical state, carbon
dioxide has both gas-like and liquid-like qualities so it can fill any size of container, like a
gas, and dissolve materials like a liquid.
Carbon dioxide is prominent in comparison with other supercritical fluids because its critical
temperature is remarkably low at only 31.1°C, so high temperatures are not necessary.
This means supercritical carbon dioxide can be used as a solvent for materials that would
be decomposed at higher temperatures. Hence, the essential oils produced from this
method possess good quality. However, this method requires high-cost system and
technical skills to perform.
2.3.4 Cold pressing method
The cold pressing uses pressure to physically squeeze the oil from the plant tissue. It is
used to obtain citrus fruit oils such as pomelo, mandarin, and orange oils. This is a simple
method that uses machines that apply a centrifugal force for the purpose of separation of
essential oil from other substances. In consequence, the essential oil is collected
(Sawamura, 2010).
The technique is a purely mechanical process while the hydro-distillation use steam from
boiling water for carrying and extracting volatile oils. In comparison with hydro-distillation
method, cold pressed extraction is carried out without applying heat to avoid the loss,
chemical changes in the constituents, and formation of artifacts during hydro-distillation
process of essential oil extraction. As a result, cold pressing method produces the essential
15
oil with native quality. On the other hand, this method produces low yield. Also, it is
important to note that oil extracted using this method have a relatively short shelf life.
There are reports in literature on the significance of extraction methods. Charles and
Simon (1990) approved the hydro-distillation is a simpler and more rapid method for oil
isolation. In comparison between hydro-distillation and ethyl acetate extraction, the study
proved that the extraction yields of hydro-distillated oils and ethyl acetate extracts from
fresh peels of citrus spp. widely varied depending on citrus cultivars. For each cultivar, the
production yields of the hydro-distillated oils were much lower than that from extraction
with ethyl acetate. In addition, several authors have compared the composition of essential
oil obtained by hydro-distillation and the product obtained by super critical fluid extraction.
They
found
that
hydro-distillated
oil
contained
higher
percentages
of
terpene
hydrocarbons. In contrast, the super critical extracted oil contained a higher percentage of
oxygen compounds (Reverchon, 1997; Donelian et al., 2009). Khajeh et al. (2004)
reported variation in the chemical composition of Carum copticum essential oil isolated by
hydro-distillation and supercritical fluid extraction methods.
2.4
Gas chromatography
Chromatography is the general name for separation technology whereby components in a
mixture are separated through continuous repetition of concentration equilibration. When a
gas is used as the mobile phase the technology is called gas chromatography (GC).
The sample mixture is injected and instantaneously vaporized at the column inlet. The
vaporized sample is then carried through the column by the carrier gas. While passing
through the column, each component in the sample is adsorbed or is partitioned to the
stationary phase according to its characteristic concentration ratio. As a result, the level of
adsorption or partition for each component causes differences in the rate of movement for
each component within the column. The components therefore elute separately from the
column outlet (Sawamura, 2010).
Figure 2.1 Diagram of Gas chromatography
(Source: http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/gaschrm.htm)
16
The rate at which a compound travels through a particular GC system depends on
the factors listed below:
Volatility of compound: Low boiling (volatile) components travel faster through
the column than high boiling point components.
Polarity of compounds: Polar compounds move more slowly, especially if the
column is polar.
Column temperature: Raising the column temperature speeds up all the compounds
in a mixture.
Column packing polarity: Usually, all compounds move slower on polar columns,
but polar compounds will show a larger effect.
Flow rate of the gas: Through the column, speeding up the carrier gas flow
increases the speed with which all compounds move through the column.
Length of the column: The
longer
the
column,
the
longer
it
will
take
all
compounds to elute. Longer columns are employed to obtain better separation
Figure 2.2 Principle of Gas Chromatography
Gas chromatography–mass spectrometry (GC-MS) is another method based on the
principle of GC. However, it combines the features of gas-liquid chromatography and mass
spectrometry to identify different substances within a test sample. The gas chromatograph
utilizes a capillary column which depends on the column's dimensions (length, diameter,
film thickness) as well as the phase properties. The difference in the chemical properties
between different molecules in a mixture will separate the molecules as the sample travels
the length of the column. The molecules are retained by the column and then elute from
the column at different times, and this allows the mass spectrometer downstream to
capture, ionize, accelerate, deflect, and detect the ionized molecules separately.
Today, GC and GC-MS are common instruments in most laboratories because they are
sensitive, accurate, and convenient.
17
There is plenty of literature on determination of chemical composition of essential oils
using gas chromatography (GC). Column and detection are two important elements in GC
system. Capillary column with flame ionization detection (FID), are, in most cases, the
method of choice for quantitative determinations because they enable more complex
mixtures to be separated and resolved. Fisher and Phillips (2006) used GC system with
capillary column and FID detector to analyze the composition of lemon, orange and
bergamot essential oils. The results indicated three components, in which limonene was
more abundant than citral or linalool in the oils tested. Also, Wungstintaweekul et al.
(2010) selected capillary column and FID for the GC system to identify components of
Citrus hystrix oil. Many researchers make use of mass spectrometers (MS), coupled with
GC, to determine the identities of components. In a study, 67 components were identified
in Citrus hystrix oils through GC-MS (Kirbaslar et al., 2009). Moreover, GC-MS analysis of
Citrus sinensis (L.) Osbeck peel oil led to identification of 10 components (Sharma and
Tripathi, 2008). Most of studies chose the capillary column with over 50 m in length to
obtain better separation. Sokovic et al. (2007) analyzed 88 compounds included in Citrus
limon and Citrus aurantium essential oils through GC-MS with capillary column (50m x
0.2mm i.d, 0.5 µm film thickness). Capillary columns selected, in most cases, are HP-5ms,
DB-5 (cross-linked 5% diphenyl/95% dimethyl siloxane) or DB-1, also known as SE-30,
(polydimethyl siloxane) stationary phases. These more non-polar stationary phases are
often complimented by the use of a more polar stationary phase, such as polyethylene
glycol (Cavaleiro et al., 2004). The composition of Citrus Turkish peel oils was analyzed by
GC with DB-5 column (60m x 0.25mm i.d, 0.25µm film thickness) (Kirbaslar et al., 2009).
2.5
Food poisoned microorganisms
2.5.1 Staphylococcus aureus
Staphylococcus aureus is facultative anaerobic gram-positive cocci which occur singly, in
pairs, and irregular clusters. Typical colonies of S. aureus are usually large (6-8 mm in
diameter), yellow to golden yellow in color, smooth, entire, slightly raised, often with
hemolysis, when grown on blood agar plates. S. aureus is nonmotile, non-spore forming.
The cell wall contains peptidoglycan and teichoic acid. The organisms are resistant to
temperatures as high as 50°C, to high salt concentrations, and to drying.
S. aureus usually affects on foods requiring hand preparation, such as potato salad, ham
salad and sandwich spreads. It is frequently found as part of the normal skin flora on the
skin and nasal passages. In normal, the bacteria do not cause disease. However, breached
skin or other injury may allow the bacteria to overcome the natural protective mechanisms
of the body, leading to infection such as pimples, impetigo, boils (furuncles), carbuncles,
scalded skin syndrome and abscesses, bacteremia and septicemia. In infants, S. aureus
18
infection can cause a severe disease - staphylococcal scalded skin syndrome (SSSS) (John
et al., 1980)
In some researches, S. aureus was found to be the most susceptible bacterium for several
essential oils. Upadhyay et al. (2010) reported that Citrus lemon and Azadirachta indica
essential oils where inhibition zone of 23.10 mm and 23.23 mm, respectively, was
recorded. Growth of this microorganism was completely inhibited by all of the citrus oils
with 100% of reduction of inoculums (Dabbah et al., 1970). In addition, Turkish citrus peel
oils also showed strong antimicrobial activity against S. aureus (Kirbaslar et al., 2009).
Figure 2.3 Scanning electron micrograph of S. aureus.
(Source: http://en.wikipedia.org/wiki/Staphylococcus_aureus)
2.5.2
Bacillus cereus
Bacillus cereus is a Gram-positive, catalase, beta hemolytic bacterium that can be
frequently isolated from soil and some food. B. cereus is aerobe, rod-shaped, and has the
ability to form protective endospore, allowing the organism to tolerate extreme
environmental conditions. Thus, B. cereus is more resistant to heat and chemical
treatments than vegetative pathogens such as Salmonella, E. coli, Campylobacter, and
Listeria monocytogenes (Jesen et al., 2003).
Spores of B. cereus can be found widely in nature, including samples of dust, dirt, cereal
crops, water, etc. Starchy foods such as rice, macaroni and potato dishes are the best
environment for development of B. cereus. Bacillus food borne illnesses occur due to
survival of the bacterial endospores when food is improperly cooked. Cooking temperatures
less than or equal to 100 °C (212 °F) allows some B. cereus spores to survive (McKillip,
2000). Bacterial growth results in production of enterotoxins, one of which is highly
resistant to heat and to pH between 2 and 11; ingestion leads to two types of illness: one
type characterized by diarrhea and the other, called emetic toxin, by nausea and vomiting.
Several reports studied on antibacterial activities of essential oils on B. cereus. Citrus
lemon and Azadirachta indica essential oils showed high inhibition on this bacterium with
41.3mm and 45.63mm inhibition zone, respectively (Upadhyay et al., 2010). According to
19
the result of the other report, all of the citrus peel oils were more effective towards B.
cereus (Kirbaslar et al., 2009). Chanthaphon et al. (2008) demonstrated that the extract
from lime peel (Citrus aurantifolia Swingle) showed broad spectrum inhibitory against all
Gram positive bacteria including B.cereus. The lime extract exhibited MIC value against B.
cereus at 0.56 mg/ml. This result was correlated to the report of Chaisawadi et al. (2005)
which cited Citrus aurantifolia displaying antibacterial activities on this microorganism.
Figure 2.4 Rod-shaped Bacillus cereus.
(Source: Courtesy of Frederick C. Michel, ASM MicrobeLibrary)
2.5.3 Salmonella typhi
Salmonella typhi is gram-negative, motile, facultatively anaerobic bacterium, made up of
nonspore-forming rods (Pelczar et al., 1993). It has a complex regulatory system, which
mediates its response to the changes of external environment. In order to survive in the
intestinal organs of its hosts where there are low levels of oxygen, Salmonella typhi has to
be able to learn to use other sources other than oxygen as an electron acceptor. The
electron acceptor of this strain is nitrogen such as nitrate, nitrite, fumarate, and
dimethlysulphoxide.
It is a food born pathogen and the most common source of infection is high protein foods
such as meat, poultry, fish and eggs. S. typhi causes systemic infections, typhoid fever in
humans (Doughari et al., 2007). It usually invades the surface of the intestine in humans,
but have developed and adapted to grow into the deeper tissues of the spleen, liver, and
the bone marrow. It is also able to inhibit the oxidative burst of leukocytes, making innate
immune response ineffective. Symptoms most characterized by this disease often include a
sudden onset of a high fever, a headache, and nausea. Other common symptoms include
loss of appetite, diarrhea, and enlargement of the spleen (depending on where it is
located) (Shah, 2012). The encounter of humans to S. typhi is made via fecal-oral route
from infected individuals to healthy ones. Poor hygiene of patients shedding the organism
can lead to secondary infection, as well as consumption of shellfish from polluted bodies of
water.
20
There are many reports in literature regarding the antimicrobial activity of essential oils on
S. typhi. Among the various essential oil treatments, the Citrus reticulata var. Tangarin
exhibited the highest antibacterial activity on S. typhi (Ashok et al., 2011). Suganya et al.
(2012) cited that the essential oil of Coriandrum sativam has the highest antibacterial
activity against this bacterium with inhibition zone of 15mm in diameter. The essential
oils distilled from Syzygium neesianum Arn, Elaeocarpus lanceifolius and Citrus sinesis also
showed a significant inhibition on S. typhi (Maridass, 2010; Ashok et al., 2011).
Figure 2.5 Flagella stain of Salmonella typhi. (approx. 1000 X)
(Source: the Wistreich Collection, appearing exclusively on MicrobeWorld).
2.5.4 Pseudomonas aeruginosa
Pseudomonas aeruginosa is a Gram-negative rod measuring 1-5 µm long and 0.5-1.0 µm
wide. Almost all strains are motile by means of a single polar flagellum (Ryan and Ray,
2004). P. aeruginosa is an obligate respirer. It grows in the absence of oxygen and use
nitrate as a respiratory electron acceptor. The pathogen is widespread in nature, inhabiting
soil, water, plants, and animals (including humans). It is a common bacterium that can
cause disease in animals, including humans (Iglewski, 1996).
Vegetables, meats, milk and water are suitable environments for the infection of P.
aeruginosa. Once infecting to human body, P. aeruginosa causes urinary tract infections,
respiratory system infections, dermatitis, soft tissue infections, bacteremia, bone and joint
infections, gastrointestinal infections and a variety of systemic infections, particularly in
patients with burn and in cancer and AIDS patients who are immune-suppressed. P.
aeruginosa infection is a serious problem in patients hospitalized with cancer, cystic
fibrosis, and burns (Ryan and Ray, 2004).
A number of publications demonstrated that various essential oils possessing antibacterial
activities on P. aeruginosa. This microorganism was inhibited by the lemongrass
(Cymbopogon citratus) oil with MIC value at 1% (v/v) whereas the lime (Citrus
aurantifolia) oil showed the lower effect with MIC value at 2% (v/v) (Hammer et al.,
1999). The essential oils Ammy visnaga L. exhibited strong inhibition effect of P.
aeruginosa with 25 mm inhibition zone diameter (Khalfallah et al., 2011)
21
Figure 2.6 Pseudomonas aeruginosa
(Source: http://fuckyeahmedicine.tumblr.com/post/455450061/pseudomonas-aeruginosainfections)
2.5.5 Aspergillus flavus
Aspergillus flavus is a plant, animal, and human fungal pathogen. It grows by producing
thread like branching filaments known as hyphae. Filamentous fungi such as A. flavus are
sometimes called molds. Conidia are globose to subglobose (3-6 um in diameter), pale
green and conspicuously echinulate. When young, the conidia of A. flavus appear yellow
green in color. As the fungus ages, the spores turn a darker green. Conidial heads are
typically radiate, mostly 300-400 µm in diameter, later splitting to form loose columns
(Amaike and Keller, 2011). A network of its hyphae known as the mycelium secretes
enzymes that break down complex food sources.
Aspergillus flavus has a world-wide distribution and normally occurs in soil and on many
kinds of decaying organic matter. The fungus infects seeds of corn, peanuts, cotton, and
nut trees. It produces significant quantities of toxic compounds known as mycotoxins,
commonly aflatoxin which is a toxic and carcinogenic compound. Aflatoxin is also the
second leading cause of aspergillosis in humans (Agrios and George, 2005). Patients
infected with A. flavus often have reduced or compromised immune systems (Amaike et
al., 2011).
Many plant oils showed inhibition abilities on A. flavus. The essential oils of lemon (Citrus
lemon L.), mandarin (Citrus reticulata L.), orange (Citrus sinesis L.) and grapefruit (Citrus
paradisi L.) possessed the capacity to reduce or inhibit the growth of the mold A. flavus.
The total inhibition of growth was obtained with all citrus essential oils at the concentration
of 0.94% (Martos et al., 2008). There was a highly marked inhibitory effect of all
treatments EOs including marjoram, mint, basil, coriander, thyme, dill and rosemary on A.
flavus strain growth. The highest growth inhibition rate of A. flavus was observed with the
thyme Eos (Habib, 2012).
22
Figure 2.7 Aspergillus flavus colony surface
(Source: William McDonald, 2011)
2.5.6 Fusarium solani
Fusarium solani is a pathogenic fungus and is an important causal agent of several crop
diseases, such as root and stem rot of pea, sudden death syndrome of soybean, foot rot of
bean and dry rot of potato. It produces asexual spores which can be spread by air,
equipment, and water (Cho et al., 2001). Colonies growing rapidly, 4.5 cm in 4 days, aerial
mycelium white to cream, becoming bluish-brown when sporodochia are present. When
young, the conidia of F. solani appear white to cream in color. Colonies grow rapidly, 4.5
cm in 4 days. As the fungus ages, the spores become a bluish-brown.
The predominant hosts for Fusarium solani are potato, pea, bean, and members of the
cucurbit family such as melon, cucumber, and pumpkin.
It produces trichothecene
mycotoxin that inhibits DNA and protein synthesis (Ueno, 1989; Thompson and
Wannemacher, 1990). This mycotoxin also causes impairment of ribosome function and
immune-suppression, allowing secondary and opportunistic bacterial infections and
possibly delayed hypersensitivity (Ueno, 1989).
There are numerous studies demonstrated that the the growth of F. solani and its potential
pathogenic activity can be controlled. Origanum vulgare EOs showed strong antifungal
activity against this mold (Laubach et al., 2012). Aziz et al. (2010) cited that the essential
oil of Thymus serpyllum L. grown in the State of Jammu and Kashmir showed significant
antifungal activity against Fusarium solani and moderate phytotoxic activity. Fungicidal
activity against F. solani was observed at 5% concentration for essential oils from Pinus
resinosa and Pinus strobus (Krauze-Baranowska et al., 2002)
23
Figure 2.8 Fusarium solani colony surface
(Source: http://gahru-on.blogspot.com/)
2.6
Antimicrobial activity of essential oils
Essential oils and other naturally occurring antimicrobials are attractive to the food
industry for the following reasons:
1. It is highly unlikely that new synthetic compounds will be approved for use as food
antimicrobials due to the expense of toxicological testing (Burt, 2004).
2. There exists a significant need for expanded antimicrobial activity both in terms of
spectrum of activity and of broad food applications (Feng and Zheng, 2007).
3. Food processors are interested in producing “green” labels, i.e., ones without
chemical names that apparently confuse consumers (Burt, 2004).
4. There are potential health benefits that come with the consumption of some
naturally occurring antimicrobials (Ayoola et al., 2008).
Recently, essential oils and extracts of certain plants have been shown to have
antimicrobial effects, without effects on flavor of foods (Burt, 2004). Some citrus essential
oils have shown promise as potential food safety interventions when added to minimally
processed fruits (Lanciotti et al., 2003).
There are many reports regarding the antimicrobial activity of essential oils (Kofidis et al.,
2004; Singh et al., 2005). Martos et al. (2007) evaluated the antibacterial functions of the
citrus essential oils from four varieties of lemon (Citrus lemon L.), mandarin (Citrus
reticulata L.), grapefruit (Citrus paradisi L.), and orange (Citrus sinesis L.) and found that
all of these essential oils displayed strong antibacterial activity against the strains tested.
Testing and evaluation of antimicrobial activity of essential oils is difficult because of their
characteristics such as volatility, water insolubility and complexity. Essential oils are
hydrophobic and high viscosity compound. These properties may reduce the dilution ability
24
or cause unequal distribution of the oil in the medium even if a proper solvent agent is
used. It has to be checked whether the applied concentrations of the emulsifier or solvent
do not affect the growth and differentiation of tested microorganisms. Moreover, essential
oils are very complex mixtures of volatile components. Consequently, long incubation
periods may result in the evaporation or decomposition of some of the components during
the testing period (Kalemba and Kunicka, 2003).
For the antimicrobial assessment of essential oils, conventional methods of testing
antibiotic abilities are usually applied. There are two basic techniques for the assessment
of both antibacterial and antifungal activities of essential oils (Kalemba and Kunicka,
2003):
The agar diffusion method (paper disc or well): The diffusion method is the most
widespread technique of antimicrobial activity assessment. According to this
method, Petri dishes of 5-12 cm diameter (usually 9 cm) are filled with 10-20 ml of
agar and inoculated with microorganisms. Two ways of essential oil incorporation
are possible: on a paper disc (Simic et al., 2000; Omer et al., 1998) or into the well
(hole) made in the agar medium. When a filter paper disc impregnated with
essential oil is placed on agar or essential oil is added into the hole, the essential oil
will diffuse from the disc or well into the agar. This diffusion will place the essential
oil in the agar only around the disc or well. The solubility of essential oil and its
molecular size will determine the size of the area of infiltration around the disc or
well. If an organism is placed on the agar, it will not grow in the area around the
disc or well because it is susceptible to the essential oil. This area of no growth
around the disc or well is known as a “zone of inhibition” (Kalemba and Kunicka,
2003). This method is mostly used as a screening method when large numbers of
essential oils and/or large numbers of bacterial isolates are to be screened (Deans
et al., 1990; Dorman and Deans, 2000). In literature, both disc diffusion (Renzini
et al., 1999; Senatore et al., 2000) and well diffusion (Dorman and Deans, 2000;
Ruberto et al., 2000) assays are reported to evaluate the antimicrobial activity of
essential oils.
The dilution method (agar or liquid broth): The serial dilution agar method is used
for bacteria and fungi, but its modification with liquid broth is mostly applied for
fungi. The broth micro dilution assay has become quite popular lately (Shapiro et
al., 1994).
The aim of broth and agar dilution methods is to determine the lowest
concentration of the assayed antimicrobial agent (minimal inhibitory concentration,
MIC) that, under defined test conditions, inhibits the visible growth of the
bacterium,
fungi
being
investigated.
MIC
values
are
used
to
determine
susceptibilities of bacteria, fungi to drugs and also to evaluate the activity of new
25
antimicrobial
agents.
Agar
dilution
involves
the
incorporation
of
different
concentrations of the antimicrobial substance into a nutrient agar medium followed
by the application of a standardized concentration of organisms to the surface of
the agar plate. For broth dilution, studies a number of different procedures exist for
determining the MIC
and
MBC. The most
common
methods
are that
of
measurement of optical density and the enumeration of colonies by viable count
(Skandamis et al., 2001; Ultee and Smid, 2001). MIC was often determined in 96well microtiter plate format, bacteria are inoculated into a liquid growth medium in
the presence of different concentrations of an antimicrobial agent. Growth is
assessed after incubation for a defined period of time and the MIC value is read.
2.7
Antioxidant activity of essential oil
From a biological point of view, antioxidants have been defined as substances that when
present in concentrations lower than the oxidation substrate are capable of delaying or
inhibiting oxidative processes (Choi, 2010).
To extend the shelf-life of foods, phenolic compounds, such as butylated hydroxyanisole
(BHA) butylated hydroxyl toluence (BHT), have been widely used as synthetic antioxidants
in the food industry (Choi, 2010). However, over the past two decades, there is great
public concern about the safety of the synthetic antioxidants in food preservation besides
health implications. Some opinions concerned the safety and side effects of synthetic
antioxidants as food additives. These synthetic antioxidants are known to have toxic and
carcinogenic effects on human and food systems. Especially, they may cause liver swelling
and influence liver system activities and cerebro-vascular diseases (Choi et al., 2007).
Therefore, recent studies interest in developing safer antioxidants based on natural
sources, as alternatives, to prevent the deterioration of foods. Essential oils and extracts
from botanical materials are known to have varying degrees of antioxidant activities
(Descalzo and Sancho 2008). Recent publications showed antioxidant activities of essential
oils and extracts which have been reported to be more effective than some synthetic
antioxidants (Hussain et al., 2008; Bendini et al., 2002).
DPPH radical scavenging assay is the most popular method used for the determination of
antioxidant activity of essential oils and plant extracts. The presence of an odd electron in
the DPPH free radical gives a strong absorption maximum at 517 nm. As this electron
becomes paired off in the presence of a hydrogen donor, the absorption strength is
decreased, and the resulting decolonization is stoichiometric with respect to the number of
electrons captured (Choi, 2010). Due to its simplicity and sensitivity, some authors only
use DPPH method for evaluating the antioxidant activities of essential oils.
Another method is to evaluate the antioxidant capacity of essential oils based on the
reduction of Fe3+ to Fe2+. This method is known as Ferric Thiocyanate (FTC) method. In
26
this method, linoleic acid is added into mixture. Then, oxygen reacts with linoleic acid,
which is unsaturated lipids (LH), through free radical initiation, propagation and
termination processes (Frankel, 1980). Initiation takes place by loss of a hydrogen radical
in the presence of trace metals, light or heat. The resulting lipid free radicals (L-) react
with oxygen to form peroxy radicals (LOO-). In this propagation process, LOO- reacts with
more LH to form lipid hydroperoxides (LOOH), which are the fundamental primary products
of autoxidation (Frankel, 1984).
LH L-+HL-+O2 LOOLOO- + LH LOOH + LAntioxidants (AH) can break this chain reaction by reacting with LOO- to form stable
radicals (A-) which are either not too reactive or form non-radical products (Frankel,
1984).
LOO-+AH A- +LOOA-+LOO- Non radical products
A-+A- Non radical products
In reaction of oxygen with linoleic acid, lipid hydroperoxides are formed, then oxidize Fe2+
to Fe3+. The latter ions form a complex with thiocyanate, and this complex has a maximum
absorbance at 500 nm. Therefore, high absorbance indicates high linoleic acid oxidation.
As a literature review, essential oils are extracted by four methods hydro-distillation,
solvent
extraction,
supercritical
carbon
dioxide
and
cold
pressing
method.
Their
compositions are usually analyzed by GC or GC-MS instruments because they are
sensitive, accurate, and convenient. Diffusion and dilution are two basic techniques for the
assessment of both antibacterial and antifungal activities of essential oils. There are many
methods used to define the antioxidant capacity of essential oils. However, DPPH radical
scavenging and FTC method are two common assays. In this study, two extraction
methods, cold pressing and vacuum distillation methods, will be employed to extract
essential oils from the Vietnamese citrus peel and DPPH radical scavenging and FTC
method will be used to determine antioxidant capacity of these EOs as well as diffusion and
dilution will be used to check antimicrobial activities of these EOs.
27
3
3.1
Chapter 3: Materials and methods
Experimental design
Material collection and
preparation
Extraction essential oils
by cold pressing
method
Evaluation of
antimicrobial
activities
Evaluation of
antimicrobial
activities by
dilution
method
Evaluation of
antimicrobial
activities by
diffusion
method
Extraction essential oils
by vacuum distillation
method
Evaluation of
antioxidant
activities
Evaluation of
antioxidant
activities by
DPPH assay
Evaluation of
antioxidant
activities by
lipid
peroxidation
assay
Figure 3.1 Flow chart of experimental process
3.2
Materials collection and preparation:
The time from flowering and fruiting to harvesting, which citrus fruits are in the major
stage, is 8 months. It is the best time to collect materials for the study because the citrus
fruits produce the highest amount of essential oils with the highest quality at this age.
Therefore, citrus fruits were collected at mature stage (after fruiting 8 months) from farms
in the provinces from North, Center and South region of Vietnam from September, 2012 to
December, 2012, which is the main season of citrus fruits in Vietnam. The fruits were in
earlier-ripe stage. After collecting, the samples were transferred to International University
laboratory by airplane in same day. Therefore, the quality of fruits was not affected. All
samples are shown in Table 3.1.
28
Table 3.1 Citrus samples in the study
Common
Scientific name
Place collection
Citrus sinensis Osbeck
Lap Vo District, Dong Thap
Image of whole fruit
Image of cross section
name
Xoan orange
Province
Vinh orange
Citrus sinensis Osbeck
Quy Hop District, Nghe An
Province
Phu Tho
Citrus hybrid - Citrus
orange
reticulata x Citrus
Viet Tri City, Phu Tho Province
maxima
Tan Trieu
Citrus grandis Osbeck
pomelo
Thanh Tra
Vinh Cuu District, Dong Nai
Province
Citrus grandis Osbeck
Thuy Bieu Ward, Hue City
pomelo
29
Doan Hung
Citrus grandis Osbeck
Pomelo
Long An lime
Da Lat lime
Doan Hung District, Phu Tho
Province
Citrus aurantifolia
Ben Luc District, Long An
Swingle
Province
Citrus limonia Osbeck
Da Lat City, Lam Dong
Province
Dao lime
Citrus hybrid – Citrus
Thai Binh City, Thai Binh
spp.
Province.
30
The specimens were further identified and authenticated by Southern horticultural
research institute (SOFRI) Vietnam. Subsequently, samples were primarily treated by
washing with tap water to remove the surface adherents. Fruits were then sliced into 6 8 equal portions. The fruits albedo layers are peeled off carefully and discarded while the
flavedo were kept for extracting the essential oils.
3.3
Essential oils extraction
3.3.1 Cold pressing method
Peel oil were extracted by hand pressing of the flavedo layer with exposed oil sacs
and collected in brine solution (saturated concentration: 40%) kept on ice, according
to the method reported by Lan-Phi et al. (2006). The extract was centrifuged at
4000g for 15 minutes. The resulting supernatant was dried in anhydrous sodium
sulphate at 5oC for 24 hours and then filtered. The oils were stored at -21oC until
analyzed. The yield of extracted essential oil was calculated base on following formula
(Njoroge and Sawamura, 2010):
Yield (%)=
x 100
3.3.2 Vacuum distillation method
In order to determine the best conditions for citrus essential oils extraction. The boiling
temperature and extraction duration were investigated and Tan Trieu pomelo was used
to extract its essential oil at the appropriate conditions.
3.3.2.1 Temperature appropriate
Samples of Tan Trieu pomelo peels were grinded in a blender to obtain small size (0.5
cm in diameter) for being extracted by a modified Clevenger-type apparatus (Figure
3.2).
The temperature of boiling flask was controlled by a thermostatic bath (Daihan Scientific
Co., Ltd; Wise Circu WCB-22), which replaced the chauffe ballon in the conventional
hydro-distillation system to provide more efficient heat transfer to the flask and avoid
localized overheating. In addition, the maintenance of the system pressure was made by
a vacuum pump (KNF Neuberger, N811 KN.18).
By fixing time extraction time for 3 hours and pressure of system at 0.7bar, 200g of
grinded materials and 400ml distilled water were then submitted to the apparatus at
60oC, 70oC, 80oC, and 90oC. Subsequently, the distillates of essential oils were separated
from water. Chemical compositions of essential oils extracted at different temperature
conditions were further analyzed using gas chromatography and compared to the cold
31
pressed oil. The temperature at which the chemical compositions of extracted essential
oils were not significant different from cold pressed oils was selected.
Figure 3.2 Model system used in extraction citrus essential oils in vacuum distillation
process.
3.3.2.2 Time appropriate
The extraction procedures were repeated as described at the best temperature condition
optimized previously for varying extraction times from 2.5 hours to 4 hours. After
extraction, the yields were determined. The time at which the extraction yield reached
the highest level was selected.
3.3.2.3 Extraction process
After determination of the best conditions, these conditions were applied for extraction of
the essential oils of all citrus samples as follow. 200g of citrus peels were grinded in a
blender to obtain small size. The grinded materials and 400ml distilled water were then
submitted to the apparatus for the optimal time, under 0.7bar and the optimal
temperature. The distillates of essential oils were separated from water and stored at
-21oC prior to analysis.
3.4
Chemical composition analysis
The analysis of the essential oils was performed by Network GC system Agilent
Technologies 6890N equipped with flame ionization detector (250oC FID) and a DB-1
column (30m x 0.25mm i.d, film thickness of 0.25µm). The column temperature was
initially maintained at 70oC for 2 min, and gradually increased at the rate 2oC per min to
32
240oC, at which the temperature was held for 20 minutes. Nitrogen was used as carrier
gas at flow rate of 0.7 ml/min.
3.5
Antimicrobial activities of citrus essential oils
3.5.1 Microbial strains
In order to determine antimicrobial activities of citrus essential oils, four bacteria and
two fungi were used. These microorganism included two gram-positive bacteria:
Staphylococcus aureus with source from Institute of Drug Quality Control – Ho Chi Minh
City and Bacillus cereus (VTCC-B-1005) obtained from Vietnam National University
Institute of Microbiology and Biotechnology, two gram-negative bacteria: Salmonella
typhi and Pseudomonas aeruginosa obtained from Institute of Drug Quality Control – Ho
Chi Minh City and two fungi Aspergillus flavus (VTCC-F-824) and Fusarium solani (VTCCF-827) were obtained from the Vietnam National University Institute of Microbiology and
Biotechnology.
The concentration of bacteria tested in experiment was 106 colony forming unit (CFU)/ml
while that of fungi was 105 CFU/ml.
3.5.2 Microorganisms counting
A loop of microorganisms from a stock culture was placed into a 10mL tube of
sterilized broth medium, and incubated for 24 h at 37oC. Then, 1mL aliquot was
transferred into a fresh sterile 9mL of Tryptone Soybean Broth (TSB) to form a 1/10 or
10-1 dilution. The 1ml of 10-1 dilution was shake vigorously and transferred to the second
9ml TSB. This second dilution represented a 10 -2 dilution of the original sample. To be
continued, a wide series of dilutions was performed (from 10-1 to 10-8). Subsequently,
100µl aliquot of microorganism suspension of each dilution was spread on plates then
incubated at 37oC for 24 hours. Only the plates which contained between 30 and 300
colonies were selected (Breed, 1916). The number of colonies from plates of selected
dilutions was used to determine the number of microorganisms in original sample based
on the following formula (Benson, 2002):
ρ
Where
N 1
VS D
N:
Number of colonies on plate (Colony forming unit: CFU)
V S:
Volume pipetted onto Petri plate (ml)
D:
Dilution factor for test tube plated out
:
Concentration of cells in original sample (CFU/ml)
33
3.5.3 Diffusion technique
The antimicrobial activity of the selected essential oils was determined by the disc
diffusion method (NCCLS, 1997). Briefly, 100µl suspension of bacteria and fungi were
spread on petri dishes containing Tryptone Soybean Agar (TSA, Himedia, India) and
Potato Dextrose Agar (PDA, Titan, India) medium, respectively. The agar plates were
prepared in 90 mm petri dishes with 22mL of agar medium giving a final depth of 3 mm.
The wells (9 mm in diameter) were punched in the culture media with essential oils and
100µl of extracts diluted in absolute ethanol to obtain a concentration 50% were added
into the wells (Armando et al., 2009). Wells without samples were used as controls. The
plates were incubated at 37 ºC for 24 hours for bacteria and at 28 °C for 48 hours for
fungal strains. Antimicrobial activity was assessed by measuring the diameter of the
growth-inhibition zone in millimeters (including well diameter of 9 mm) for the test
organisms comparing to the controls.
3.5.4 Dilution technique:
For minimum inhibitory concentration (MIC), a broth dilution susceptibility assay was
used (Takhi et al., 2011). A serial twofold dilution of essential oils in absolute ethanol
was prepared to obtain concentration of essential oils: 42mg/ml, 21mg/ml, 10.5mg/ml,
5.25mg/ml, 2.63mg/ml, 1.31mg/ml, and 0.66mg/ml. 500µl of diluted essential oils were
transferred to the test tubes. Subsequently, a fixed volume 4ml of liquid culture medium
was distributed into the test tubes and inoculated with 500µl of bacterial or fungal
suspension (106CFU/ml for bacteria and 105CFU/ml for fungi) (Takhi et al., 2011). The
experiment included three controls: the negative control tube containing culture medium
and essential oils only, the positive control tube containing culture medium and
microorganisms, and the solvent control
tube containing ethanol, medium and
microorganism. During the incubation period, the tubes were submitted to a manual
agitation every hour. The test tubes were incubated for 24 hours at 37°C for bacteria
and 48 to 25°C for fungi. After incubation, 100µl from each tube was spread on agar
medium and incubated for 24 hours to determine MIC. The lowest concentration
demonstrating no apparent growth was recorded as MIC.
3.6
Antioxidant activities of citrus essential oils
3.6.1 DPPH assay
The antioxidant activity of the essential oils was assessed by measuring their ability to
scavenging 2, 2׳-diphenyl-1-picrylhydrazyl stable radicals (DPPH) based on the method
of Ghasemi et al. (2010). Depend on the activities, the essential oils were diluted in
absolute methanol to obtain different concentrations: 5, 10, 15, 20, 25, 30, 40, 50, 60,
34
70, 80, 100, 120 and 140 mg/ml. The mixtures of essential oils were added, at an equal
volume, to methanolic solution of DPPH (100 µM). After 15 minutes at room
temperature, the absorbance of blank and resulting solutions was recorded. The control
contained methanol and DPPH solution. The disappearance of DPPH was read
spectrophotometrically at 517 nm using a spectrophotometer. DPPH inhibition (%) by
essential oil was calculated in following way (Ghasemi et al., 2010):
Inhibition (%) =
x 100
Where A control is the absorbance of the negative control and A sample is the
absorbance of the test compound. The concentration of essential oils causing 50%
inhibition (IC50) was calculated from the graph-plotted scavenging percentage against
essential oils concentration.
3.6.2 Ferric thiocyanate (FTC) assay
The inhibitory capacity of extracts was tested against oxidation of linoleic acid by FTC
method as previously reported by Hoa et al. (2007) and Sadaf et al. (2009). Essential
oils were initially diluted into different concentrations depending on the activities of
essential oils at 5 , 10 , 15 , 20, 25 , 30 ,40 , 50 , 60 , 70, 80 , 100 , 120, 140, 150,
200, 250, and 300mg/mL in absolute ethanol. Each concentration was added to a
solution mixture of 4ml 2.51% linoleic acid, 8mL absolute ethanol and 8mL of 0.2 M
sodium phosphate buffer (pH 7). All were tightly capped and kept at 40oC in the dark for
48 hours. During incubation, for every 24 hours, 0.1mL of each prepared samples above
were dissolved in 9.7 mL of 75% ethanol, 0.1mL of 30% ammonium thiocyanate, 0.1ml
of 20mM ferrous chloride in 3.5% HCl. Precisely 3 minutes after the addition of ferrous
chloride to the reaction mixture, the absorbance of red color was measured at 500 nm.
The investigation continued for every 24 hours until the absorbance of the control
reached its maximum (48 hours). A mixture without a plant extract is used as a negative
control. Synthetic antioxidant, BHT was used as positive control.
The degree of linoleic acid peroxidation was calculated using the following formula
(Pitchaon, 2011):
Antioxidant activity =
× 100
Where A control is the absorbance of the negative control after 48 hours and A sample is
the absorbance of the test compound after 48 hours. The antioxidant activity was plotted
against sample concentration in order to determine the concentration required to achieve
a 50% inhibition of linoleic acid oxidation [AA50] (Pitchaon, 2011).
35
3.7
Data analysis
Each parameter was tested in triplicate. Microsoft excel software is used to calculated
means and standard deviations. Analysis of variance (ANOVA) was applied to the data to
determine differences (p < 0.05). Statistical data analysis was undertaken using the
Statistical Package for the Social Sciences (SPSS).
36
4
4.1
Chapter 4: Results and Discussion
Optimization conditions for vacuum distillation extraction
4.1.1 Temperature optimization
Table 4.1 lists the volatile compounds detected from various extraction methods.
Detection and quantification of nine compounds were determined by GC.
In order to optimize heating temperature for the vacuum distillation, a range of
temperature from 60oC to 90oC with 10oC interval was employed. As a result, the heating
temperature for vacuum distillation could not be lower than 70 oC or higher than 80oC
because of low efficiency of essential oil extraction obtained. The power of vacuum pump
in this study could only provide vacuum pressure of 0.7bar. At 0.7 bar of vacuum
pressure, the boiling temperature of materials was only reduced up to 70 oC. At 60oC and
0.7 bar, the mixture of citrus peels and distilled water was not boiled to produce steam
for extraction of essential oils. In case of 90oC, this temperature was high and closed to
100oC. The composition of citrus essential oils was affect seriously and most of
compounds were degraded. Therefore, the compositions of essential oils extracted at
60oC and 90oC were not shown in Table 4.1.
Table 4.1 Volatile compositions (%w/w) of Tan Trieu peel EOs extracted at 70oC and
80oC by vacuum distillation method and cold pressing method.
No.
Compounds
Relative concentration (%)
Cold pressing
Vacuum distillation
o
Vacuum distillation
at 70 C
at 80oC
1
α-pinene
1.32±0.01
1.08±0.04
1.28±0.00
2
Sabinene
0.20±0.00
0.18±0.00
0.19±0.00
3
β-pinene
0.97±0.00
0.82±0.01
---
4
myrcene
2.16±0.01
1.82±0.01
1.84±0.00
5
α-terpinene
0.33±0.01
0.25±0.00
0.27±0.00
6
limonene
80.55±0.06
80.08±0.46
81.89±0.01
7
terpinolene
0.03±0.00
0.04±0.00
0.05±0.00
8
٧-terpinene
10.90±0.01
10.82±0.04
12.29±0.01
9
linalool
Tr
Tr
---
Compositions of the Tan Trieu pomelo oil extracted by cold pressing method and the
vacuum distillation at 70oC were mostly similar. There were 9 components detected in
the oils extracted by these two methods. The most abundant compound in Tan Trieu
pomelo essential oil was limonene. No significant differences were observed in the
37
concentration of limonene identified in the oils obtained by the cold pressing method
(80.55%) and the vacuum distillation at 70oC (80.08%). In addition, the concentration
of sabinene, β-pinene, α-terpinene and ٧-terpinene were 0.20%, 0.97%, 0.33% and
10.90%, respectively in cold pressed oil. Similar results were found in oils extracted by
vacuum distillation at 70oC with sabinene (0.18%), β-pinene (0.82%), α-terpinene
(0.27%) and ٧-terpinene (10.82%).
The heating temperature of the vacuum distillation affected the composition of the
essential oil. Although 9 main compounds were identified in the cold pressed oil and
distillated oil at 70oC, two of them (β-pinene and linalool) were not detected in the
essential oil extracted by vacuum distillation at 80 oC. These components were identified
to strongly affect antioxidant and antimicrobial activities of essential oils (Belletti et al,
2009; Dorman and Deans, 2000). Linalool, in previous studies, plays an important role
in inhibition ability of peroxidation and caused essential oils possessing antioxidant
activity (Hussain et al., 2008; Malhotra et al., 2009). The lost of β-pinene and linalool
might be due to high heating temperature. High temperature caused the missing of βpinene, linalool and moderate the amount of other compounds. Moreover, the
concentration of the identified compounds was also apparently different. The oil
extracted by distillation at 80oC contained higher percentage of limonene (81.89%) than
did oil obtained by cold pressing method (80.55%). However, the concentrations of
component of cold pressed oil such as ٧-terpinene (10.90%) and myrcene (2.16%) were
significant different compared to those of vacuum distillated oil. In general, chemical
compositions of extracted sample at 70oC were close to chemical compositions of cold
pressed sample. As a result, the optimal temperature for vacuum distillation of citrus
essential oil was 70oC.
4.1.2 Time optimization
Figure 4.1 Effect of extraction time on yield (%) of essential oil
38
Figure 4.1 shows effect of extraction time on the yield of essential oil from peel of Tan
Trieu pomelo. It was observed that by increasing the extraction time between 2.5 and
3.5 hours, the amount of oil extracted also increased from 0.19% to 0.29% whereby the
maximum yield of extracted oil was achieved at 3.5 hours of process. Further increase in
extraction process after 3.5 hours did not significantly increase in the oil yield. Thus, the
optimal time for vacuum distillation of citrus essential oil was 3.5 hours.
4.2
Yield of citrus essential oils:
The extraction yields of cold pressed and vacuum distillated essential oils from fresh
peels of citrus spp. widely varied depending on citrus cultivars. For each cultivar, the
production yields of the cold pressed essential oils were much lower than those of
extraction by the vacuum distillation. The production yields of citrus essential oils
extracted from 2 methods are given in Figure 4.2.
Figure 4.2 Extraction yield of citrus peel extracts using different extracting methods.
( ) EOs extracted by the cold pressing method;
( ) EOs extracted by the vacuum distillation method.
Vacuum distillation extraction of Long An lime, Da Lat lime, Dao lime, Xoan orange, Phu
Tho orange, Vinh orange, Tan Trieu pomelo, Thanh Tra pomelo and Doan Hung pomelo
peels provided the production yields of 0.14, 0.19, 0.16, 0.45, 0.59, 0.39, 0.30, 0.25
and 0.04%, whereas only 0.02, 0.04, 0.03, 0.24, 0.25, 0.14, 0.16, 0.08 and 0.03 %
yield, respectively were obtained by the cold pressing method. Phu Tho orange peel
yielded the highest amount of vacuum distillated and cold pressed essential oils
39
comparing to other citrus cultivars. The lowest yields of essential oils extracted by both
methods were obtained from Doan Hung pomelo peel. Thus, the variety of EOs yield
depends on several parameters including the locations where the plants grew and
extraction method (Uribe-Hernandez et al., 1992). Viljoen et al. (2006) and Chalchat et
al. (1995) reported variations in the yield of essential oils from Mentha longifolia (L.) and
Tagetes minuta populations, collected from different geographical locations, respectively.
The yield and of essential oils from Origanum vulgare ssp. hirtum
essential oils from
twenty three localities, scattered all over Greece were varied significantly (Vokou et al .,
1993).
4.3
Chemical compositions of citrus essential oils
4.3.1 Chemical compositions of lime essential oils
The main components of lime oils extracted by cold pressing and vacuum methods were
identified as presented in Table 4.2. The result indicated that nine components were
detected in lime EOs extracted by cold pressing and vacuum distillation methods
Limonene was the most abundant compound in lime essential oils.
Among three different lime varieties, LLA oil showed the lowest content of limonene
(40.29 and 50.64%). However, the LLA peel oils had the highest amounts of β-pinene
(14.58 and 21.89%) and sabinene (2.54 and 3.89%). The high content of β-pinene was
matched with various studies on key lime (Citrus aurantifolia) oils reported by Dugo et
al. (2002). Besides, the portion of γ-terpinene was the lowest among 3 kinds of lime EOs
(2.61 and 2.25%). Other components of LLA oils such as α-pinene (1.12 and 1.98 %),
myrcene (2.54 and 3.89%), α-terpinene (0.08 and 0.02%), terpinolene (0.57 and
0.25%) and linalool (0.31 and 0.24%) also present significant distinctions as compared
with LD and LDL oils. The differences in chemical compositions between 3 lime oils could
be linked to the different geographical locations. Variable soil textures result in different
chemical products (Hussain et al., 2008).
Climatic factors such as heat and drought
were also related to the essential oil profiles obtained (Uribe-Hermandez et al., 1992;
Milos et al., 2001).
Moreover, genetic make-up of the plant shows a greater influence
on the chemical profile of the oil produced (Graven et al., 1990; Milos et al., 2001).
For the volatile components of Da Lat EOs, nine compounds were also identified and
quantified. The limonene was also found to be prominent in EOs extracted by cold
pressing and vacuum distillation methods with 53.41% and 56.04%, respectively. The
proportion of limonene was higher than that in the literature reported for Ben Tre lime
(Citrus aurantifolia Swingle) oil (41.40%) (Lan-Phi, 2010). Among constituents, only γterpinene, which made up 12.14 and 2.25% in LDL-CP and LDL-VA, and α-terpinene,
which represented 0.18 and 11.41%, were found to indicate considerably distinctions
between these kinds of extracts. However, the total of 2 compounds in both LDL-CP and
40
LDL-VA was not considerably different. The distinction may not cause remarkable
difference in activities of these 2 essential oils because the antioxidant and antimicrobial
abilities of essential oils was stimulated by certain combination of the volatiles
(Filtenborg et al., 1996). β-pinene (9.95 and 11.02%), α-pinene
(1.74 and 1.91%),
sabinene (1.76 and 1.85%), myrcene (1.50 and 1.36%), and terpinolene (0.21 and
0.17%) were also determined in LDL-CP and LDL-VA EOs. In addition, the only member
of alcohol group, linalool, just occupied (0.30 and 0.26%) of the total content.
In case of Dao lime EOs, both LD-CP and LD-VA had high content of monoterpene
hydrocarbons with limonene (70.56% and 72.82%, respectively), γ-terpinene (10.40 %
and 8.52%) and β-pinene (6.10% and 6.73%). In addition, the percentage of myrcene
obtained in LD-CP and LD-VA accounted for 1.78% and 1.68%, followed by α-pinene
(1.50 and 1.38%), sabinene (1.27 and 1.32%), terpinolene (0.30 and 0.26%),
α-
terpinene (0.19 and 0.06%) and linalool (0.17 and 0.13%). Most of contents in LD-CP
EO were not significantly differently from LD-VA EO with exception of α-terpinene. The
amount of α-terpinene obtained in LD-CP exceeded three times larger than in LD-VA EO.
α-terpinene is one of components contributes to the antioxidant activities of essential
oils (Rudback et al., 2012). Therefore, the low proportion of α-terpinene also affects on
the quality of LD-VA.
41
Table 4.2 Volatile compositions (%w/w) of Vietnamese lime peel EOs extracted by cold pressing and vacuum distillation method
No
Compound
Long An lime
Da Lat lime
Dao lime
LLA-CP
LLA-VA
LDL-CP
LDL-VA
LD-CP
LD-VA
1
α-pinene
1.12±0.02
1.98±0.01
1.74±0.02
1.91±0.02
1.50±0.02
1.38±0.02
2
Sabinene
2.54±0.02
3.89±0.03
1.76±0.02
1.85±0.04
1.27±0.00
1.32±0.01
3
β-pinene
14.58±0.31
21.89±0.26
6.73±0.07
11.02±0.06
6.10±0.02
6.73±0.03
4
myrcene
0.96±0.02
1.15±0.04
1.68±0.01
1.36±0.03
1.78±0.02
1.68±0.02
5
α-terpinene
0.08±0.00
0.02±0.00
0.18±0.00
11.41±0.13
0.19±0.00
0.06±0.00
6
limonene
40.29±0.38
50.64±0.44
53.41±0.29
56.04±0.28
70.56±0.15
72.82±0.17
7
terpinolene
0.57±0.00
0.25±0.01
0.21±0.00
0.17±0.00
0.30±0.00
0.26±0.00
8
٧-terpinene
2.61±0.02
0.84±0.01
12.14±0.21
2.25±0.07
10.40±0.04
8.52±0.41
9
Linalool
0.31±0.00
0.24±0.01
0.30±0.00
0.26±0.00
0.17±0.00
0.13±0.00
Total
63.06±0.77
80.90±0.81
77.15±0.62
86.27±0.63
92.27±0.25
92.9±0.66
LLA-CP: Long An lime extracted by cold pressing method; LLA-VA: Long An lime extracted by vacuum distillation method.
LDL-CP: Da Lat lime extracted by cold pressing method; LDL-VA: Da Lat lime extracted by vacuum distillation method.
LD-CP: Dao lime extracted by cold pressing method; LD-VA: Dao lime extracted by vacuum distillation method
42
4.3.2 Chemical compositions of orange essential oils
EOs of three different orange varieties were analyzed and their volatile components were
identified. Chemical compositions of these EOs are displayed in Table 4.3. Orange EOs
mainly consisted of limonene with more than 90 % in chemical profiles of these EOs. In
case of Xoan orange EOs extracted by cold pressing and vacuum distillation methods
(OX-CP and OX-VA), limonene (94.25 and 94.43%), myrcene (2.10 and 2.11 %), αpinene (0.61 and 0.52%), sabinene (0.32 and 0.28%) and linalool (0.30 and 0.18%)
were the main constituents. Furthermore, α-terpinene (0.15 and 0.14 %) and β-pinene
(0.02 and 0.02%) occupied small amount in OX-CP and OX-VA EOs. Terpinolene and ٧terpinene were also detected in both OX-CP and OX-VA EOs at trace amount. Alphapinene and linalool were identified to strongly inhibit growth of bacteria (Dorman and
Deans, 2000; Fisher and Phillips, 2006). The difference in percentage of α-pinene and
linalool between OX-CP and OX-VA EOs cause the distinction in antimicrobial capacities
of these oils.
Alcohols including linalool were reported to be the most important to orange flavor
(Shaw, 1979). The amount of linalool in Vinh orange extracted by cold pressing and
vacuum distillation methods (OV-CP and OV-VA) was 0.39% and 0.43%. This compound
was identified at the higher level of 0.73% in Xa Doai orange (Citrus sinesis (L.) Osbeck)
in previous study (Lan-Phi, 2010). β-pinene (0.02 and 0.02%), terpinolene (0.02 and
0.02%) and ٧-terpinene (0.02 and 0.01%) represented at low content in cold pressed
and vacuum distillated oils.
Only eight compounds were indentified in Phu Tho orange EOs extracted by cold pressing
and vacuum distillation methods (OPT-CP and OPT-VA). This caused difference from
other orange EOs.
Limonene was the most abundant compound in both OPT-CP and
OPT-VA EOs with 95.78 and 95.61%, respectively. In addition, myrcene (2.16 and 2.12
%), α-pinene (0.59 and 0.57%), linalool (0.43 and 0.02%), sabinene (0.15 and 0.17%)
and β-pinene (0.14 and 0.03%) were obtained in both OPT-CP and OPT-VA EOs. The
results show that linalool and β-pinene contents of OPT-CP were significantly higher than
those of OPT-VA EOs. These components cause the inhibition of EOs on pathogens
(Dorman and Deans, 2000; Fisher and Phillips, 2006). Also, terpinolene, which was
found to be one of factors affecting on radical scavenging effect of EOs (Choi et al.,
2010), made up small amount in cold pressed and vacuum distillated oils with the same
percentage at 0.02%.
All three kinds of orange EOs showed the high percentage of limonene. However,
antioxidant and antimicrobial activities of these orange EOs were low because limonene
was considered that it would not play principle role in determining antioxidant and
antibacterial
activities
of
essential
oils
(Choi,
2010;
Sokovic
et
al.,
2010).
43
Table 4.3 Volatile compositions (%w/w) of Vietnamese orange peel EOs extracted by cold pressing and vacuum distillation method
No
Compound
Xoan orange
Vinh orange
Phu Tho orange
OX-CP
OX-VA
OV-CP
OV-VA
OPT-CP
OPT-VA
1
α-pinene
0.61±0.02
0.52±0.02
0.53±0.02
0.51±0.02
0.59±0.03
0.57±0.01
2
Sabinene
0.32±0.01
0.28±0.01
0.28±0.01
0.27±0.01
0.15±0.00
0.17±0.00
3
β-pinene
0.02±0.00
0.02±0.00
0.02±0.00
0.02±0.00
0.14±0.00
0.03±0.00
4
myrcene
2.10±0.10
2.03±0.08
2.67±0.08
2.11±0.05
2.16±0.05
2.12±0.08
5
α-terpinene
0.15±0.00
0.14±0.00
0.42±0.00
0.27±0.00
---
---
6
limonene
94.25±1.37
94.43±0.18
94.35±1.76
95.04±1.21
95.78±1.69
95.61±1.35
7
terpinolene
Tr
Tr
0.02±0.00
0.02±0.00
0.02±0.00
0.02±0.00
8
٧-terpinene
Tr
Tr
0.02±0.00
0.01±0.00
Tr
Tr
9
Linalool
0.30±0.00
0.18±0.00
0.39±0.39
0.43±0.00
0.43±0.02
0.02±0.00
Total
97.75±1.50
97.60±0.29
98.70±2.26
98.68±1.29
99.27±1.79
98.54±1.44
Tr: Trace, weight percent quantified less than 0.01%
OX-CP: Xoan orange extracted by cold pressing method; OX-VA: Xoan orange extracted by vacuum distillation method.
OV-CP: Vinh orange extracted by cold pressing method; OV-VA: Vinh orange extracted by vacuum distillation method.
OPT-CP: Phu Tho orange extracted by cold pressing method; OPT-VA: Phu Tho orange extracted by vacuum distillation method
44
4.3.3 Chemical compositions of pomelo essential oils
Table 4.4 show the data of chemical compositions of the pomelo essential oils
extracted by two methods. In case of Tan Trieu pomelo EOs extracted by cold
pressing and vacuum distillation methods (PTT-CP and PTT-VA), the most abundant
compound was limonene (80.55 and 80.08%), followed by ٧-terpinene (10.90 and
10.82%) and α-pinene (1.32 and 1.08%). The contents of myrcene (2.16 and
1.82%), sabinene (0.20 and 0.18%) and α-terpinene (0.33 and 0.25%) were in
agreement with Nam Roi pomelo EOs in previous study (Lan-Phi, 2010). Linalool,
which plays an important role in antioxidant and antimicrobial ability of EOs (Alma et
al., 2004; Hussain et al., 2008), was detected in both PTT-CP and PTT-VA EOs at trace
amount.
A total of nine main compounds were found in Thanh Tra pomelo extracted by cold
pressing and vacuum distillated methods (PTTR-CP and PTTR-VA). Limonene (58.44
and 64.06%), myrcene (26.43 and 24.29%) and ٧-terpinene (7.63 and 6.57%) were
the major components. Subsequently, α-pinene (0.93 and 0.78%), β-pinene (0.85
and 0.77%) and α-terpinene (0.17 and 0.07%) were also obtained in PTTR-CP and
PTTR-VA EOs. The contents of α-pinene, β-pinene and α-terpinene of cold pressed oil
were significantly higher than those of vacuum distillated oil. High amount of these
compounds in EOs were corresponding to the high antioxidant and antimicrobial
activities (Dorman and Deans, 2000; Choi, 2010; Rudback et al., 2012).
Only 7-8 compounds were detected in Doan Hung pomelo EOs extracted by cold
pressing and vacuum distillation methods (PDH-CP and PDH-VA). Limonene (42.93
and 47.26%) was the most abundant compound, followed by myrcene (41.95 and
38.22%), α-pinene (0.33 and 0.04%) and β-pinene (0.29 and 0.46%). The contents
of ٧-terpinene (0.10 and 0.08%) in Doan Hung EOs were lower than those in Tan
Trieu and Thanh Tra EOS. α-terpinene was not detected in both PDH-CP and PDH-VA
EOs.
Generally, chemical compositions of cold pressed EOs presented better quality
compared with vacuum-distillation EOs. The differences in volatile compositions of
these pomelo peels EOs were due to differences in varieties and extraction methods
(Njoroge et al., 2006; Lota et al., 2000). Cold pressed EOs offered better quality
because this technique did not affect by heat, pressure, or solvent. On the contrary,
70oC from vacuum hydro-distillation apparatus still had effect on EOs. At this
temperature, chemical transformation may happen during distillation.
45
Table 4.4 Volatile compositions (%w/w) of Vietnamese pomelo peel EOs extracted by cold pressing and vacuum distillation method
No
Compound
Tan Trieu pomelo
Thanh Tra pomelo
Doan Hung pomelo
PTT-CP
PTT-VA
PTTR-CP
PTTR-VA
PDH-CP
PDH-VA
1
α-pinene
1.32±0.01
1.08±0.04
0.93±0.00
0.78±0.00
0.33±0.00
0.04±0.00
2
Sabinene
0.20±0.00
0.18±0.00
0.20±0.00
0.19±0.00
0.16±0.00
0.17±0.00
3
β-pinene
0.97±0.00
0.82±0.01
0.85±0.00
0.77±0.00
0.29±0.00
0.46±0.00
4
myrcene
2.16±0.01
1.82±0.01
26.43±0.01
24.29±0.01
41.95±0.02
38.22±0.00
5
α-terpinene
0.33±0.01
0.25±0.00
0.17±0.01
0.07±0.00
---
---
6
limonene
80.55±0.06
80.08±0.46
58.44±0.02
64.06±0.30
42.93±0.04
47.26±0.23
7
terpinolene
0.03±0.00
0.04±0.00
0.20±0.00
0.17±0.00
0.07±0.00
0.01±0.00
8
٧-terpinene
10.90±0.01
10.82±0.04
7.63±0.00
6.57±0.18
0.10±0.00
0.08±0.00
9
Linalool
Tr
Tr
0.11±0.00
0.01±0.00
0.04±0.00
---
Total
96.46±0.10
95.09±0.56
94.96±0.04
96.91±0.49
85.87±0.06
86.24±0.23
Tr: Trace, weight percent quantified less than 0.01%.
PTT-CP: Tan Trieu pomelo extracted by cold pressing method; PTT-VA: Tan Trieu pomelo extracted by vacuum distillation method.
PTTR-CP: Thanh Tra pomelo extracted by cold pressing method; PTTR-VA: Thanh Tra pomelo extracted by vacuum distillation method.
PDH-CP: Doan Hung pomelo extracted by cold pressing method; PDH-VA: Doan Hung pomelo extracted by vacuum distillation method.
46
4.4
Antioxidant activities of citrus essential oils:
4.4.1 DPPH assay
Antioxidant activity of the essential oils of citrus fruits, as assessed by DPPH radical
scavenging assay as well as expressed in terms of 50% inhibition concentration (IC50) is
given in Figure 4.3. The use of the DPPH free radical is more beneficial in evaluating
antioxidant activity because it is more stable than hydroxyl and super oxide (LayinaPathirana et al., 2006). DPPH is a stable free radical having maximum absorption at 517
nm that accepts an electron or hydrogen atom to become a stable diamagnetic molecule.
In the presence of a substance capable of donating a hydrogen atom, its free radical
nature is lost and hence the reduction in DPPH radical was determined by the decrease
in its absorbance at 517nm. In this assay, the violet color of DPPH was reduced to a
yellow color due to the abstraction of hydrogen atom from antioxidant compound. The
more antioxidant occurred in the extract, the more DPPH reduction will occur. High
reduction of DPPH is related to the high scavenging activity performed by particular
sample (Blois, 1958). IC50 values denote the concentration of sample, which is required
to scavenge 50% of DPPH free radicals. The lower IC50 value is, the higher antioxidant
activity is.
Figure 4.3 IC50 values of investigated citrus oils by DPPH method.
( ) EOs extracted from cold pressing method;
(
) EOs extracted from vacuum distillation.
47
As can be clearly seen from the figure, all citrus EOs tested possed radical scavenging
activities. Also, the radical scavenging capacity varied significantly (p0.05) according to Tukey’s
Values followed by the same
according to Tukey’s test.
zone (mm) including well diameter of 9 mm) are mean ± standard
small letter within the same column are not significant different
test.
letter within the same line are not significant different (p>0.05)
In case of EOs extracted by cold pressing method, Long An lime EO, Doan Hung pomelo
and Tan Trieu pomelo EOs showed the highest effects with the inhibition zone diameter
of 27.67, 27.17 and 26.83 mm, respectively. From the results, other citrus EOs were
found highly susceptible to S. aureus as growth inhibition zone diameters were obtained
in a range from 20.00 to 24.00 mm. Three orange EOs (Xoan, Vinh and Phu Tho orange)
possessed the least effects on this bacterium. The results were in agreement with
previous reports. Sharma and Tripathi (2006) cited that orange oils did not showed
powerful activity against bacteria. In a report, Thailand pomelo EO obtained from cold
51
pressing method had inhibition zone with 9.50 mm in diameter against S. aureus
(Mungdee et al., 2012), whereas 3 kinds of pomelo essential oils in this study possessed
larger zone ranging from 21.58 to 27.17 mm. This indicated that Vietnamese pomelo
EOs in this assay had higher effect on this strain than Thailand pomelo EO.
Among vacuum distillation samples, Long An lime, Da Lat lime, Dao lime and Doan Hung
pomelo EOs also exhibited appreciable antimicrobial activities with inhibition zones (IZ)
of 25.17, 21.08, 18.33 and 18.17 mm against this strain, respectively. Two kinds of
pomelo, Tan Trieu and Thanh Tra pomelo essential oils showed the lowest antibacterial
activities against S. aureus with zone diameters 14.42 and 15.08 mm, respectively.
Three orange EOs presented moderate effects.
The MIC values of different citrus oils are presented in Table 4.6. Most of cold pressed
oils showed high susceptibility against S. aureus, as MIC values obtained were very low.
The lowest MIC values among cold pressed extracts were Long An lime, Tan Trieu
pomelo and Doan Hung pomelo with the same value at 2.63 mg/ml, followed by Dao
lime, Da Lat lime and Thanh Tra pomelo EOs with 5.25, 10.5, 10.5 mg/ml in MIC values,
respectively. Three kinds of orange essential oils exhibited the highest MIC values (21
mg/ml). In other word, these orange extracts possessed the lowest antimicrobial activity
on this bacterium. These results were suitable with the above data from zone of
inhibition (mm) of citrus essential oils.
As regards essential oils extracted by the vacuum distillation, most of essential oils
showed low antimicrobial capacity on this strain with MIC values were higher than 42
mg/ml except for Long An and Da Lat lime EOs. The reason may be due to the
degradation of some active compounds of essential oils extracted by vacuum distillation.
As a literature review, peel extracts of Citrus aurantifolia and Citrus hystrix DC exhibited
antibacterial activities on S. aureus with the inhibition zone of 15.5 and 16 mm,
respectively (Chaisawadi et al., 2005). Some authors proved that ethyl acetate extracts
of pomelo peel from Khao-nahm-peung and Khao-paen varieties exhibited better
inhibition activities against this bacterium. At concentration 200 mg/ml, the inhibition
zones of these oils affecting on S. aureus were 10 and 7.5 mm (Suklampoo et al., 2012).
Additionally, Citrus sinesis peel of aqueous extracts showed a very good antimicrobial
capacity when compared to Citrus limon. The inhibition zone of Citrus sinesis was 9mm,
whereas Citrus limon did not showed antibacterial activity on this strain (Ashok et al.,
2011). Upadhyay et al. (2010) reported that Citrus lemon and Azadirachta indica
essential oils had inhibition zones of 23.10 mm and 23.23 mm, respectively, were
recorded. In one research, S. aureus was found to be the most susceptible bacterium
for Citrus limon and Citrus aurantium essential oils with MIC values of 6.0 µg/ml for both
(Sokovic et al., 2017). Growth of this microorganism was completely inhibited by all of
the citrus oils with 100% of reduction of inoculums (Dabbah et al., 1970). In addition,
52
Turkish citrus peel oils also showed strong antimicrobial activity against S. aureus
(Kirbaslar et al., 2009).
The growth of S. aureus was affected by the appearance of α-pinene in the composition
of essential oils (Dorman and Deans, 2000). In addition, linalool was also significantly
effective inhibitory on this bacterium (Fisher and Phillips, 2006). Possible action
mechanisms by which microbial growth may be reduced or totally inhibited have been
suggested. It is commonly accepted that it is the toxic effects of the EO components on
the functionality and structure of the cell membrane that is responsible for the
antimicrobial activity. Uribe et al. (1985) related the respiration and alter the
permeability of the microbe cell membrane would be induced by low EO concentrations
with changes in the cell structure, while high concentrations would prompt severe
damage to the membrane and the loss of homeostasis, resulting in cell death (Carson et
al., 2002 ). Connerand and Beuchat (1984) recommended that interactions caused by
EOs in the enzymatic systems related with energy production and in the synthesis of
structural components of the microbial cells produces the antimicrobial capacity of EOs.
On the other hand, Omidbeygi et al. (2007) proposed that components of the essential
oils cross the cell membrane, interacting with the enzymes and proteins of the
membrane, so producing a flux of protons towards the cell exterior which creates
changes in the cells and, ultimately, their death. Cristani et al. (2007) reported that the
antimicrobial activity is related to ability of terpenes to affect not only permeability but
also other functions of cell membranes, these compounds might cross the cell
membranes, thus penetrating into the interior of the cell and interacting with critical
intracellular sites.
Table 4.6 MIC values (mg/ml) of citrus essential oils for S. aureus
Methods
Cold pressing
Vacuum distillation
Long An lime
2.63
5.25
Da Lat lime
10.5
10.5
Dao lime
5.25
42
Xoan orange
21
>42
Vinh orange
21
>42
Phu Tho orange
21
42
Tan Trieu pomelo
2.63
>42
Thanh Tra pomelo
10.5
>42
Doan Hung pomelo
2.63
42
Essential oils
53
4.5.2 B. cereus
Antimicrobial activities against pathogenic B. cereus of the cold pressed extracts and the
vacuum distillated extracts were investigated and compared. According to the results of
table 4.7, all EOs tested showed inhibition activity on B. cereus. For cold pressed
extracts, most of citrus EOs exhibited good effects against this baterium. Only Xoan
orange and Phu Tho orange EOs showed the least inhibition capacity on B. cereus with
the inhibition zones of 17.17 and 18.42 mm, respectively. The highest antibacterial
ability of cold pressed oils belonged to Long An lime EO (IZ=28.17mm). Da Lat lime,
Dao lime, Vinh orange, Thanh Tra pomelo and Doan Hung pomelo EOs displayed
potential effects with the inhibition zones of 22.42, 26.67, 24.17, 24.83, 26.17 mm, in
the order given.
Table 4.7 Zone of inhibition (mm) of citrus essential oils against B. cereus
Methods
Cold pressing
Vacuum distillation
Long An lime
28.17±0.29gB
23.83±0.14gA
Da Lat lime
22.42±0.38dA
22.08±0.14fA
Dao lime
26.67±0.29fB
20.33±0.29eA
Xoan orange
17.17±0.29aB
12.67±0.29aA
Vinh orange
24.17±0.29eB
17.67±0.14dA
Phu Tho orange
18.42±0.14bB
13.83±0.14bA
Tan Trieu pomelo
20.42±0.14cB
16.00±0.00cA
Thanh Tra pomelo
24.83±0.14eB
22.25±0.25fA
Doan Hung pomelo
26.17±0.29fB
22.17±0.14fA
Essential oils
Values (Diameter of inhibition
deviation.
Values followed by the same
(p>0.05) according to Tukey’s
Values followed by the same
according to Tukey’s test.
zone (mm) including well diameter of 9 mm) are mean ± standard
small letter within the same column are not significant different
test.
letter within the same line are not significant different (p>0.05)
With regard to EOs extracted by vacuum distillation method, the strongest inhibitory
effect was Long An lime EO with IZ of 23.83 mm, whereas the least effect was recorded
in Xoan orange (IZ=12.67 mm). Other citrus EOs showed antibacterial abilities against
this strain with their inhibition zones ranging from 13.83 to 22.25 mm in diameters.
These results indicated that different EOs had diverse antibacterial activities. The
difference may be due to the various chemical compositions of EOs due to geographical
regions (Njoroge et al., 2006).
Additionally, most of cold pressed EOs were more effective than vacuum distillated EOs.
Only Da Lat Lime EO was special case. A report cited the antibacterial ability of citrus
54
essential oils on B. cereus caused by the presence of linalool in EOs (Fisher and Phillips,
2006). The percentages of linalool component in cold pressed oils were higher than
those in vacuum distillated oils. This reason may explain for the higher inhibition
capacities of oils extracted by cold pressing method.
MIC of the investigated citrus essential oils obtained by dilution method are shown in
Table 4.8. Among cold pressed oils, Long An lime EO showed the best effect on B. cereus
with MIC value at 1.31mg/ml. The lowest effect were recored in Xoan orange and Phu
Tho orange EOs (MIC at 42 mg/ml). Three kinds of pomelo EOs possessed MIC values
which were larger than 2.63 mg/ml. The results were matched with the conclusion of the
study of Chanthaphon et al. (2008) who revealed that MIC value of pomelo essential oil
on B. cereus was larger than 2.25 mg/ml.
As regards citrus oils extracted by vacuum distillation method, Long An lime exhibited
the lowest MIC (5.25 mg/ml). Therefore, it maintained the highest effect against B.
cereus. Xoan orange, Phu Tho orange and Tan Trieu pomelo EOs possessed the less
antimicrobial activities with MIC values more than 42 mg/ml. The results of MIC values
was corelated with the data of inhibition zone.
Table 4.8 MIC values (mg/ml) of citrus essential oils for B. cereus
Methods
Cold pressing
Vacuum distillation
Long An lime
1.31
5.25
Da Lat lime
10.5
10.5
Dao lime
2.63
21
Xoan orange
42
>42
Vinh orange
5.25
42
Phu Tho orange
42
>42
Tan Trieu pomelo
21
>42
Thanh Tra pomelo
5.25
10.5
Doan Hung pomelo
2.63
10.5
Essential oils
Several researches studied on antibacterial activities of essential oils on B. cereus. Citrus
lemon and Azadirachta indica essential oils showed high inhibition on this bacterium with
41.3mm and 45.63mm inhibition zone, respectively (Upadhyay et al., 2010). According
to the result of the other report, all of the citrus peel oils were more effective towards B.
cereus (Kirbaslar et al., 2009). Mungdee et al. (2012) reported that only extracts from
pomelo peel indicated inhibition activities against B. cereus (IZ=8.17 mm) while those
from pomelo albedo and seed showed no inhibition activities. Chaisawadi et al. (2005)
cited EOs of Citrus aurantiifolia Swing and Citrus hystrix DC showed antimicrobial effect
against this strain. Chanthaphon et al. (2008) demonstrated that the extract from lime
55
peel (Citrus aurantifolia Swingle) showed broad spectrum inhibitory against all Gram
positive bacteria including B. cereus. The lime extract exhibited MIC value against B.
cereus at 0.56 mg/ml. This result was correlated to the report of Chaisawadi et al.
(2005)
which
cited
Citrus
aurantifolia
displaying
antibacterial
activities
on
this
microorganism.
4.5.3 S. typhi
Screening of antibacterial activities using well diffusion method of peel extracts from
citrus varieties performed various degree of growth inhibition against S. typhi. The
results are shown in Table 4.9. The cold pressed EOs showed stronger antimicrobial
activities than the ones obtained from vacuum distillation technique. The variance in
chemical compositions of EOs due to extraction methods may be the main cause in
difference in antibacterial activities (Njoroge et al., 2006).
Table 4.9 Zone of inhibition (mm) of citrus essential oils against S. typhi
Methods
Cold pressing
Vacuum distillation
Long An lime
26.33±0.29eB
24.17±0.14gA
Da Lat lime
23.17±0.14dA
23.00±0.00fA
Dao lime
28.83±0.29fB
18.33±0.29cA
Xoan orange
17.08±0.14aB
16.17±0.29aA
Vinh orange
23.00±0.00cdB
20.08±0.14dA
Phu Tho orange
20.33±0.29bB
18.17±0.29cA
Tan Trieu pomelo
20.42±0.14bB
17.17±0.29bA
Thanh Tra pomelo
22.42±0.14cB
20.50±0.25deA
Doan Hung pomelo
22.58±0.38cdB
21.08±0.14eA
Essential oils
Values (Diameter of inhibition
deviation.
Values followed by the same
(p>0.05) according to Tukey’s
Values followed by the same
according to Tukey’s test.
zone (mm) including well diameter of 9 mm) are mean ± standard
small letter within the same column are not significant different
test.
letter within the same line are not significant different (p>0.05)
The highest antibacterial ability of cold pressed oils belonged to Dao lime EO
(IZ=26.17mm). The other citrus EOs also possessed high potential against this
bacterium with inhibition zone in a range between 17.08 and 26.33 mm. There were not
significantly different in inhibtion zones among Da Lat lime, Vinh orange and Doan Hung
pomelo with zone diameters of 23.17, 23.00 and 22.58 mm, respectively. In addition,
Phu Tho orange and Tan Trieu pomelo EOs displayed the same effects on S. typhi.
56
For samples from vacuum distillation method, most of EOs exhibited low ability against
this strain with inhibition zone lower than 20.50 mm. The highest effect was replaced by
Long An lime EO with zone diameter of 24.17 mm.
The result of MIC values of citrus essential oils are presented in Table 4.10. Among cold
pressed oils, Long An lime essential oil displayed the highest capacity in inhibition on this
pathogen (MIC value at 1.31 mg/ml). The lowest effect belonged to Xoan orange with
MIC at 42 mg/ml. The MIC values of cold pressed oils were in wide range from 1.31 to
42 mg/ml, while those of vacuum distillated extracts between 5.25 and more than 42
mg/ml. These data demonstrated that the inhibition abilities of citrus extracted by cold
pressing method were higher than those extracted by vacuum distillation method on S.
typhi. However, Da Lat lime EO was exception. The LDL-CP and LDL-VA had the same
effect on this strain with the MIC value at 5.25 mg/ml.
Table 4.10 MIC values (mg/ml) of citrus essential oils for S. typhi
Methods
Cold pressing
Vacuum distillation
Long An lime
2.63
5.25
Da Lat lime
5.25
5.25
Dao lime
1.31
42
Xoan orange
42
>42
Vinh orange
5.25
21
Phu Tho orange
21
42
Tan Trieu pomelo
21
42
Thanh Tra pomelo
10.5
21
Doan Hung pomelo
10.5
10.5
Essential oils
There are many reports regarding the antimicrobial activity of essential oils on S. typhi.
Among the various essential oil treatments, the Citrus reticulata var. Tangarin exhibited
the highest antibacterial activity on S. typhi (Ashok et al., 2011). The essential oils
distilled from Syzygium neesianum Arn, Elaeocarpus lanceifolius and Citrus sinesis
showed a significant inhibition on S. typhi (Maridass, 2010; Ashok et al., 2011). Citrus
sisnesis (Linn.) also possessed potential activities against S. typhi with inhibition zone of
10 mm and MIC at a concentration of 0.25 mg/ml (Lawal et al., 2013). This strain was
also found to be more susceptible to the Citrus aurantifolia and Citrus hystrix DC
essential oils (Chaisawadi et al., 2005).
57
4.5.4 P. aeruginosa
The efficiency of two extraction methods for antimicrobial activities of various citrus
peels on P. aeruginosa was investigated. The results are shown in Table 4.11. Overall,
citrus extracts from the cold pressing method exhibited the stronger inhibitory effects
than those from the vacuum distillation method on P. aeruginosa. However, LDL-CP and
LDL-VA had not significant difference in inhibition zone on P. aeruginosa.
For cold pressed EOs, the antibacterial activity of Dao lime was predominant against P.
aeruginosa with inhibition zone of 28.67 mm in diameter. This bacterium was also
estimated as more sensitive to Long An lime, Da Lat lime, Vinh orange and Doan Hung
pomelo with zone diameters of 23.33, 22.33, 23.58, 22.33 mm. The lowest effect was
seen at Xoan orange EO (IZ=19.00 mm).
Among the vacuum distillation samples, most citrus essential oils tested exhibited low
effect with the inhibition zones in a range from 18.08 to 20.58 mm. The highest
activities against P. aeruginosa were demonstrated at Dao lime EO (IZ=23.08 mm) and
Da Lat lime EO (IZ=22.08 mm).
Table 4.11 Zone of inhibition (mm) of citrus essential oils against P. aeruginosa
Methods
Cold pressing
Vacuum distillation
Long An lime
23.33±0.29dB
20.58±0.14cA
Da Lat lime
22.33±0.38cA
22.08±0.14dA
Dao lime
28.67±0.29eB
23.08±0.14eA
Xoan orange
19.00±0.25aB
18.08±0.14aA
Vinh orange
23.58±0.14dB
20.33±0.29cA
Phu Tho orange
20.58±0.14bB
17.42±0.38aA
Tan Trieu pomelo
20.08±0.14bB
17.33±0.29aA
Thanh Tra pomelo
21.83±0.14cB
19.33±0.29bA
Doan Hung pomelo
22.33±0.29cB
19.83±0.14cA
Essential oils
Values (Diameter of inhibition
deviation.
Values followed by the same
(p>0.05) according to Tukey’s
Values followed by the same
according to Tukey’s test.
zone (mm) including well diameter of 9 mm) are mean ± standard
small letter within the same column are not significant different
test.
letter within the same line are not significant different (p>0.05)
As shown in Table 4.12, cold pressed oils displayed higher MIC than oils extracted by
vacuum distillation method. In case of cold pressed oils, Dao lime EO exhibited the
strongest effect on P. aeruginosa with MIC value of 1.31 mg/ml, whereas Xoan orange,
Phu Tho orange and Tan Trieu pomelo were recorded as the weakest with the same MIC
values of 21 mg/ml.
58
Turning to extracts from the vacuum distillation method, Dao lime EO had the lowest
MIC (5.25 mg/ml). In addition, 3 kinds of lime EOs showed very high susceptibility
against this pathogen, as MIC values obtained were very low. The rest of citrus EOs
possessed low inhibition capacities on P. aeruginosa because of high MIC values
A
number
of
publications
demonstrated
that
various
essential
oils
possessing
antibacterial activities on P. aeruginosa. Antimicrobial activity of the peel oils was found
to be directly concerning with the components that they contained. The studies showed
β-pinene, α-terpinene and geraniol were effective toward P. aeruginosa (Dorman and
Deans, 2000). Several reports investigated the antibacterial activities of essential oils on
P. aeruginosa. The Turkish citrus peel oils exhibited strong inhibition effect of P.
aeruginosa with 10-12 mm inhibition zone diameter (Kirbaslar et al., 2009). In another
research, cold water extract of orange peel performed a high antimicrobial effect against
bacteria tested, especially P. aeruginosa with zone diameter of 13 mm (Jwanny et al.,
2012). The essential oils Ammy visnaga L. exhibited strong inhibition effect of P.
aeruginosa with 25 mm inhibition zone diameter (Khalfallah et al., 2011). This
microorganism was inhibited by the lemongrass (Cymbopogon citratus) oil with MIC
value at 1% (v/v) whereas the lime (Citrus aurantifolia) oil showed the lower effect with
MIC value at 2% (v/v) (Hammer et al., 1999).
Table 4.12 MIC values (mg/ml) of citrus essential oils for P. aeruginosa
Methods
Cold pressing
Vacuum distillation
Long An lime
5.25
21
Da Lat lime
10.5
10.5
Dao lime
1.31
5.25
Xoan orange
21
42
Vinh orange
5.25
21
Phu Tho orange
21
42
Tan Trieu pomelo
21
42
Thanh Tra pomelo
10.5
21
Doan Hung pomelo
10.5
21
Essential oils
4.5.5
A. flavus
The antimicrobial activity of citrus extracts against A. flavus employed and its activity
potentials were assessed by the presence or absence of inhibition zones. The data
obtained from well diffusion method indicated that citrus EOs displayed a variable degree
of antimicrobial activity on this fungus.
59
As regards EOs extracted by cold pressing method, Dao lime EO showed the strongest
activity against A. flavus (IZ=25.42 mm), while Xoan orange and Tan Trieu pomelo
extracts possessed the least antifungal capacities with zone diameters of 17.17 mm for
both EOs. Long An lime, Da Lat lime, Thanh Tra pomelo and Doan Hung pomelo samples
exhibited zone diameters of 24.25, 23.67, 22.33 and 23.08 mm, respectively.
Among vacuum distillated EOs, the Xoan orange and Phu Tho orange EOs exhibited the
lowest effects on A. flavus. This strain was recorded to be resistant to these oils with no
inhibition zone, while the zone diameters of other EOs were between 14.17 and 23.42
mm. The strongest inhibitory effect belonged to Dao lime EO (IZ=24.17 mm). Moreover,
all cold pressed oils exhibited larger inhibition zones than vacuum distillated oils.
Limonene showed the most abundant content in citrus oils. This component in vacuum
distillated oils was predominant that in cold pressed oils. However, it had showed the
lowest effect against microorganisms (Fisher and Phillips (2006). α-pinene and linalool
which are found in appreciable amounts in cold pressed oils showed considerable
antifungal activity (Chutia et al., 2009).
Table 4.13 Zone of inhibition (mm) of citrus essential oils against A. flavus
Methods
Cold pressing
Vacuum distillation
Long An lime
24.25±0.25fB
23.42±0.29eA
Da Lat lime
23.67±0.14eA
23.17±0.14eA
Dao lime
25.42±0.14gB
24.17±0.14fA
Xoan orange
17.17±0.14aB
9.00±0.00aA
Vinh orange
18.33±0.14bB
15.08±0.14cA
Phu Tho orange
17.67±0.14aB
9.00±0.00aA
Tan Trieu pomelo
17.17±0.29aB
14.17±0.29bA
Thanh Tra pomelo
22.33±0.29cB
15.17±0.29cA
Doan Hung pomelo
23.08±0.14dB
17.08±0.14dA
Essential oils
Values (Diameter of inhibition
deviation.
Values followed by the same
(p>0.05) according to Tukey’s
Values followed by the same
according to Tukey’s test.
zone (mm) including well diameter of 9 mm) are mean ± standard
small letter within the same column are not significant different
test.
letter within the same line are not significant different (p>0.05)
The results of dilution method are presented in Table 4.14. Most cold pressed oils
exhibited low MIC value from 0.66 to 10.5 mg/ml. Also, Dao lime EO showed the lowest
MIC value (0.66 mg/ml), followed by Long An lime, Dao lime and Doan Hung pomelo
with the same MIC of 1.31 mg/ml. 3 kinds of orange and Tan Trieu pomelo possessed
the highest MIC (10.5 mg/ml).
60
With regard to vacuum distillated oils, three kinds of lime displayed strong antimicrobial
activities on A. flavus, as MIC values obtained were very low (only 1.31 mg/ml). In
addition, Xoan orange and Phu Tho orange did not show effects against this fungus.
Few studied have examined the antifungal activity of citrus essential oils. Martos et al.
(2008) examined the antifungal activities of citrus EOs such as Citrus lemon L., Citrus
reticulata L., Citrus paradisi L. and Citrus sinesis L. against A. flavus. They reported that
Citrus reticulata L. EO was the most effective at reducing the growth of this strain. In
another study, the Nigerian lime (Citrus aurantifolia) and orange (Citrus sinesis) EOs
exhibited antifungal activities against A. flavus. The MIC values of these EOs were
recorded at 50 mg/ml (Jeff-Agboola et al., 2012).
Table 4.14 MIC values (mg/ml) of citrus essential oils for A. flavus
Methods
Cold pressing
Vacuum distillation
Long An lime
1.31
1.31
Da Lat lime
1.31
1.31
Dao lime
0.66
1.31
Xoan orange
10.5
NI
Vinh orange
10.5
21
Phu Tho orange
10.5
NI
Tan Trieu pomelo
10.5
42
Thanh Tra pomelo
2.63
21
Doan Hung pomelo
1.31
10.5
Essential oils
NI: No inhibition
Citrus essential oils are a complex mixture of volatile compounds that show, among
other properties, antifungal activity by reducing or totally inhibiting fungal growth in a
dose response manner (Sharma and Tripathi, 2006b). Several authors have cited the
antifungal ability of citrus essential oils to the presence of components such as linalool
(Alma et al., 2004). Caccioni et al. (1998) suggested that the antimicrobial activities of
EOs could be the results of the synergic effect of various compounds. French (1985)
proposed that the various components of any oils may act sinergically while several
compounds may have a stimulating action on fungal spore germination. Although, some
volatile compounds show significantly distinctive activities according to their abundance,
the synergic and additive effects of volatile compounds in citrus oils may prevail over
contrasting effects of each single compound (Caccioni et al., 1998). Daferera et al.
(2000) reported that the antimicrobial activity of EOs may have been due to formation of
61
hydrogen bonds between the hydroxyl group of oil phenolics and active sites of target
enzymes.
4.5.6 F. solani
The results of antifungal activities of EOs on F. solani tested are presented in Table 4.15.
Generally, EOs extracted from cold pressing method showed larger growth inhibition
zone diameters in comparison to those extracted by vacuum distillation technique with
the exception of Da Lat lime EO. There was no significant difference in zone diameters
between LDL-CP EO and LDL-VA EO.
For cold pressing samples, various EOs exhibited excellent antifungal activities against
this fungus with the inhibition zone more than 21.17 mm, which was the zone diameter
of Tan Trieu pomelo EO. The strongest antifungal ability was recorded in Dao lime EO
with zone diameter of 27.58 mm, followed by Long An lime, Da Lat lime, Doan Hung
pomelo, Vinh orange, Thanh Tra pomelo extracts with inhibition zones of 26.17, 25.33,
24.83, 24.58, 24.08 mm, respectively. Furthermore, there was no significant difference
in inhibition zone between Xoan orange (IZ=23.17 mm) and Phu Tho orange EOs
(IZ=23.08 mm).
Table 4.15 Zone of inhibition (mm) of citrus essential oils against F. solani
Methods
Cold pressing
Vacuum distillation
Long An lime
26.17±0.29fB
24.33±0.29eA
Da Lat lime
25.33±0.14eA
25.00±0.00fA
Dao lime
27.58±0.29gB
25.33±0.14eA
Xoan orange
23.17±0.14bB
13.17±0.14aA
Vinh orange
24.58±0.14cdB
14.67±0.14bA
Phu Tho orange
23.08±0.14bB
13.33±0.29aA
Tan Trieu pomelo
21.17±0.29aB
15.17±0.29bA
Thanh Tra pomelo
24.08±0.14cB
17.17±0.14cA
24.83±0.14deB
18.17±0.29dA
Essential oils
Doan Hung pomelo
Values (Diameter of inhibition
deviation.
Values followed by the same
(p>0.05) according to Tukey’s
Values followed by the same
according to Tukey’s test.
zone (mm) including well diameter of 9 mm) are mean ± standard
small letter within the same column are not significant different
test.
letter within the same line are not significant different (p>0.05)
The results of MIC values are shown in Table 4.16. Among cold pressed oils, the lowest
MIC value was recorded in Dao lime EO (1.31mg/ml), followed by Long An lime and Dao
lime with MIC values at 2.63 mg/ml. Tan Trieu pomelo showed the highest MIC (10.5
mg/ml). Most of citrus EOs extracted by vacuum distillation method possessed higher
62
MIC than those extracted by cold pressing method. In case of EOs extracted by vacuum
distillation method, the lowest MIC were recorded in Da Lat lime and Dao lime EOs with
the same MIC at 2.63 mg/ml.
Table 4.16 MIC values (mg/ml) of citrus essential oils for F. solani
Methods
Cold pressing
Vacuum distillation
Long An lime
2.63
5.25
Da Lat lime
2.63
2.63
Dao lime
1.31
2.63
Xoan orange
5.25
>42
Vinh orange
5.25
>42
Phu Tho orange
5.25
>42
Tan Trieu pomelo
10.5
>42
Thanh Tra pomelo
5.25
42
Doan Hung pomelo
5.25
42
Essential oils
Some author mentioned that the presence of phenolic compounds leads to the antifungal
activity of EOs (Veldhuizen et al., 2006). In fact, the hydrophilic part of the molecule
interacts with the polar part of the membrane, while the hydrophobic benzene ring and
the aliphatic side chains are buried in the hydrophobic inner part of the bacterial
membrane (Cristani et al., 2007). Furthermore, the involvement of the hydroxyl group in
the formation of hydrogen bonds and the acidity of these phenolic compounds may have
other possible explanations (Cristani et al., 2007). Lucini et al., (2006) indicated that the
monoterpenes presenting in essential oils causes mycelial growth inhibition. The increase
of quantity of lipid peroxides such as hydroxyl, alkoxyl and alkoperoxyl radicals is
induced by monoterpenes. Thus, it brings about cell death. For Sharma and Tripathi
(2006b), the EOs would act on the hyphae of the mycelium, stimulating the exit of
components from the cytoplasm, the loss of rigidity and integrity of the hyphae cell wall,
resulting in its collapse and death of the mycelium. A positive correlation between
monoterpenes other than limonene and sequiterpene content of the oils and the
pathogenic fungi inhibition was observed and reported (Caccioni et al., 1998).
Although antifungal activities of EOs were found to be related their compositions.
However, the variation of each component amount depends on several parameters
including ripeness of fruits, vegetative stage of plant, storage condition, geographical
region and extraction methods (Lota et al., 2000; Njoroge et al., 2006).
63
5
Chapter 5: CONCLUSION
In the present study, the yields of EOs extracted by the vacuum distillation method were
higher than those extracted by the cold pressing method. Three kinds of orange
essential oils possessed the highest yields among citrus EOs investigated.
Both cold pressed essential oils and vacuum hydro-distillated essential oils showed high
antioxidant and antimicrobial activities. In addition, most of vacuum hydro-distillated
essential oils of citrus varieties tested had less antioxidant and antimicrobial capacities
than the cold pressed extracts. Antioxidant activities of lime essential oils were the
highest.
Moreover,
these
lime
EOs
showed
the
strongest
activity
against
all
microorganism tested. The lowest antioxidant and antimicrobial activities were recorded
in orange EOs.
This study has some limitations. The vacuum pump does not support high power enough
to reduce temperature of boiling flask to 50oC for avoiding changes in chemical
composition and functional properties of essential oils. If we can construct the best
optimization for vacuum system, we can produce better quality of essential oils and
better biological activities as natural oils but also high yields for applications in food
industry.
The result of study provides important baseline information for the use of vacuum hydrodistillation system in further research.
64
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APPENDIX
Appendix 1. Chromatogram of LD-CP (on the top) and LD-VA EOs (on the bottom). 1:
α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7:
terpinolele, 8: ٧-terpinene, 9: Linalool
75
Appendix 2. Chromatogram of LDL-CP (on the top) and LDL-VA EOs (on the bottom). 1:
α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7:
terpinolele, 8: ٧-terpinene, 9: Linalool
76
Appendix 3. Chromatogram of LLA-CP (on the top) and LLA-VA EOs (on the bottom). 1:
α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7:
terpinolele, 8: ٧-terpinene, 9: Linalool
77
Appendix 4. Chromatogram of PTT-CP (on the top) and PTT-VA EOs at 70oC (on the
middle) and 80oC (on the bottom). 1: α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene,
5: α-terpinene, 6: limonene, 7: terpinolele, 8: ٧-terpinene, 9: Linalool
78
Appendix 9. Chromatogram of PTTR-CP (on the top) and PTTR-VA EOs (on the bottom).
1: α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7:
terpinolele, 8: ٧-terpinene, 9: Linalool
79
Appendix 10. Chromatogram of PDH-CP (on the top) and PDH-VA EOs (on the bottom).
1: α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7:
terpinolele, 8: ٧-terpinene, 9: Linalool
80
Appendix 11. Chromatogram of OV-CP (on the top) and OV-VA EOs (on the bottom). 1:
α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7:
terpinolele, 8: ٧-terpinene, 9: Linalool
81
Appendix 12. Chromatogram of OX-CP (on the top) and OX-VA EO (on the bottom). 1:
α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7:
terpinolele, 8: ٧-terpinene, 9: Linalool
82
Appendix 13. Chromatogram of OPT-CP (on the top) and OPT-VA EOs (on the bottom).
1: α-pinene, 2: sabinene, 3: β-pinene, 4: myrcene, 5: α-terpinene, 6: limonene, 7:
terpinolele, 8: ٧-terpinene, 9: Linalool
83
B. cereus
LD
LDL
LLA
PTT
PTTR
PDH
OV
OX
OPT
Appendix 14. Illustration of control and Citrus EOs on the growth of B. cereus (first row: cold-pressed EO, the second row: vacuum
distillated EO)
S. Typhi
LD
LDL
LLA
PTT
PTTR
PDH
OV
OX
OPT
Appendix 15. Illustration of control and Citrus EOs on the growth of S. typhi (first row: cold-pressed EO, the second row: vacuum
distillated EO)
84
P.aerugi
nosa
LD
LDL
LLA
PTT
PTTR
PDH
OV
OX
OPT
Appendix 16. Illustration of control and Citrus EOs on the growth of P. aeruginosa (first row: cold-pressed EO, the second row: vacuum
distillated EO)
S. aureus
LD
LDL
LLA
PTT
PTTR
PDH
OV
OX
OPT
Appendix 17. Illustration of control and Citrus EOs on the growth of S. aureus (first row: cold-pressed EO, the second row: vacuum
distillated EO)
85
A. Flavus
LD
LDL
LLA
PTT
PTTR
PDH
OV
OX
OPT
Appendix 18. Illustration of control and Citrus EOs on the growth of A. flavus (first row: cold-pressed EO, the second row: vacuum
distillated EO)
F. solani
LD
LDL
LLA
PTT
PTTR
PDH
OV
OX
OPT
Appendix 19. Illustration of control and Citrus EOs on the growth of F. solani (first row: cold-pressed EO, the second row: vacuum
distillated EO)
86
Samples
Culture
tubes
Controls
TSB
medium
&
Bacteria
TSB
medium
&
Ethanol
&
Bacteria
Concentration (mg/ml)
0.655
1.31
2.63
5.25
10.5
21
42
PTT-CP
PTT-VA
PTTRCP
PTTRVA
PDH-CP
87
PDH-VA
OX-CP
OX-VA
OV-CP
OV- VA
OPT-CP
88
OPT-VA
LLA-CP
LLA-VA
LDL-CP
LDL-VA
LD-CP
89
LD-VA
Appendix 20. Effect of Citrus EOs B. cereus at different essential oil concentrations.
90
Samples
Culture
tubes
Controls
TSB
medium
&
Bacteria
TSB
medium
&
Ethanol
&
Bacteria
Concentration (mg/ml)
0.655
1.31
2.63
5.25
10.5
21
42
PTT-CP
PTT-VA
PTTRCP
PTTRVA
PDH-CP
91
PDH-VA
OX-CP
OX-VA
OV-CP
OV- VA
OPT-CP
92
OPT-VA
LLA-CP
LLA-VA
LDL-CP
LDL-VA
LD-CP
93
LD-VA
Appendix 21. Effect of Citrus EOs S.typhi at different essential oil concentrations.
94
Samples
Culture
tubes
TSB
Controls
TSB
medium
medium
&
&
Bacteria
Ethanol
0.655
1.31
Concentration (mg/ml)
2.63
5.25
10.5
21
42
&
Bacteria
PTT-CP
PTT-VA
PTTR-CP
PTTRVA
95
PDH-CP
PDH-VA
OX-CP
OX-VA
OV-CP
OV-VA
96
OPT-CP
OPT-VA
LLA-CP
LLA-VA
LDL-CP
LDL-VA
97
LD-CP
LD-VA
Appendix 22. Effect of Citrus EOs P. aeruginosa at different essential oil concentrations.
98
Samples
Culture
tubes
TSB
Controls
TSB
medium
medium
&
&
Bacteria
Ethanol
0.655
1.31
Concentration (mg/ml)
2.63
5.25
10.5
21
42
&
Bacteria
PTT-CP
PTT-VA
PTTR-CP
PTTRVA
99
PDH-CP
PDH-VA
OX-CP
OX-VA
OV-CP
OV-VA
100
OPT-CP
OPT-VA
LLA-CP
LLA-VA
LDL-CP
LDL-VA
101
LD-CP
LD-VA
Appendix 23. Effect of Citrus EOs S. aureus at different essential oil concentrations.
102
Samples
Culture
tubes
TSB
Controls
TSB
medium
medium
& Fungi
&
0.655
1.31
Concentration (mg/ml)
2.63
5.25
10.5
21
42
Ethanol
& Fungi
PTT-CP
PTT-VA
PTTR-CP
PTTRVA
103
PDH-CP
PDH-VA
OX-CP
OX-VA
NI
OV-CP
OV-VA
104
OPT-CP
OPT-VA
NI
LLA-CP
LLA-VA
LDL-CP
LDL-VA
105
LD-CP
LD-VA
Appendix 24. Effect of Citrus EOs A. flavus at different essential oil concentrations.
106
Samples
Culture
tubes
TSB
Controls
TSB
medium
medium
& Fungi
&
0.655
1.31
Concentration (mg/ml)
2.63
5.25
10.5
21
42
Ethanol
& Fungi
PTT-CP
PTT-VH
PTTR-CP
107
PTTRVA
PDH-CP
PDH-VA
OX-CP
OX-VA
OV-CP
108
OV-VA
OPT-CP
OPT-VA
LLA-CP
LLA-VA
LDL-CP
109
LDL-VA
LD-CP
LD-VA
Appendix 24. Effect of Citrus EOs F. solani at different essential oil concentrations
110
[...]... antibacterial functions of the citrus essential oils from four varieties of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.), and orange (Citrus sinesis L.) and found that all of these essential oils displayed strong antibacterial activity against the strains tested Testing and evaluation of antimicrobial activity of essential oils is difficult because of their characteristics... regions, including three major subregions in the north of Vietnam (the mountainous midland region, the Red River Delta, and the northern central coast), in total accounting for 35-40% of citrus production in Vietnam The Mekong River Delta in the south of Vietnam accounting for 55-60% of citrus production, and the rest concentrated in the central provinces (Agro, 2006) The citrusgrowing areas have increased... the proportion of α-terpineol in this oil remained at higher level than orange and mandarin oils, the amount of linalool was low (only 0.12%) Linalool, in previous studies, plays an important role in inhibition ability of peroxidation and caused essential oils possessing antioxidant activity (Hussain et al., 2008) For the volatile components of Indian Citrus sinesis (L.) Osbeck essential oil, limonene... 2.2 shows the main volatile component of citrus essential oils from several countries Table 2.2: The main volatile components (%w/w) of several citrus essential oils No Compound Vietnamese Vietnamese Vietnamese India orange mandarin pomelo orange (Citrus sinensis) (Citrus reticulata (Citrus grandis (Citrus sinesis (Ref.71) Blanco) Osbeck) (L.) Osbeck) (Ref.71) (Ref.71) (Ref.105) 1 α-pinene 0.81 0.93... collection and preparation Extraction essential oils by cold pressing method Evaluation of antimicrobial activities Evaluation of antimicrobial activities by dilution method Evaluation of antimicrobial activities by diffusion method Extraction essential oils by vacuum distillation method Evaluation of antioxidant activities Evaluation of antioxidant activities by DPPH assay Evaluation of antioxidant activities. .. in mandarin (Citrus reticulata Blanco) essential oil were limonene (91.58%), followed by myrcene (2.79%), and α-pinene (0.93%) β-pinene was important to mandarin aroma and flavor β-pinene presented at the level of 0.60% in this oil Terpene compounds are the most reasons lead to antimicrobial activities of citrus essential oils These components pass the cell membranes, penetrates into the interior of. .. growth results in production of enterotoxins, one of which is highly resistant to heat and to pH between 2 and 11; ingestion leads to two types of illness: one type characterized by diarrhea and the other, called emetic toxin, by nausea and vomiting Several reports studied on antibacterial activities of essential oils on B cereus Citrus lemon and Azadirachta indica essential oils showed high inhibition... bacteremia, bone and joint infections, gastrointestinal infections and a variety of systemic infections, particularly in patients with burn and in cancer and AIDS patients who are immune-suppressed P aeruginosa infection is a serious problem in patients hospitalized with cancer, cystic fibrosis, and burns (Ryan and Ray, 2004) A number of publications demonstrated that various essential oils possessing antibacterial... and a DB-1 column (30m x 0.25mm i.d, film thickness of 0.25µm) The column temperature was initially maintained at 70oC for 2 min, and gradually increased at the rate 2oC per min to 32 240oC, at which the temperature was held for 20 minutes Nitrogen was used as carrier gas at flow rate of 0.7 ml/min 3.5 Antimicrobial activities of citrus essential oils 3.5.1 Microbial strains In order to determine antimicrobial. .. REVIEW Essential oils An essential oil is a concentrated hydrophobic liquid containing volatile aroma compounds from plants It is less soluble in water Essential oil is extracted from plant so it carries a distinctive scent, or essence, of the plant and is therefore applied in food flavoring and perfumery (Gunther, 1952) The occurrence of essential oils is restricted to over 2000 plant varieties from ... of citrus essential oils 39 4.3 Chemical compositions of citrus essential oils 40 4.3.1 Chemical compositions of lime essential oils 40 4.3.2 Chemical compositions of. .. yield and quality of citrus essential oils Therefore, the objective of this study is to determine chemical composition, antimicrobial and antioxidant activities of Vietnamese citrus essential oils. .. of inhibition (mm) of citrus essential oils against S aureus 51 Table MIC values (mg/ml) of citrus essential oils for S aureus 53 Table Zone of inhibition (mm) of citrus essential oils
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