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Lab-scale extraction of essential oils from Tunisian lemongrass ( Cymbopogon

flexuosus )

Article in Chemical Engineering and Processing · December 2017

DOI: 10.1016/j.cep.2017.12.012

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Accepted Manuscript

Title: Lab-scale extraction of essential oils from Tunisian

lemongrass (Cymbopogon flexuosus)

Authors: Mohamed Amine Balti, Bilel Hadrich, Karim Kriaa

PII: S0255-2701(17)30616-5
DOI: https://doi.org/10.1016/j.cep.2017.12.012
Reference: CEP 7146

To appear in: Chemical Engineering and Processing

Received date: 29-6-2017

Revised date: 22-11-2017
Accepted date: 17-12-2017

Please cite this article as: Mohamed Amine Balti, Bilel Hadrich, Karim Kriaa, Lab-
scale extraction of essential oils from Tunisian lemongrass (Cymbopogon flexuosus),
Chemical Engineering and Processing https://doi.org/10.1016/j.cep.2017.12.012

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Lab-scale extraction of essential oils from Tunisian lemongrass (Cymbopogon flexuosus)

Mohamed Amine BALTIa, Bilel HADRICHb* and Karim KRIAAc,d

(a)
University of Sfax - National School of Engineers of Sfax - Research Group Agro-Food

Process Engineering (GP2A), B.O.: 1173 - 3038 Sfax Tunisia.

(b)
Unité de Biotechnologie des Algues, Biological Engineering Department, National School

of Engineers of Sfax, University of Sfax, Sfax, Tunisia

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(c)
Department of Chemical Engineering, National School of Engineers of Gabes, University

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of Gabes, Tunisia.

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(d)
College of Engineering, Al Imam Mohammad Ibn Saud Islamic University (IMSIU), PO

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Box 5701, Riyadh 11432, Saudi Arabia.

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*Corresponding author: Email: bilelehadrich@yahoo.fr; Tel: 00216 22 423 576
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Graphical Abstract:
A
M

Time Power Temperature Desirability

3.6000

3.1050
1.
ED

1.
3.0927

Y (g/100 g d.m.)
2.4625

.5
.5
PT

1.8200

0.
0.
E

1.4000
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.99045
A

Desirability

-1. .5 1. -1. 1. -1. 1.

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Highlights:

 A very important medicinal value of essential oils extracted from lemongrass.

 Quantity and purity are improved by optimization of ultrasound pretreatment.

 Time, power and temperature have a more or less significant effect on extraction

yield.

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Abstract

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The present study is conducted to investigate the effect(s) of ultrasonic pretreatment on the

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extraction of essential oils from dried leaves of lemongrass (Cymbopogon flexuosus) by

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hydro-distillation. The drying process was carried out within a forced convective dryer at

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50°C and a drying air velocity of 1 m/s. Using a response surface methodology (RSM) based
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on central composite design with the face centered, the obtained optimum conditions were as
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follows: an ultrasound pretreatment for 25 min within a power of 250W, at 50°C. Modeling
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showed that all factors (time, power and temperature) were found to be highly significant in

the extraction process (p < 0.001), to significant in all 2-way interactions (p < 0.05) except in
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the Time×Temperature interaction. Then, a second-degree model with interactions was

retained. The maximum amount of essential oils was obtained under optimized conditions
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(≈3.093 g/100g d.m.). The major component of the extracted essential oils is citral, which
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represents 85% of the extracted mass and it is present in both cis-isomer (neral) and trans-
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isomer (geranial) (respectively at 38.89% and 45.86%).

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Keywords

Lemongrass (Cymbopogon flexuosus); drying; ultrasound; hydro-distillation; essential oils.

Practical Applications

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Essential oils extracted from lemongrass leaves have a very important medicinal value. To

improve their quantity and/or purity, the extraction conditions should be studied. The key to

this improvement is the optimization of ultrasound pretreatment operating conditions. In this

work, we posit that the ultrasound application improves the extraction yield and that each

studied factor (namely time, power and temperature of ultrasound application) has a more or

less significant effect on the essential oils’ extraction yield.

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Nomenlature

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n: number of experiments

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p: probability value (0 ≤ p ≤ 1)

t: time (s or h)

xi: independent coded factors (-) U

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X: water content (kg/kg d.m.)
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Y: extraction yield of essential oils and independent variable (response) of the model (g/100g
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d.m.)
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i , ii , ij : regression coefficients

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Subscript
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0: initial value
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i, j: ith and jth value

r: reduced value
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1. Introduction

Recently, aromatic and medicinal plants have increasingly been exploited for their therapeutic

properties. Lemongrass (Cymbopogon flexuosus) is one of the most important aromatic plants,

3
which are cultivated in tropical regions. Lemongrass culture develops on different soil types,

and usually requires no manure or pesticides (Mandal et al., 2015), which is considered one of

lemongrass’ most important advantages. Generally speaking, sandy grounds with frequent

rain and an abundance of sunshine are convenient for this produce’s cultivation (Nambiar and

Matela, 2012).

Srivastava et al. (2013) and Desai et al. (2014) argue that the essential oils of lemongrass,

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extracted from the leaves, are used in flavor and fragrance, cosmetics and toiletries,

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pharmaceutical industries and therapy (for treatment of nervous and gastrointestinal

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disturbances, as well as an element with antispasmodic, analgesic, anti-inflammatory, anti-

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pyretic, diuretic and sedative effects).

In addition, lemongrass essential oils contain some components that can be

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chemotherapeutically preventative in cases of cancerous growths. In fact, lemongrass essential
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oils, having potent cytotoxic and anti-cancer potential are considered among the traditional
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and potential treatments for many ailments (Bayala et al, 2014; Boukhatem et al., 2014;
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Sheikh et al., 2017).

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In particular, lemongrass essential oils contain citral, a vital component which has significant

anti-inflammatory, antimicrobial, antifungal and mosquito-repelling properties (Desai et al.,

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2014; Sheikh et al., 2017). In fact, a mixture of isomeric acyclic monoterpene aldehydes:

neral (cis-citral) and geranial (trans-citral) (Rauber et al., 2005), citral is the major active
E
CC

component found in lemongrass oils, whose content percentage is used as a gauge of the

essential oils quality (Lin et al., 2008; Yen and Lin, 2017).
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Conventionally, lemongrass oils have been extracted using solvent extraction, steam

distillation, or hydro-distillation (Parikh and Desai, 2011; Boukhatem et al., 2014;

Tongnuanchan and Benjakul, 2014). The choice of the adequate extraction process, largely

depends on the histological localization of oils in the plant and their uses (Tongnuanchan and

4
Benjakul, 2014). However, these conventional techniques suffer from such severe drawbacks

as the inability to resolve solvent residual problems in the finished product, the long

extraction time and the energy inefficiency of the whole extractive process (Desai et al.,

2014).

In order to meet the industrial needs while protecting the environment, modern scientific

approaches of chemical and process engineering have been suggested. Techniques such as

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“eco-extraction” and “green chemistry” are becoming more and more attractive and widely-

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used (Chemat et al., 2012; Chemat et al., 2017). In fact, according to Chemat et al. (2017), the

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“Green Food Processing” is based on the discovery and design of technical processes, which

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reduce waste, energy consumption, solvent and water, allow recycling of by-products through

bio-refinery, and ensure a safe and high-quality product. The evolution of the “Green Food
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Processing” shows this innovative aspect using ultrasounds, supercritical CO2, DIC (Détente
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Instantanée Contrôlée, in French or Instant Controlled Pressure-Drop), etc. The overall
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objective is one of optimizing the production of essential oils with important citral content,
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while at the same time reducing the extraction cost. Such innovative technologies as grinding,
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enzyme-aided extraction and, more promising, ultrasound treatment may offer more viable

substitutes, for a better use of raw materials and energy (viz. reduction in extraction time).
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Although, few applications are available concerning the extraction of aroma compounds

(Ochiai et al., 2016; Muhammad et al., 2017), the benefit of using ultrasound treatment in
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plant extraction has already been demonstrated through the use of bioactive substances (de

Barros Fernandes et al., 2016: Millan-Sango et al., 2016; Boubechiche et al., 2017).
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Ultrasound-assisted extraction is an inexpensive, green, simple and efficient alternative to

conventional extraction techniques. The principle of sonication is to create an ultrasonic

cavitation, which facilitates the extraction of compounds from the solid matrix (Morsy, 2015;

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Hadrich et al., 2017). Assami et al. (2012) found out that ultrasound pretreatment reduces the

extraction time by one third compared to conventional extraction processes.

The aims of this work are: first, to study the influence of convective drying temperature (used

for matter conservation) and second, to maximize the extraction yield of essential oils from

lemongrass (Cymbopogon flexuosus). These objectives are ideally to be attained via the

optimization of the operating conditions of the ultrasonic pretreatment (time, power and

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temperature) that takes place just before the hydro-distillation (for effective extraction). The

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optimization is established using the response surface methodology (RSM). The amount of

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citral in the extracted essential oils in the final test of the optimal conditions that are to be

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determined.

2. Material and methods

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2.1 Process diagram
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Lemongrass essential oils were extracted according to conventional methods (Figure 1)

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coupled with the ultrasonic pretreatment step.

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2.2 Material
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Lemongrass belongs to the family of Graminae (Poaceae) and the genus Cymbopogon

(Skaria, et al., 2006; Marigowda et al., 2016). Lemongrass is a tropical herbaceous plant of
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the Panicoideae subfamily, cultivated for its stems and leaves with aromatic qualities (with a

sour, citric taste). Generally, three species are identified: Cymbopogon flexuosus (Nees ex
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Steud) Wats. (2n = 20, 40); Cymbopogon citratus Stapf. (2n = 40, 60); and Cymbopogon

pendulus (Nees ex Steud) Wats (Skaria et al., 2006).

Cymbopogon flexuosus is known as East Indian, Cochin or Malabar grass. C. flexuosus is a

tufted robust perennial grass of about 90 cm to 2 m in height. The leaves are linear and

6
lanceolate. It flowers freely. The inflorescence is very large with highly branched terminal

drooping panicle bearing paired spikes on tertiary branches. The spikes bear pairs of sessile

and pedicellate spikelets. The sessile spikelet is an awned, bisexual floret whereas the

pedicellate one is an awnless staminate floret (Skaria, et al., 2006; Festy, 2008). Under this

species two varieties or types are identified based on the colour of the stem: C. flexuosus var.

flexuosus (red grass) and C. flexuosus var. albescens (white grass). The corresponding

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essential oils contain more than 75-80% of citral, exhibit good solubility in alcohol and hence

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is superior in quality (Skaria et al., 2006; Desai et al., 2014).

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Lemongrass (Cymbopogon flexuosus) is planted in Sidi Bouzid (central Tunisia) in ground

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compost and drained, completely exposed to the sun. The density of plantation equals two

seedlings per square meter (m²). It forms a strong grass tuft, which can reach more than one
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meter of height. Though it has persistent foliage, its water requirements are very important.
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The fresh leaves were collected after one year of plantation (in February).
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2.3 Drying by forced convective drier

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The drying process is a crucial step performed to preserve raw material by reducing moisture

content. Three temperature levels were tested (40, 50 and 60°C) in order to probe the
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temperature effect on essential oil yield extracted without ultrasound pretreatment. The drying

was carried out within a forced convective dryer with a velocity fixed at 1 m/s (Figure 2).
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CC

The drying kinetics were determined from the mass variation over time. The measurements

were performed using an electronic precision balance 10-4 g (Mettler AT400), which was
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linked to a computer equipped with software adapted to the measuring and recording of mass

for a period of time fixed beforehand.

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Because of the instability of the values of the initial water content (X0), all drying curves are

presented as reduced moisture content (Xr) (equation 1) as a function of drying time. The

drying rate is presented as a function of reduced moisture content using equation 2.

X
Xr  (1)
X0

dX

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 dX  dt
   dX (2)
 dt  r  

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 
 dt 0

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Just after the drying step, hydro-distillation extraction of essential oils was performed to

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specify the best drying temperature, i. e., the one resulting in the largest amount of extraction.

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The amount of extracted essential oils (Y) represents the yield expressed in g/100g of dry
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matter.
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2.4 Ultrasonic pretreatment
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The experimental procedure is presented as follows: 80g of dried leaves of lemongrass was

submerged in 1L of distilled water and was placed in an ultrasonic bath (Powersonic, Digital
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Ultrasonic Bath with basket, model UB-405, capacity: 5 liters, temperature range: from room

temperature to 50°C, power range: from 70 to 250 W, internal dimensions: 300×155×150

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mm).
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The pretreatment was studied at three temperatures (30, 40 and 50°C) and at three ultrasonic
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powers (70, 160 and 250 W (at 40 kHz)) during three different extraction times: 10, 20 and 30

minutes. The coded values (xi) of different factors’ levels are indicated by −1, 0 and +1. ‘−1’:
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lowest level, ‘0’: central level and ‘+1’: highest level of variables of the model (Table 1).

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2.5 Statistical study

The studied response of the experimental design (Y), expressed in g/100g of dry matter,

presents the yield of essential oils. The experiment design was carried out using Response

Surface Methodology (RSM). The central composite design was chosen with the face

centered using two blocks.

Our study was divided into two blocks (or two phases) for the purpose of testing the influence

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of external, uncontrolled factors and verifying the reproducibility (Goupy and Creighton,

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2006): the first block is composed of the first eleven experiments of Table 2 and the second

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block consists in the remaining experiments.

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The experimental plan includes 20 trials. Six central trials are used to test the repeatability

criterion (experiments 9, 10, 11, 18, 19 and 20). The different operating conditions of the

experimental design are presented in Table 2. U

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The chosen experimental design is suitable for the exploration of quadratic-response surfaces
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and for the construction of second-order polynomial models (equation 3), thus helping to
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optimize the response by using a small number of experimental runs.

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n n n n
Y  0   i  x i  ii  x i2   ij  x i  x j   (3)
i 1 i 1 i 1 ji
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where 0 the constant term, i , ii and ij the regression coefficients, ε the error, x i the

factors, n their number and Y the response.

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The coefficients of the extraction yield of essential oils (response Y) were performed with

STATISTICA 8.0 (Stat Soft Inc.), by multiple-linear regressions. The statistical analysis of
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the model was performed to evaluate the analysis of variance (ANOVA) at the 10%

significance level, corresponding to a confidence level of 90%.

An effect of a factor (time, power and temperature) was considered as highly significant on

response when p-value < 0.01. It was considered as significant on response when 0.01 ≤ p-

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value < 0.10. The p-value represents the probability of error that is involved in accepting our

observed result as valid. The ANOVA test helps us to find the significant factors and

interactions.

2.6 Hydro-distillation

After being ultrasonically pretreated, 80g of the dried sample was cut into small pieces of 2×2

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cm² and subjected to hydro-distillation. A Clevenger-type apparatus was used twice for

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extracting essential oils from raw plant materials for 5 hours each time (Figure 3). During the

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hydro-distillation, the increase of temperature causes an increase in pressure within the plant

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cells containing the essential oils. Once the pressure increases above a certain level, the cell

walls break, and the essential oils are released. The extracted volatile compounds are then
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weighted and the yield is calculated. Thereafter, the extracted essential oils were dried using
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anhydrous sodium sulfate and stored in sealed vials at low temperature (4°C) for future use.
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2.7 Localization and identification of stationary point (maximum, minimum or saddle point)
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Equation (3) can also be presented as follows:

ˆ 
YY
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with Ŷ representing the second-order model. So, Ŷ can be written like (Myers and
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Montgomery, 1995):
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Ŷ  0  x ' b  x ' B  x (4)

where  0 , b and B contain estimates of the intercept, linear, and second-order coefficients,
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respectively. In fact, x '   x1 , x 2 , x 3  and B is the k×k symmetric matrix comporting the

model coefficients (Myers and Montgomery, 1995).

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The signs of the eigenvalues of B ( 1 ,  2 ,  3 ) determine the nature of the stationary point of

the system x s , and the relative magnitude of the eigenvalues helps the user to gain a better

understanding of the response system:

- If 1 ,  2 ,  3 are all negative, the stationary point is one of maximum response;

- If 1 ,  2 ,  3 are all positive, the stationary point is one of minimum response;

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- If 1 ,  2 ,  3 are mixed in sign, the stationary point is a saddle point.

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2.8 Gas chromatography (GC) analysis of the lemongrass essential oils

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Samples of lemongrass essential oils are analyzed on a Hewlett Packard gas chromatograph

6390 HP equipped with Flame Ionization Detector (FID) and an HP-5MS 5% capillary

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column (30.00 m × 0.25 mm, 0.25 µm film thickness). Oven temperature was initially kept at
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60°C for 4 minutes, and then it has been raised at the rate of 4°C/min to 260°C.
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Injector and detector temperatures are set respectively at 290°C and 300°C. Helium is used as
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carrier gas at a flow rate of 2 ml/min, and 0.1 µl samples are injected manually in the split
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mode. Peak areas are used for quantifying the constituent percentage in the total oils volume.

The gas chromatograph is coupled with a Hewlett Packard 5973 HP mass selective detector.
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Operating parameters for the EI-MS are: ionization voltage, 70 eV; ion source temperature,

200°C.
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2.9 Scaling up
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For the scaling up study, we need to calculate the absolute ultrasonic power P, as the heat

energy received by the treated sample versus time (equation 5), the ultrasound intensity UI

(equation 6) and acoustic energy density AED (equation 7), using the following equations

(Tiwari and Mason, 2012):

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  dT 
P  m  Cp    (5)
 dt  t 0

P
UI  (6)
S

P
AED  (7)
V

where P is the power (W); V is the volume of treated sample (L); t is applied time (s); m is the

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mass of the solvent (kg), Cp is the specific heat capacity (J/kg K); (dT/dt) is the initial

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variation of temperature (K/s) and S is the emitter surface (m²).

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3. Results and discussion

3.1 Drying results U

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The initial water content of fresh leaves of lemongrass (X0) varies from 2.303 to 2.333 kg/kg
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d.m. (about 70% on wet basis) and was reduced to a final moisture content equal to 0.001
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kg/kg d.m. (about 0.1%). Other studies presented a higher value of dried lemongrass leaves’
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moisture content (e.g. 6% in Desai and Parikh, 2015). This value was obtained by drying

under a shed for 48 hours at ambient temperature. This difference is essentially due to
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differences in the climatic conditions respective to the plants used as well as to the adopted

drying protocol. While seeking the lowest possible moisture content, drying aims to preserve
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CC

raw material from fermentation.

The curves of the reduced water content as a function of time (Xr = f (t)) for the tested
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temperatures are shown in Figure 4-a. Indeed, an increase in temperature induced a decrease

in the drying time from 2.69 h at 40°C to 2 and 1.47 h at 50°C and 60°C, respectively. This

was due to the increase of the air heat supply rate to the product, and also to the acceleration

of water migration inside it.

12
The shape of the drying kinetics of lemongrass leaves is the same for different temperatures

(Figure 4-a; Figure 4-b) and shows that the water content of the product decreases. The

reduced drying rate decreases during drying time (Figure 4-b). This confirms that the drying

of the lemongrass leaves is obtained in only one phase (phase I of the drying: decreasing rate

phase). Temperature change has an effect on water content (Figure 4-a) and drying rate

(Figure 4-b).

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The essential oils extraction yield obtained from dried lemongrass leaves (without

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pretreatment) varies between 0.70 and 0.95 g/100g d.m. (Figure 5). Our results are in

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agreement with previous studies which ranged from 0.66 to 0.90 g/100g d.m. Gautam and

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Agrawal, 2017; Yen and Lin, 2017).

According to Figure 5, it can be noticed that the extraction yield of essential oils depends on
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the drying temperature. The highest value of extraction yield of non-ultrasonically pretreated
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essential oils was obtained at 50°C and 1 m/s (0.95±0.01 g/100g d.m.). That is why, for the
A

rest of the study, the conservation of lemongrass is performed at 50°C and 1 m/s.
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3.2 Effect of ultrasound pre-treatment on the composition of the extracted lemongrass

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essential oils

Table 3 shows the composition of the essential oils extracted by two methods: direct
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extraction without pre-treatment (E0) and pre-treated extraction by ultrasound at different

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conditions (from E1 to E9) corresponding to tests 1 to 9 of Table 2.

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Chromatographic analysis show 23 compounds (mainly cis- and trans-citral with 75.4-96.9%)

that can be present in the essential oils of lemongrass. These results are considered important
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compared to those previously reported in the literature (Ganjewala, 2009; Chowdhury et al.,

2010; Desai et al., 2014). The proportion of essential oil compounds varies with the condition

of pre-treatment. It is noted that at high ultrasound power (E3, E4, E7 and E8), a maximum

13
number of compounds was noted (19, 23, 11 and 15 compounds, respectively). Based on this

criterion the better condition will be defined with the higher ultrasonic power, the longer time

and the lowest pre-treatment temperature.

However, if the selectivity is sought such us the second criterion, the minimum of compounds

is obtained in two cases: extraction without ultrasonic pretreatement (E0, with citral content

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of 96,9%), and extraction with the lower ultrasonic power, the lower temperature, and the

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reduced time of pre-treatment (E1, with citral content of 90,3%). In these conditions it is

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possible to have only 11 or 8 of 23 compounds, respectively.

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A third criterion can be a maximum extraction yield. In this case, we are interested to

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maximize the total extract yield. This criterion will be the subject of the following work and
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an experimental design will be adopted to study the effect of pre-treatment parameters on the
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extraction yield.
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3.3 Ultrasonic pretreatment effects through factorial design of experiments

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Table 2 shows the experimental extraction yields of essential oils from dried lemongrass

leaves pretreated by ultrasounds for different tested conditions of time, ultrasonic power, and
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temperature. The obtained extraction yields vary from 1.820 to 3.105 g/100g d.m., which are

patently better results than extraction without ultrasonic pretreatment (0.95±0.01 g/100g
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d.m.). In fact, there is an increase in efficiency over ordinary extraction from 2 to 3.3-times.
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The ultrasounds pre-treatment show a better performance compared to the literature results.

For example, the obtained yield is 1.5 to 2.6 times better than hydro-distillation extraction
A

(Desai et al., 2014), and 1.1 to 1.85 times better than microwave-assisted extraction (Desai

and Parikh, 2015).

A statistical test established on the effect of ultrasound proves a highly significant effect of

the ultrasonic pretreatment (p < 0.001).

14
This result can be attributed to the specific mechanism of the ultrasounds which discussed in

literature. Indeed, ultrasonic waves propagation in liquid medium, generate cavitation bubbles

due to the pressure variation, inciting a breakdown of cell walls and then facilitate the

washing out of the cell content (Tiwari and Mason, 2012; Hadrich et al., 2017). For this,

ultrasonic-assistance extraction (UAE) allowed better yields and a shorter extraction time

compared to the conventional methods. Moreover, the final product usually showed less

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thermal degradation than traditional methods, since the UAE is used for short time (Chemat et

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al., 2011; Morsy, 2015).

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The UAE influences has been developed previously, for essential oils from different aromatic

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plants (Chemat et al., 2011; Assami et al., 2012; Morsy, 2015; Boubechiche et al., 2017).

The two series of tested central points (tests 9, 10 and 11 for the first block and tests 18, 19
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and 20 for the second) in Table 2 show an important repeatability criterion in experiments. In
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fact, the corresponding standard error was calculated, and it showed a very low value (±0.006
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g/100g d.m.).
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The Analysis of Variance (ANOVA) (Table 4) shows the comparison between variance (or
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mean square) due to the factors effects (and interactions effects) and the error variance. This

comparison was established via Fisher-ratio (F). In fact, the variance is defined by the ratio
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between the sum of squares and the corresponding degree of freedom (Table 4). The

probability (p), which was calculated following the Fisher table, indicates the significance of
E
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factors and interactions if p < 0.10, and their highest significance if p < 0.01.

Table 4 shows that all studied effects (time, power and temperature) are highly significant (p
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< 0.001) on extraction yield. All interactions (except Time × Temperature interaction) are also

significant on the same response (p < 0.05), even the Time × Time term (p < 0.07). Hence, the

extraction yield of essential oils was influenced principally by the chosen factors and their

interactions: time, power and temperature. Obtaining Time × Time term as significant

15
explains the parabolic criterion of the extraction yield when the extraction duration varied

from 10 to 30 minutes.

The same results were obtained with the ultrasound-assisted extraction oils from different

matters (Tian et al., 2013; Desai et al., 2014; Morsy, 2015). This proves that chosen factors in

this work are very important to optimize essential oils extraction.

Working in blocks was not significant (p > 0.10). This confirms the reproducibility of the

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response. This result can be considered as important because of the possibility to obtain

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reproducible results, whatever the external environmental factors, for example.

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Figure 6 represents the comparison between the standard effects and the critical value of the

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Student test at p < 0.10 (vertical line). A standard effect exceeding the critical value means

that the corresponding factor (or interaction) is significant. Additionally, it confirms the
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obtained results of ANOVA. It similarly points out that the main factors (time, power and
N
temperature) and the Power × Temperature interaction have positive and important effects on
A

the extraction yield. To recap, the highest value of extraction yield was obtained at the highest
M

levels of the different factors.

ED

The quality of the model was tested with the coefficient of determination (R² = 94.79%) and

the adjusted coefficient of determination (R²(adj) = 89%). The obtained values are very high
PT

and confirm that the model fits very well with the experimental data.

Figure 7 presents the residual values versus the observation order. Compared to the
E
CC

experimental data, the residual values are very low (-0.1740 ≤ Residual ≤ 0.0970 g/100g d.m.

versus 1.820 ≤ Y ≤ 3.105 g/100g d.m.). The distribution of obtained points is random. The
A

homoscedasticity (same experimental error throughout the study area) was verified. A second-

degree model with interactions can then be retained. Equation (9) presents the final form of

the model:

16
 Ŷ  2.656  0.182  Time  0.244  Power  0.193  Temperature  0.107  Time2
0.090  Time  Power  0.100  Power  Temperature

(9)

Figure 8 shows the form of yield evolution as a function of factors in 3D forms. These results

confirm that the yield is maximum with high levels of all three factors (time, power and

T
temperature). In fact, working with longer time (20-30 min), higher power (250 W) and

IP
higher temperature (50°C) during the ultrasounic preteatement step creates facilities to extract

R
the essential oils from dried lemongrass leaves with a maximal yield.

SC
The nature of the stationary point was identified using examination of the second-order

eigenvalues: 1 = – 0.0031; 2 = – 0.0763 and 3 = – 0.1512. All eigenvalues are negative and
U
N
1 is close to zero. This demonstrates that the response surface presents a stationary point as a
A
maximum though localized outside of the experimental region (Myers and Montgomery,
M

x s1   9.094 x s2  26.219
1995). In fact, the corresponding coordinates are: ; and

x s3  29.947
ED

and the corresponding calculated response is Ys = 7.895 g/100g d.m. It is

obvious that this point is far outside the region of exploration to fit the second-order model
PT

x s1 x s2 x s3 x s1  1 x s2  1 x 1
(i.e. , and exceed the experimental region, ; and s3 ). In this
E

case, the surface may be a rising ridge (Montgomery, 1991). The most important point giving
CC

maximum yield in the experimental region is depicted in Figure 9. Results were calculated by
A

the STATISTICA Software. Figure 9 portrays the profiles of yield and desirability as a

function of each factor separately. First of all, it is clear that the time factor presents a

parabolic distribution for response Y. However, the power and the temperature factors have

almost linear influences on the response. This confirms the results previously obtained. The

17
resultant maximum value in this case is 3.093 g/100g d.m. and it is obtained with a

desirability equal to 99%. This value is very important compared with the ones obtained by

means of hydro-distillation or other techniques: from 0.942 to 1.85 g/100g d.m. (Desai et al.,

2014; Desai and Parikh, 2015; Gautam and Agrawal, 2017; Yen and Lin, 2017). The obtained

value of extraction yield of lemongrass could equally be generated through the application of

T
ultrasonic pre-treatment during 25 min (level +0.5), the highest ultrasonic power 250 W (level

IP
+1) and the highest temperature 50°C (level +1) (Figure 9). This result is very pivotal from a

R
technical point of view. In fact, applying an ultrasonic radiation such as pre-treatment, on the

SC
matter just before the effective extraction, during a reduced time of 25 min, with a high power

U
and a high temperature, results a maximum yield of extraction with reduced energy
N
consumption.
A
M
ED

3.4 Industrial application

Citral is widely used in the food industry (as a foods flavor, beverages, sweets, frozen dairy
PT

desserts, baked foods, gelatins and puddings, meat and meat products and fats and oils etc.)
E

and in cosmetics and perfumery for its lemon scent. It is also used to enrich lemon oil. It is
CC

used also in the pharmacy for the synthesis of vitamins A and E, as well as carotenoid. It has

strong antimicrobial, antiviral, sedative, antifungal, anti-tumor properties.

A

Worldwide production of essential oils is in the range of 50 to 100 t/year (such as lemon,

peppermint, citronella etc..) for prices ranging from 6 to 45 €/kg of essential (Pascual-

Villalobos et al., 2017). The prices of Cymbopogon flexuosus essential oils is about 17 €/kg

(Ultra International B.V., 2017)

18
According to Mr. Kardi, Producer of stevia and aromatic plants in Tunisia, production of

lemongrass is estimated at 180 tons in 2017, and more depending on the grounds and the

fertilizer. Production is therefore in improving way, and a transition to an industrial scale is

necessary.

4. Conclusion

Forced convective drying conditions at 50°C and 1 m/s were preferred to store lemongrass

T
(Cymbopogon flexuosus) in order to be used for extraction in our study. After that, the

IP
extraction of essential oils started by an ultrasonic pretreatment at three ultrasonic power

R
levels (70, 160 and 250W) and three temperature levels (30, 40 and 50°C) and during three

SC
time intervals (10, 20 and 30 minutes). An ordinary hydro-distillation, which lasted for 90

minutes, was subsequently performed. The objective of the present study was to optimize the
U
ultrasound pretreatment effect on the final extraction yield and on the citral amount. To
N
achieve this aim, a response surface methodology (RSM) based on central composite design
A

with face centered was used to specify the optimal conditions at which extraction yield
M

reaches its maximum, which is found to be 3.093 g/100g d.m. (250W, 50°C and 25 min).
ED

It would be a crucial step to improve the amount and the quality of citral contained in the

extracted essential oils. The obtained amount of citral in essential oils was about 85%,
PT

composed essentially of cis-citral (neral with 38.89%) and trans-citral (geranial with 45.86%).

Other optimizations can still be applied on extraction of essential oils, such as the influence of
E
CC

micro-wave assistance, the influence of instant controlled pressure drop pretreatment, the

influence of sample size.

A

Acknowledgement

We would like to thank Mr. Neji KADRI, Producer of stevia and aromatic plants in Tunisia,

for providing raw material.

19
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U
N
A
M
ED
E PT
CC
A

24
Figures:

T
R IP
SC
FIGURE 1.

PROCESS DIAGRAM

U
N
5 6 7 8 Reference Designation
A
1 Bend T
9
13 2 Horizontal test section
M

3 Vertical test section

4 4 Bend T
10 5 and 7 Cylinder
ED

2 6 Cone
Element of passage
8
14
3
section
PT

9 Bend blade
10 Diffuser
11
11 Ventilator
12 Right element
E

15 12 1
13 Heating coil
CC

14-15 Dilatation

FIGURE 2.
DRYING SYSTEM
A

25
 T
R IP
SC
U
N
A
M
ED

FIGURE 3.
SYSTEM OF HYDRO-DISTILLATION - CLEVENGER-TYPE APPARATUS
E PT
CC
A

26
 1
0.9 a
T = 40°C
0.8
T = 50°C
0.7 T = 60°C
0.6
Xr

0.5

T
0.4

IP
0.3
0.2

R
0.1

SC
0
0 1 2 3 4 5 6
Time (h)
1 U
N
0.9 b
A
0.8
M

0.7
0.6
ED
(dX/dt)r

0.5
0.4
PT

0.3
T = 40°C
0.2
T = 50°C
E

0.1 T = 60°C
CC

0
0 0.2 0.4 0.6 0.8 1
Xr
A

FIGURE 4.
EFFECT OF DRYING CONDITIONS TEMPERATURE ON THE DRYING KINETICS OF
LEMONGRASS LEAVES. (a): Xr = f(t) ; (b): (dX/dt)r = f(Xr)

27
 1

0.9

0.8
Yield (g/100 g d.m.)

0.7

0.6

0.5

T
0.4

IP
0.3

R
0.2

SC
0.1

FIGURE 5.
T = 40°C T = 50 °C
U T = 60 °C
N
EVOLUTION OF THE YIELD OF LEMONGRASS ESSENTIAL OILS EXTRACTION
A
ACCORDING TO THE TEMPERATURE OF DRYING.
M
ED
E PT
CC
A

28
 Power 7.624341

Temperature 6.050021

Time 5.708866

Power × Temperature 2.799432

Time × Power -2.51949

T
Time × Time -2.06158

IP
Block -1.6445

R
Power × Power -1.57962

SC
Temperature × Temperature -1.07356

Time × Temperature .2099574

p=.1
U
N
Standardized Effect Estimate (Absolute Value)
A
FIGURE 6.
PARETO CHART OF STANDARDIZED EFFECTS (p < 0.10)
M

0.15
ED

0.10

0.05
PT

0.00
Raw Residuals

-0.05
CC

-0.10

-0.15
A

-0.20

-0.25
-2 0 2 4 6 8 10 12 14 16 18 20 22
Case Number

FIGURE 7.
RESIDUAL VALUES VERSUS CASE NUMBER

29
 (c)

(a)
(b)

T
R IP
SC
FIGURE 8.
SURFACES PLOTS OF ESSENTIAL OILS EXTRACTION YIELD AS FUNCTION OF
U
DIFFERENT FACTORS. a: Y = F(POWER, TIME); b: Y = F(TEMPERATURE, TIME)
N
AND c : Y = F(TEMPERATURE, POWER).
A
M
ED
E PT
CC
A

30
 Time Power Temperature Desirability
3.6000

3.1050
1.
1.
3.0927

Y (g/100 g d.m.)
2.4625
.5
.5

1.8200
0.
0.

T
1.4000

IP
.99045

R
Desirability
SC
U
N
-1. .5 1. -1. 1. -1. 1.
A
FIGURE 9.
M

PROFILES FOR PREDICTED VALUES AND DESIRABILITY

ED

pA

900

800
PT

700

600

500 17.123

400
E

16.182
300

200
CC

19.975
100 21.512
11.782 19.271 20.324 26.692 27.875
11.982 21.115 25.854 27.032 29.254
23.021 28.432 29.596
5 10 15 20 25 min
A

FIGURE 10.
CHROMATOGRAM OF OBTAINED ESSENTIAL OILS

31
Tables:

TABLE 1.

CODED VALUES FOR FACTORS’ LEVELS

Level
Factor Coded Factor
-1 0 +1
Time (min) x1 10 20 30
Ultrasonic Power (W) x2 70 160 250

T
Temperature (°C) x3 30 40 50

IP
TABLE 2.

R
EXPERIMENTAL DESIGN AND RESULTS

SC
Exp. Y
Blocks x1 x2 x3
Number (g/100g d.m.)
1
2
1
1
-1
1
-1
-1 U -1
-1
1.820±0.000
2.000±0.000
N
3 1 -1 1 -1 2.290±0.014
4 1 1 1 -1 2.805±0.007
A
5 1 -1 -1 1 2.450±0.000
6 1 1 -1 1 2.595±0.007
M

7 1 -1 1 1 2.495±0.007
8 1 1 1 1 3.105±0.007
9 1 0 0 0 2.700±0.009
ED

10 1 0 0 0 2.710±0.008
11 1 0 0 0 2.700±0.002
12 2 -1 0 0 2.423±0.015
PT

13 2 1 0 0 2.517±0.012
14 2 0 -1 0 2.197±0.015
15 2 0 1 0 2.803±0.015
E

16 2 0 0 -1 2.290±0.017
CC

17 2 0 0 1 2.773±0.006
18 2 0 0 0 2.707±0.012
19 2 0 0 0 2.713±0.012
20
A

2 0 0 0 2.720±0.017
x1 : coded variable of time; x2 : coded variable of power ; x3 : coded variable of temperature

32
 R I
SC
TABLE 3.
COMPOSITIONS OF THE ESSENTIAL OILS EXTRACTED AT DIFFERENT CONDITIONS

U
Retention Concentration (%)
N° Compounds
time (min) E0 E1 E2 E3 E4 E5 E6 E7 E8 E9

N
1 8.4 α-Pinene 0.3 4.44 0.78 3.99 3.09 2.84 1.65 - - -
2 11.84 Myrcene 1.02 - - 1.38 1.04 0.55 2.16 - 0.04 -

A
3 12.18 Linalool oxide 0.06 0.97 0.65 0.69 0.59 - - - 0.41 -
4 13.25 (Z)-Linalool oxide 0.3 - - - 0.38 - - - - -

M
5 13.82 Linalool - - - - 0.31 - 0.4 - - -
6 14.36 1-Hexene, 4,5-dimethyl 0.03 - - 0.15 0.7 - 0.35 - - -
7
8
16.29
17.24
ED cis-Citral(Z)
trans-Citral (E)
40.36
56.52
39.91
50.4
37.03
45.22
36.47 38.17 38.19 38.32
43.2 43.06 51.77 47.54
34.56
44.99
34.58
40.85
36.3
48.17
9 17.76 β-Citronellol 0.3 - 3.82 1.47 1.67 - - - 2.98 -
10 19.25 Nerol 0.02 - 2.22 2.45 2.15 - 1.96 5.03 4.28 -
PT

2-Methyl-6-methylene-oct-3,7-dien-2-
11 19.96 - - 2.31 2.97 2.82 0.69 - 2.7 3.26 3.07
ol
12 20.3 Geraniol formate 0.01 2.03 2.26 2.87 2.09 4.32 2.18 6.61 6.85 2.03
E

13 22.99 3-Hexene-1-ol - 0.29 0.31 0.24 0.13 0.32 3.31 1.05 0.45 4.8
CC

14 25.2 Caryophyllene oxide - - - 0.22 0.13 - 0.24 - - -

15 26.15 α-Sinensal - - - - 0.13 - - - - -
16 30.65 β-Elemene - - - - 0.11 - - 0.45 - -
- - 0.29 0.22 0.16 - 0.75 - 0.51
A

17 31.87 Trans caryophyllene

18 32.69 2,3-Epoxygeranial - - 0.83 0.57 0.36 0.52 0.74 0.94 0.68 1.11
19 35.89 Myrcenol - - - 0.12 0.06 - - - 0.17 -
20 36.45 Linalyl acetate - 0.55 0.36 0.5 0.19 - - - 0.48 0.39
21 37.1 Nerolidol 0.02 - 0.93 0.6 0.56 0.46 0.83 1.7 1.44 1.4
22 37.27 β-Costol - 0.14 0.75 0.47 0.46 0.34 0.68 1.23 1.08 1.02
23 37.58 β-Farnesene - - 0.27 0.14 0.15 - - - 0.21 -

33
 R I
SC
TABLE 4.

U
ANALYSIS OF VARIANCE FOR THE FACTORS AND THE INTERACTION MODEL OF THE RESPONSES

N
Sum of Degree of Mean
Factor F p
Square freedom Square

A
Blocks 0.027607 1 0.027607 2.70438 0.134484
Time 0.332698 1 0.332698 32.59115 0.000291

M
Time × Time 0.043386 1 0.043386 4.25012 0.069302
Power 0.593410 1 0.593410 58.13057 0.000032
ED Power × Power 0.025472 1 0.025472 2.49520 0.148651
Temperature 0.373649 1 0.373649 36.60275 0.000190
Temperature × Temperature 0.011765 1 0.011765 1.15254 0.310958
Time × Power 0.064800 1 0.064800 6.34783 0.032796
Time × Temperature 0.000450 1 0.000450 0.04408 0.838377
PT

Power × Temperature 0.080000 1 0.080000 7.83682 0.020732

Error 0.091874 9 0.010208
Total 1.763235 19
E

R² = 94.79% ; R²(adj) = 89 %
CC
A

34

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