EXTRACTION OF CAROTENOID

A Seminar Report submitted in partial fulfilment of the

requirements for award of the degree

BACHELOR OF ENGINEERING

In

CHEMICAL ENGINEERING
Of

SAVITRIBAI PHULE PUNE UNIVERSITY

By

1.PRAVIN KALE Roll No: 306A018

2.SONALI SURYAVANSHI Roll No:306A058

Under the guidance of

DR. J. G. GUJAR

Department of Chemical Engineering

Sinhgad College of Engineering,
Vadgaon (Bk.), Pune-411041
2020-2021
 CERTIFICATE

This is certified that the Seminar Report entitled

EXTRACTION OF CAROTENOID

Submitted by
1.PRAVIN KALE

2.SONALI SURYAVANSHI
Have successfully completed their Seminar under the supervision of DR. J. G. GUJAR for the
partial ful fillment of Bachelor of Engineering - Chemical of Savitribai Phule Pune
University. This work has not been submitted elsewhere for any degree.

Name Dr. S. R. Shirsath Dr. S. D. Lokhande

Guide Head of Department Principal
 DECLARATION
We declare that this written submission represents our ideas in our own words and where others’
ideas have been include, we have adequately cited and referenced the original sources. We also
declare that we have adhered to all principles of academic honesty and integrity and have not
misrepresented or fabricated or falsified any idea/data/fact/source in our submission. We
understand that any violation of the above will be cause for disciplinary action by the Institute and
can also evoke penal action from the sources which have thus not been properly cited or from whom
proper permission has not been taken when needed.

PRAVIN KALE

SONALI SURYAVANSHI
 ACKNOWLEDGEMENT

We hereby acknowledge all the help and guidance we received throughout the course of the
preparation of this seminar from various quarters. We cannot overstate the importance of the
guidance received from our guide DR. J. G. GUJAR & Head of the Chemical Engineering
Department Dr. S. R. Shirsath. Not only did they guide us in the choice of the topic but also
went out of his/her way to provide the background study material for the same. For all this and
more, we thank them.

Our sincere gratitude goes also to Dr. S. D. Lokhande, Principal, Sinhgad College of
Engineering, for the support and encouragement they provide to everyone in this department.
A word of thanks also for all the staff, teaching and non-teaching, because whether it be a
seminar or any other student activity, there’s always a contribution from them.

Lastly we would like to thank all our classmates for their support during this seminar.

PRAVIN KALE

SONALI SURYAVANSHI
 LIST OF CONTENT

Sr. Topics Page

No.
No.

1. Abstract 1

2. Introduction 2

3. Literature review 4

1. Conventional methods 5

2. Novel methods 8

4. Case Study

1.Column Chromatography 20

2.Thin Layer Chromatography 21

3.Spectrometory 22

5. Result 27

6. Conclusion 28

7. Reference 29
 LIST OF FIGURES AND TABLE

LIST OF FIGURES
Sr. No. Figure Caption Page No.

3.1 Batch ultrasound – assisted extraction 11

3.2 The process of enzyme-assisted extraction method from the natural 14

products

3.3 (a) Closed type microwave system & (b) open type microwave system 17

4.3 HPLC-UV chromatogram of carrot extract using PFE 23

LIST OF TABLE
Sr. No. Table Caption Page No.

Table 1. Carotenoids Content in Major Plant 23

Table 2. Types and Analysis 24

Table 3. Qualitative comparison of extraction methods 24

Table 4. Physical methods used in establishing the chemical structure of 25

carotenoids.

Table 4.3 Energy values according to the spectral region 26

 Extraction of carotenoid

CHAPTER – 1

ABSTRACT

Carotene as a pigment that naturally contained in most vegetables is important source of

nutrients. carotenoids are one of the most important classes of plant pigments and play a crucial
role in defining the quality parameter of different types of natural products, fruits and
vegetables. They possess a large numbers of important biological activities such as antioxidant
properties, anticarcinogenic, precursor of vitamin A and preventive in a large number of chronic
diseases[1]. Carotenoids are the most powerful nutrients on earth due to their potent antioxidant
properties. The main constituent of the carotenes which is beta-carotene was investigated. Two
variables were varied in order to investigate the effect of drying time and solid-to-solvent ratio
on the extraction and beta-carotene yield[2]. carotenoids have been extracted from plant
materials and agro-industrial by product using different solvents, but these procedures result in
contaminating the target compound (carotenoids) with extraction solvents. The aim of this
study was to extract carotenoids from those by products. Furthermore, some utilized solvents
are not safe and hence are harmful to the environment. This has attracted criticism from
consumers, ecologists, environmentalists, and public health workers. However, there is clear
consumer preference for carotenoids from natural origin without traces of extracting solvent.
Therefore, In this project, some of the most promising techniques such as the theoretical aspects
and recent developments of various conventional and nonconventional methods used for the
extraction of carotenoids, including ultrasound-assisted extraction (UAE), pressurized liquid
extraction (PLE), and supercritical fluid extraction (SFE). Recent applications of non-toxic and
environmentally safe solvents (green solvents) and ionic liquids (IL) for carotenoid extraction
are also described. Proper choice of standard methods also influences the measurement of
extraction efficiency[3].

Key words: Antioxidants Extraction, Conventional Methods, Natural products,

ultrasoundassisted extraction (UAE), pressurized liquid extraction (PLE), and supercritical
fluid extraction (SFE).

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CHAPTER -2

INTRODUCTION

Carotenoids belong to the isoprenoid group of pigments that are produced by both
photosynthetic plants and some non-photosynthetic fungi and bacteria. Most animals cannot
synthesize carotenoids and have to obtain them from foods . Carotenoids are important
phytochemicals and have been studied extensively for their health benefits. Moreover, they are
valuable to the food industry because they can be used as natural food colorants to provide a
range of pigments, from yellow to red[1].

The uncontrollable nature of production results in a quarter of the harvest to be regarded as

byproduct. Pressurized fluid extraction (PFE) utilizes conventional solvents at controlled
temperatures and pressure, it requires less solvent and shorter extraction times, and the set up
of the technique maintains samples in an oxygen and light-free environment, which makes it
preferable for use in the nutraceutical industry[2].

Carotenoids are known for their antioxidant activity and therefore have a neuroprotective effect.
Fruits and vegetables found to possess several non-digestible components and
phytoconstituents in the form of antioxidants, which significantly contribute towards the
several added health benefits. These antioxidants are nowadays widely used as functional
ingredients in many processed foods. In developed countries, alimentation is more focused on
complimentary aspects than merely covering major component needs. Because of this, the so
called red fruits, or berries, have recently attracted a lot of attention for their antioxidant
properties, which are related to the high concentration of polyphenols present in them. In
addition, their consumption worldwide has notoriously increased, and red fruits are nowadays
not only consumed fresh but also used in cosmetics and dietary supplements[2].

A process for the extraction of carotenoid dyes from pre dried natural starting materials is
described using com pressed gases such as propane and/or butane in which organic entrainlng
agents can be additionally added in order to facilitate and complete the extraction process. With
the aid of this process highly concentrated carotenoid dyes are obtained in high yield. The
methods of TLC and UV were used for the separation and identification of carotenoid pigment
extracted from carrot through the application of different organic solvent shown Table 1.[4].

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The advantage of applying pressure is not only that temperatures above the atmospheric boiling
point of the solvents can be used, but also that it forces the extracting solvent into the matrix
and therefore may improve the extraction efficiency. Using pressure reduces the solvent surface
tension, which facilitates the penetration of solvent into the matrix pores. It results in matrix
disruption and therefore enhances the mass transfer of the analyte from the sample to the
solvent. In summary, PFE improves and therefore results in efficient extraction processes. The
extraction of carotenoids from different food matrices has recently been reviewed in. In this
paper, we have used PFE for its reported merits[5].

The aim of this study was to make beneficial use of by-product by extracting high-value
carotenoids from them. Established methods for the extraction of carotenoids require the use
of organic solvents that include n-hexane, propanol, methanol, tetrahydrofuran or ethyl acetate.
Liquid-liquid extraction of carotenoids using organic solvents is reviewed in. These organic
solvents are regarded as costly, environmentally hazardous, and require expensive disposal
procedures. There are however several studies pointing at the use of supercritical carbon
dioxide as extraction solvent for carotenoids. The objective of the study is not only to estimate
the amount of carotenoids but to also compare the estimated amounts with the other samples
present in the study. The primary objective of this study is to estimate the concentration of
carotenoids (carotenes and xanthophylls) using simple solvent extraction ant thin layer
chromatography in the Natural Products, Fruits and vegetables[6].

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CHAPTER 3

LITERATURE REVIEW

Conventionally, carotenoids are extracted using organic solvents, such as acetone, chloroform,
hexane, isopropanol, methanol, methylene chloride and diethyl ether. A wide variety of solvent
combinations have also been used, which provides a synergistic effect on extraction of
carotenoids. The choice of appropriate solvent or solvent combination is one of the most critical
factors for efficient extraction of carotenoids. However, selection of the appropriate one is not
always easy, as the functional group and chain length of the existing carotenoids the sample
matrix and its components, and moisture content play important roles[6]. Lycopene and
βcarotene are highly lipophilic non-polar carotenoids due to their conjugated hydrocarbon
structure lacking polar functional groups. The addition of polar functional groups, such as
epoxy (violaxanthin and neoxanthin), hydroxyl (lutein and zeaxanthin), keto (canthaxanthin)
or keto with hydroxy (astaxanthin) groups, increases the polarity of carotenoids. Usually,
acetone and hexane are frequently selected for extraction of polar and nonpolar carotenoids,
respectively. On the other hand, a mixture of acetone/ ethanol/hexane is most frequently applied
for the simultaneous extraction of polar and nonpolar carotenoids. Owning to water miscible
properties, acetone and ethanol are preferred for efficient extraction of carotenoids from plant
material containing a high amount of moisture[7].

Chromatographic methods are commonly employed to separate, identify, and quantify

carotenoids in food, blood, and tissue samples. Prior to analysis, carotenoids must first be
extracted from the sample matrix. Due to the inherent instability of these compounds, it is
important that this step minimizes isomerization, oxidation, and degradation to ensure accurate
analytical results. Carotenoids are highly conjugated, making them susceptible to degradation
by heat, light, oxygen, and metal ions. For example, b-carotene is rapidly degraded under
ultraviolet and visible light in an aerobic environment. Therefore, samples should be handled
under dim or red light, solvent evaporated with gas (nitrogen, argon, or helium), and stored in
the dark at temperatures below –20 C. The pH of the solutions used during extraction and
analysis should also be considered, as 5,6-epoxides of cyclic carotenoids can rearrange to
5,8epoxides in the presence of acid . Carotenoids are generally less stable in extracts than in
food or biological matrices, and therefore samples should be analyzed immediately following
extraction to limit the formation of artifacts shown table 2.[8].

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Methods of Extraction of Antioxidants:

There are two types of extraction techniques based on their ease to extract solute from mixture:-

1. Conventional methods

2. Novel methods

3.1 Conventional Methods of Extraction:

Classical/conventional extraction techniques are being used at a small scale level to extract
antioxidants components from several plant materials. These techniques are usually based on
the extraction efficiency of different solvents, which are being used for this purpose. It is a
three-process approach, which consists of[3].

1. Soxhlet extraction

2. Maceration process

3. Hydro distillation (HD).

3.1.1. Soxhlet Extraction:

The soxhlet extraction technique has been widely used for extracting several bioactive
compounds from various plant materials. Dry plant material sample needs to be kept in the
thimble. The thimble is then placed in the distillation flask, which contains selective solvent
when overflow level of solvent is reached; the solution of the thimble-holder is aspirated by a
siphon. Siphon unloads the solution back into the distillation flask. This solution carries
extracted solutes into the bulk liquid. Solute is remained in the distillation flask and solvent
passes back to the solid bed of plant. The process runs repeatedly until the extraction is
completed[3].

Example: Soxhlet extraction of curcumin from turmeric.

The Soxhlet extraction was implemented as the reference method to evaluate the effectiveness
of the enzyme-assisted ionic liquid extraction. Soxhlet extraction is a type of atmospheric liquid

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extraction, utilizing solvents at boiling temperature and low pressures (ambient pressure), for
the selective extraction of targeted compounds. Soxhlet extraction is a conventional technique
providing the highest recovery of carotenoids. Thus, it is commonly used for evaluating the
performance of other methods . However, it’s time consuming, and also uses significant
amounts of solvents, thus increasing the cost of extraction. Additionally, the high temperature
and long extraction time increases the possibilities of thermal degradation and cis-trans
isomerization of carotenoids. Thus, the authors suggested that ASE can replace the time
consuming Soxhlet extraction for industrial scale extraction[6].

3.1.2. Maceration Process:

It is solvent extraction techniques . Maceration step was used at homemade level for preparing
tonic since from olden days. This becomes popular and also one of the cheapest ways to obtain
several essential oils and bioactive compounds from different plant materials. For smaller scale
of extraction, maceration process generally consists of several steps. First, the materials are
ground, i.e. their size is being reduced in order to increase the surface area for uniform mixing
with chosen solvent. As a second step, in maceration process, appropriate solvent, i.e.
menstruum, is added in a closed vessel. Third, the liquid is strained off but the marc, which is
the solid residue of this extraction process is pressed to recover large amount of occluded
solutions. The obtained strained and the press out liquid are mixed and separated from
impurities by filtration. Effective cell disruption, either by physical, chemical or mechanical
means, is a prerequisite for the efficient extraction of intracellular carotenoids. Cell wall
disruption facilitates the entry of solvents into the cell to solubilize intracellular carotenoids.
Among the extraction methods, the highest yield of total carotenoids from pink shrimp was
obtained by maceration using acetone, followed by Soxhlet extraction with hexane/isopropanol,
as compared to ultrasound-assisted extraction. Subsequently, Soxhlet also presented a
satisfactory yield of carotenoids, probably due to the low viscosity and surface tension at
boiling temperature, which improves the diffusion and solubilization of carotenoids.. In a recent
study, the highest total carotenoids and antioxidant potential from the lyophilized peel of Gac
fruit were obtained using ethyl acetate compared to acetone, ethanol and hexane. With
increasing evidence of the carotenoid composition of Gac fruit peel (containing 54, 31 and 13%
of β-carotene, lycopene and lutein respectively), the higher yield of total carotenoids obtained
by ethyl acetate compared to hexane might be due to the higher polarity of ethyl acetate, which
can extract both carotene and xanthophylls simultaneously[9].

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In this study, the authors have not investigated the extraction efficiency of hexane and ethyl
acetate mixture, which can possibly yield higher carotenoids, as described by Strati and
Oreopoulou (2011) for highest extraction of carotenoids from tomato waste. Carotenoid
extraction with organic solvents thus provides good extraction yields without utilizing
sophisticated instruments. However, owing to the cost, toxic nature and the residue
contamination of final products, there are limitations for its application in food and feed
products.

Occasional shaking in maceration process will facilitate extraction by means of two ways:

at first, it will increase diffusion and second, it will remove concentrated solution from the
sample surface for bringing new solvent to the menstruum for achieving more extraction
yield[2].

Example: Extraction of total phenolic and total flavonoid compounds from grapes

Wine pomace is characterized by the presence of natural antioxidants and is characterized by

high-phenolic contents due to poor extraction during winemaking, and thus making their
utilization worthwhile.

3.1.3. Hydrodistilation (HD):

Hydrodistilation, which is one of the traditional method used for extracting bioactive
compounds and essential oils from several plant materials. In this process, organic solvents are
not involved, and it can be performed before dehydration of any plant materials [2].

There are three types of HD:

1. water distillation

2. water and steam distillation

3. direct steam distillation

In HD, 1) the plant materials are packed in a still compartment; 2) water is added in sufficient
amount and then brought to boil. Alternatively, direct steam is also injected into the plant
sample. Both hot water and steam can act as the main influential factors for releasing bioactive
compounds from several plant tissues. Indirect cooling by water condenses the vapour mixture

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of water and oil. Condensed mixture flows from condenser to a separator, where oil and
bioactive compounds were separated automatically from the water. HD involves three main
physicochemical processes: 1. Hydro diffusion 2. Hydrolysis, and 3. Decomposition by heat.
At higher extraction temperature, some of the volatile components will be lost. Therefore, this
drawback limits its use for extraction of various thermolabile compounds from different plant
tissues[4].

The extraction rate of any conventional method mainly depends on the choice of using selective
solvents.

Criteria for selecting solvents:

1. Solvent polarity

2. Molecular affinity between chosen solvent and solute

3. Solvents environmental safety

4. Toxicity

5. Economic feasibility.

Disadvantages:

1. longer extraction time

2. Requirement of costly and high purity solvent

3. Evaporation of the huge amount of solvent

4. Low extraction selectivity

5. Thermal decomposition of thermolabile compounds.

3.2 Novel Methods of Extraction:

To overcome limitations of conventional extraction methods, new and promising extraction

techniques are introduced. These techniques are referred as novel extraction techniques. Some
of the most promising techniques are

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1. Ultrasound-assisted extraction (UAE),

2. Microwave-assisted extraction (MAE),

3. Pressurized liquid extraction (PLE),

4. Supercritical fluid extraction (SFE), Some of these techniques are considered as ‘green
techniques’ as they comply with standards set by Environmental Protection Agency
(2015) [3].

Advantage:

1. Less hazardous chemical synthesis

2. Designing safer chemicals

3. Safe solvents auxiliaries

4. Design for energy efficiency

5. Use of renewable feedstock

6. Reduce derivatives

7. Green techniques

8. Catalysis

9. Design to prevent degradation

10. Atom economy

11. Time analysis for pollution prevention

12. Inherently safer chemistry for the prevention of accident.

3.2.1 Ultrasound-assisted extraction (UAE):

An effective technique of food processing, ultrasound can be applied to many other processes,
including intracellular metabolite extraction and microbial inactivation. The major impact of

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ultrasound in a liquid medium is attributed to the acoustic cavitation, leading to cell rupture that
enhances the mass-transfer of extractants. Ultrasound is a special type of sound wave beyond
human hearing, and its frequency ranges from 20 kHz to 100 MHz. Like other waves, it can
pass through a medium by creating compression and expansion. Therefore, this process
produces a phenomenon known as cavitation, which further leads in production, growth, and
collapse of bubbles. A large amount of energy can be produced during the conversion of kinetic
energy of motion, and thereby, it helps in heating the contents of the bubble. Bubbles have
temperature about 5000 K, pressure of 1000 atmosphere, heating, and cooling rate above 1010
K/s. Only liquid and liquid containing solid materials have cavitation effect. The effects caused
by the ultrasonic waves are compression and expansion cycles during the passage through the
fluid. The expansion can create bubbles or cavities in a liquid. The process by which vapor
bubbles form, grow, and undergo implosive collapse is known as cavitation. This condition of
cavitation produces conditions very high shear energy waves and turbulence in the cavitation
zone. The combination of these factors (pressure, heat, and turbulence) is used to accelerate
mass transfer in the extraction process[10] .

The extraction mechanism by ultrasound process involves two main types of physical
phenomena:

1. The diffusion across the cell wall

Fig.3.1 Batch ultrasound – assisted extraction

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2. Rinsing the contents of cell after breaking the walls Important factors for obtaining
efficient and effective extraction;

Moisture content of sample, milling degree, particle size, solvent, temperature, pressure,
frequency, and time of sonication[4].

Process system:

UAE experiments can be performed with a sonotrode and a glass reaction tank. The
doublelayered mantle of the reactor, which allows the control of extraction temperature with a
cooling system by means of water circulation. The transducer is connected to the horn with a
‘booster’ installed in amplification mode and finally the sonotrode, which needs to be immersed
into the middle of the liquid and samples have to be filled in the tank (Figure 3.1).

Examples:

Using response surface methodology (RSM), solvent (acetone)/CDW ratio of 67.38 µl/mg,
power 27.82% (total power 500 W), pulse length of 19.7 s and extraction time of 13.48 min
were optimized to obtain the highest yield of zeaxanthin (11.2 mg/g) and βcarotene (4.98 mg/g)
from the green microalgae Chlorella saccharophila. In another experiment, comparison among
five different solvents yielded highest carotenoid content (79.61%) from Rapeseed meal, using
petroleum ether/acetone, when compared to petroleum ether, acetone, chloroform and
methanol.Additionally, an extraction temperature of 49.6 °C, ultrasonic power 252.9 W, liquid
to material ratio 41.4 ml/g and duration of 48.5 min, was optimized using RSM for the highest
recovery of carotenoids. The use of an optimized range of ultrasound intensity is a crucial
parameter to obtain the highest yield of carotenoids in UAE. Above the optimum range, the
higher ultrasound intensity can lead to formation and accumulation of OH and H radicals during
the cavitation process, which can lead to the significant degradation of the antioxidant
compounds including carotenoids obtained the highest recovery of carotenoids (80%) from the
microalgae Heterochlorella luteoviridis using 50% ultrasound intensity (total power 664 W),
at 30 °C temperature and 75% of ethanol concentration. The authors observed a significant
decrease (59%) in the extraction yield at an ultrasound intensity of 100%. Using UAE, the
highest extractability of xanthophylls from lyophilized Aristeus antennatus shrimp was
achieved using acetone as a solvent (10 ml/g), compared to N,Ndimethylformamide (DMF),
isopropanol/n-hexane (1:1 v/ v) petroleum ether/acetone (1:1 v/v) and petroleum ether/acetone/
ethanol mixture (2:1:1 v/v/v). Interestingly, nhexane/acetone/ethanol mixture (2:1:1) with a

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liquid to material ratio of 20 ml/g, was the most suitable solvent for MAE of xanthophylls from
the same samples. These results can be explained by the heating efficiency of solvents that
absorb microwaves and provide high extraction yields. Other UAE (5 min, 600 W), and MAE
(7 min, 30 W) parameters were also optimized using RSM for the highest yields of 60% and
105% recovery of carotenoids, respectively. The power 27.82% (total power 500 W),
solvent/CDW ratio of 67.38 ml/mg, a pulse length of 19.7 s for 13.48 min, were found to be
optimum ASE conditions for the highest yield of zeaxanthin and β-carotene from Chlorella
Saccharophila[11].

Extraction process:

1. Box-Behnken design (BBD) was employed to optimize the

ultrasoundassisted extraction of polysaccharides from guava leaves (GLP).
The ultrasound-assisted extraction for GLP is effective and environmentally
friendly and GLP has wide application potentials as natural antioxidants and
α-glucosidase inhibitors [3].

2. In a study conducted by Cho et al. (2006), the impact of UAE on resveratrol

recovery from grapes was evaluated. With UAE, they obtained a significant
resveratrol yield (up to 24 and 28% in Campbell and Gerbong grape,
respectively) and a decrease of the extraction time compared to traditional
solvent extraction at 60°C for 30 min [4].

3. Corrales et al. (2008b) studied the effects of UAE combined with thermal
treatment (70°C) to recover antioxidant bioactive compounds from grape
byproducts. They observed a significant increase in total phenolic
compounds of the extracts obtained after applying USN treatments compared
to conventional water extraction [4].

Advantage:

1. Reduction in extraction time, energy, and use of solvent.

2. Facilitates more effective mixing, faster energy transfer, reduced thermal gradients

3. Efficacious extraction temperature and selective extraction , reduced equipment size

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4. Faster response to process extraction control and increased prod/uction

5. Common extraction techniques for by-products of fruits and vegetable

3.2.2 Enzyme-assisted extraction:

Enzyme-assisted extraction (EAE) methods utilize hydrolytic enzymes to break down the
structural integrity of cell walls to expose intracellular materials for improved extraction yield.
EAE of carotenoids from plant products appears to have a high potential, and is promising for
commercial applications Cellulase and pectinase enzymes are generally employed in a pre
treatment step before solvent extraction. Cellulase hydrolyzes the 1,4-βdglycosidic linkages of
the cellulose present in the primary cell wall of plant cells. Similarly, pectinase breaks down
the pectic substances and pectin found in the middle lamella and primary cell walls[5].

Some phytochemicals in the plant matrices are dispersed in cell cytoplasm, and some
compounds are retained in the polysaccharide-lignin network by hydrogen bonding or
hydrophobic interactions, which are not accessible with a solvent in a routine extraction
process. Enzymatic pre-treatment has been considered as a novel and an effective way to release
bounded compounds and also to increase overall yield. The addition of specific enzymes such
as cellulose, α-amylase, and pectinase during extraction enhances recovery by breaking the cell
wall and hydrolyzing the structural polysaccharides and lipid bodies. The oil extracted by
enzyme assisted methods was found to contain higher amount of free fatty acids and
phosphorus contents than traditional hexane extracted oil[12].

Extraction process:

There are two approaches for EAE process:

1. Enzyme-assisted aqueous extraction (EAAE)

EAAE methods have been developed mainly for the extraction of oils from various
seeds.The process of EAE method from the natural products was shown in Figure. The
breakdown of cell walls is the critical step for the extraction of many bioactive
compounds, which are existing inside the cell walls. EAE is based on the ability of
enzymes to hydrolyze cell wall components and disrupt the structural integrity of the
plant cell wall under mild process conditions, thereby allowing the efficient extraction

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and release of the bioactive compounds. The EAAE is recognized as eco-friendly

technology for extraction of bioactive compounds from oil because it uses water as
solvent instead of organic chemicals [4].

2. Enzyme assisted cold pressing (EACP)

In EACP technique, enzymes is used to hydrolyze the seed cell wall because in this
system polysaccharide-protein colloid is not available, which is obvious in EAAE.
EACP as an ideal alternate for extracting bioactive components from oilseed because
of its nontoxic and non-inflammable properties [5].

Key factors for extraction:

• Enzyme composition

• Concentration of enzyme

• Particle size of plant materials

• Solid to water ratio

• Hydrolysis time

• Moisture content of plant materials

Figure 3.2: The process of enzyme-assisted extraction method from the natural products

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Example: Enzyme – assisted ionic liquid extraction of antioxidant compound from

turmeric. (Curcuma longa L.)

Curcumin is the bioactive compound of turmeric with wonderful therapeutic properties;

however, is slight aqueous solubility has limited its biological applications. Advanced
extraction techniques such as enzyme assisted help to overcome the low solubility of curcumin
in water, extraction of curcumin from turmeric using a developed carbamate ionic liquid was
investigated. N,N-Dipropyl ammonium -dipropylcarbamate (DPCARB) was synthesized from
dry ice and dipropylamine and used for extraction of curcumin from turmeric which resulted in
extraction yield of 3.58%, at room temperature within 2 h.To enhance the curcumin extraction
yield, a simple enzymatic pretreatment step was utilized. Use of a mixture of amylase and
amyloglucosidase enzymes to destruct the turmeric cell wall prior to the extraction process
remarkably improved the extraction yield to 5.73%. The superior performance of the
synthesized DPCARB over conventional extraction solvents was confirmed when acetone was
used for extraction of curcumin from pretreated turmeric at the same operation condition(25 ◦C
and 2 h) which resulted in extraction yield of only 3.11%.[13].

Industrial limitations:

The EAE method has several technical limitations when it is being commercially adopted. The
enzymes used for extraction is quiet expensive for large-scale industrial production process.
Certain enzymes cannot breakdown the plant cell walls completely. Therefore, the EAE is not
always feasible method to be applied on industrial scale basis because enzymes behavior was
limited by environmental conditions rigidly, and the above limitations were needed to be
considered in order to make full use of enzymes of potential value in the exploitation and
utilization of natural products[3] .

3.2.4 Microwave-assisted extraction

Microwave-assisted extraction (MAE) is a simple, rapid and economic method for extraction
of carotenoids, requiring a very short extraction time with low amount of solvents .Ho, Ferruzzi,
Liceaga, and San Martín-González (2015) optimized the MAE of all-E-lycopene from tomato
peels using ethyl acetate in the solid to liquid ratio of 1:20 (w/v), and 400 W power (24 kJ
equivalents) for 60 s. Interestingly, all-Z-lycopene yields increased, as the solid to liquid ratio
was decreased and the ethyl acetate proportion was increased compared to hexane. Overall,
ethyl acetate was suggested to be a better MAE solvent for highest recovery of all-Elycopene,

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compared to hexane. Although MAE involved a significantly shorter extraction time, the
carotenoid yield was lower than Soxhlet extraction. Extending the extraction time in MAE
causes the thermal degradation of carotenoids. Hiranvarachat and Devahastin (2014) used an
alternative procedure of intermittent microwave radiation to enhance the yield of β-carotene
and carotenoids from carrot waste, without causing excessive thermal degradation, by
minimizing the high-temperature environment due to the presence of the off period. The
fraction of the radiation time to the total processing time in one cycle is designated by
intermittency ratio (α), such as α = 1/2, 1/3 or 1/4. Combined use of lower microwave power
(180 W) and solvent volume (75 ml), or higher microwave power (300 W) and solvent volume

(150 ml), along with a lower intermittency ratio (α = 1/4), resulted in higher contents of
β-carotene and total carotenoids, compared to the continuous MAE under similar conditions.
Additionally, prolonging the off period (α = 1/4, compared to 1/2 and 1/3) resulted in higher
antioxidant activity of the extracts. The structure of the material is the key factor influencing
the extraction efficiency of carotenoids. Certain pre-treatments of the sample before MAE were
found to be beneficial in improving the yield of carotenoids investigated the various influences
of sample pre-treatment prior to extraction, on the sample structure and subsequent
microwave-assisted extraction (MAE) of carotenoids from carrot. Results show that carotenoid
recovery and antioxidant activity from carrots blanched in water and citric acid were
significantly higher, when compared to untreated carrots. These pretreatments damaged and
softened the sample structure, possibly by heat mediated solubilization of pectin. Pasquet et
al.compared the efficiency of MAE and Vacuum-MAE (VMAE) to conventional processes
(cold and hot soaking and ASE), to extract the carotenoids from frustulated and unfrustulated
microalgae. A frustule is the external hard silica layer of diatoms, which acts as a mechanical
barrier to pigment extraction. VMAE facilitates an efficient extraction of chlorophyll “a” and
fucoxanthin from C. closterium, at moderate irradiation power (75 W). However, the extraction
yield was higher with MAE; also, VMAE is technically more complex due to the requirement
of vacuum. Cold soaking was an efficient, simple and rapid method to extract pigments from
D. tertiolecta, as the absence of frustule allows easy penetration of solvents. However, cold
soaking was not efficient for extracting carotenoids from frustulated microalgae. The authors
further demonstrated that temperature is a crucial parameter determining the yield of microalgal
carotenoids. Thus, it is important to accurately define the highest extraction temperature for
rapid extraction for highest yields, without damaging the thermolabile carotenoids. In this
regard, MAE at 60 °C was found to be optimum for the extraction of carotenoids in

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nondenaturating conditions. The authors used 100% acetone for the pigment extraction, which
helps in minimizing ‘chlorophyll a’ hydrolysis by the chlorophyllase enzyme, as this enzyme
will actively degrade ‘chlorophyll a’ in aqueous acetone or methanol. The authors also
suggested that acetone is an efficient solvent for the extraction of major and minor pigments of
different polarities from the studied microalgae species. However, the authors have not studied
the comparative efficiency of acetone with other solvents (e.g., hexane and ethyl acetate), which
possibly can yield higher carotenoids. MAE is a simple and economical method for a large
variety of samples. However, thermal degradation and cis-trans isomerization cannot be ruled
out. In this regard, intermittent radiation has proven efficient for the higher recovery of
carotenoids and improved antioxidant activities, minimizing the thermal degradation. Thus, the
MAE procedure can be further optimized with various levels of microwave power, solvent
volume and intermittency ratio. The MAE is also considered as a novel method for extracting
soluble products into a fluid from a wide range of materials using microwave energy.
Microwaves are electromagnetic fields in the frequency range from 300 MHz to 300 GHz. They
are made up of two oscillating fields that are perpendicular such as electric field and magnetic
field. The principle of heating using microwave is based on its direct impacts on polar
materials[9].

Electromagnetic energy is converted to heat following ionic conduction and dipole rotation
mechanisms. During ionic conduction mechanism, heat is generated because of the resistance
of medium to flow ion. On the other hand, ions keep their direction along field signs, which
change frequently. This frequent change of directions results in collision between molecules
and consequently generates heat. In the case of extraction, the advantage of microwave heating
is the disruption of weak hydrogen bounds promoted by the dipole rotation of the molecules.

A higher viscosity of the medium lowers this mechanism by affecting molecular rotation. The
migration of dissolved ions increases solvent penetration into the matrix and thus facilitates the
solvation of the analytically . The extraction mechanism of MAE is supposed to involve three
sequential steps [13].

• Separation of solutes from active sites of sample matrix under increased temperature
and pressure
• Diffusion of solvent across sample matrix

• Release of solutes from sample matrix to solvent .

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Process system:

The MAE systems are classified into multi-mode system and focused-mode system
(monomode). Multi-mode system allows random dispersion of microwave radiation in cavity
by a mode stirrer, while focused system (mono-mode) allows focused microwave radiation on
a restricted zone in cavity. The schematic diagrams of closed system and open system are
illustrated in Figure. Extraction is normally carried out under uniform microwave heating. High
working pressure and temperature of the system allow fast and efficient extraction. The pressure
inside the extraction vessel is controlled in such a way that it would not exceed the working
pressure of the vessel, while the temperature can be regulated above the normal boiling point
of the extraction solvent. Recent advancements in the closed system have led to the
development of high pressure MAE. The increase in temperature and pressure accelerates MAE
due to the ability of extraction solvent to absorb microwave energy. Despite the fact that the
closed system offers fast and efficient extraction with less solvent consumption, but it is
susceptible to losses of volatile compounds with limited sample throughput. Open system is
developed to counter the shortcomings of closed system such as the safety issues, and it is
considered more suitable for extracting thermolabile compounds. This system has higher
sample throughput, and more solvent can be added to the system at any time during the process.
Basically, open system operates at more mild condition[2].

(a) (b)

Figure 3: (a) Closed type microwave system (b) open type microwave system

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Example:

Blueberries and bilberries emerge as the berries with the highest antioxidant content and
capacity. The intrinsic moisture of fruit is used and, therefore, mass and heat transfer
phenomena take place in the same direction, from the berry matrix to the liquid medium. Cells
are damaged and intracellular content is released to the medium. MAE’s greatest advantage is
the fast heating, with a reduced equipment size. This reduces both the extraction time and
amount of solvent needed, which automatically causes a lower CO2 emission to the atmosphere
Some authors designed an optimal solid-to-solvent ratio of 30%–34%. Higher or lower ratio
cause poor extraction [2].

The determination of antioxidant content can be done either by chromatography methods, such
as High Performance Liquid Chromatography (HPLC) coupled with a Diode-Array-Detector
(DAD), Mass Spectroscopy (MS) or fluorescence detector; or using other less specific
colorimetric methods.

Total Polyphenol Content, (TPC) can be determined by colorimetric spectrophotometry with

the Folin-Ciocalteu reagent method. This reagent contains complexes of
fosfomolibdic/fosfotungstic acid. The chemical reaction is based on the electron transfer from
phenolic compounds and the measurement of the absorbance of the blue colored complexes at
725 nm. Gallic acid is used as a standard, and results are usually expressed as mass of Gallic
Acid Equivalents (GAE) per gram of mass of the sample or extract[13] .

One of the main disadvantages is that the TPC method is not specific, as the reagent can be
reduced by other compounds other than phenolic. The high values obtained for TPC could result
from interference of other reducing substances, such as ascorbic acid or reducing sugars[9] .

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Chapter 4

CASE STUDY

4.Analysis of Extraction:

There are three types of extraction Analysis based on their ease to extract solute from mixture:-

4.1.Column chromatogaphy

4.2.Thin layer chromatography

4.3.Spectophotometric analysis (shown Table 2).

4.1.Column chromatogaphy

Нe most important method for carotenoid separation, column

chromatography, was recently replaced with TLC by its adsorption characteristics. Нis method
is used by its improvement along with the discovery of high performance liquid
chromatography (HPLC), method that presents the advantages of using low quantities of
substance and high separation speeds, but limited to a certain scale, not being able to be used
in preparative purposes. According to the nature of the carotenoid, the adsorbent will be chosen
and the adequate developing solvent. Of the adsorbents used in column chromatography for the
separation of carotenoids, one can add 2.Thin layer chromatography cellulose, calcium
hydroxide (separation of carotenoid hydrocarbons), saccharose, starch, silicagel, aluminium
oxide (separation of monohydroxicarotenoids), calcium carbonate and magnesium oxide
(separation of intermediate polarity carotenoids, such as xanthophylls). On the same column
there can be used di ‫ٴو‬erent adsorbents placed according to the adsorption power, the most
powerful being placed in the superior side of the column. Нe solvent used in column
chromatography for carotenoids are weak eluents. Usually it is worked with a mix of solvents,
such as acetone–ethylic ether, ethylic alcohol–ethylic ether, ethyl acetate–benzene, etc. Нe
adsorption column must be loaded with adsorbent in proportion of 3, a part being leі for the
solvent and the extract. Нe adsorbent must be very well compacted in the column, for this
reason the column fill being done with small portions of adsorbent that must be pressed. An

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example of high-performance liquid chromatography analyses of concentrated methanolic

extracts could be conducted using an HPLC (with an 18 column). Carotenoids could be detected
at 470 nm by using an online photodiode array detector with a 600 nm reference wavelength.
Peak areas generated by an HPLC auto-integrator could be used for carotenoid quantification
as all detected carotenoids shared similar absorption maxima and their absorption coeficients
were unknown. Нe carotenoid extract must be very concentrated for being able to be added to
the column once. $іer the pigment mix is introduced in the column, the developing is started
with di‫ٴ و‬erent solvents, starting with the most nonpolar. In column chromatography there can
be distinguished three base processes for carotenoid separation, as follows[10]:

• zone chromatography

• stage elution chromatography

• gradient elution chromatography

Zone chromatography: Нe carotenoid mix that is in a nonpolar solvent is applied on the upper
side of the column. For the separation of zones on the column there is used an adequate solvent
for developing. Нe zones are pulled from the column, each zone being then eluted individually
with a polar solvent. Stage elution chromatography: In this method it is applied the general
principle of column chromatography, the introduction of carotenoid mix in the superior side of
the column, the applying the solvents, according to the polarity. Нe pigments are eluted in the
order of growth of their asdorbtion anity and collected. Each eluted zone is then evaporated,
redissolvedi a small volume of solvent, in view of spectrophotometrication[10].

4.2.Thin layer chromatography

TLC is a technique used in the separation and purification of carotenoids. Нe degree of

purification by this method is higher, compared to column chromatography. Depending on the
nature of the carotenoid the stationary phase is chosen, also the mobile phase and the plate
elution system. In TLC for carotenoids there are some precautions that must be taken: • Fast
application of the sample • Application of the sample in low light • Immediate developing of
the chromatogram • Developing the chromatogram in the dark • Using a nitrogen atmosphere
Нe biological materials that contain carotenoids or their solutions must be protected from the
action of light; because by exposing carotenoids to light, especially direct solar light or UV

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light, the cis– trans photoisomerization is done, that can lead to their photodestruction. Many
carotenoids are thermolabile, especially xanthophylls, their heating up being done only in the
situation when it is absolutely necessary. Separation of carotenoids must be done at room
temperature, up to –20°C and in dark [32]. In the case of warm saponification, the solutions
must be protected by using solvents with boiling points not very high (30–60°C). In the
presence of oxygen (air) or peroxides, many carotenoids can be oxidized, being sensitive to
oxygen in adsorbed state (in chromatograms, on column and thin layer)[10].

4.3.Spectophotometric analysis

Нe absorption of electromagnetic waves in the visible field (400– 800 nm) and ultraviolet (140–
400 nm) is produced with changes of energy of valence electrons. Нe spectra obtained at the
absorption of radiation with wavelength between 120 and 1000 nm in the UV, visible and near
IR field are named electronic spectra, because are due to electronic transitions in orbitals σ, π
or n, from a low energy state to an excited state richer in energy. Нe electronic transitions
happen from a link orbital σ or π, occupied with electrons in fundamental state or with electrons
that do not participate in an anti–link orbital σ* or unoccupied in fundamental state, but
occupiable in excited state[11].

• σ→σ* transitions are manifested at the carotenoid units with σ links, require high
energies, for which the corresponding bands are at low wavelengths in vacuum UV.

• n→σ* and n→π* transitions are characteristic to carotenoid units that contain atoms
with unparticipating electrons, require lower energies, the corresponding absorption bands
being at wavelengths higher than those corresponding to the σ→σ* transitions .

• π→π* transitions are specific to carotenoid units with double bonds and determine the
absorptions at wavelengths who’s values depend on the nature of the substituents.spectrometry

Characterizing with precision of carotenoid chromophore is possible due to the correlation

between the electronic structure and absorption, position of absorption maximums being a
function of the number of the conjugated double bonds in the molecule. In the case in which
the chromophore contains triple bond instead of the double bond, λ max will decrease. For the
determination of the chemical structure it is important to know the level of influence on the
spectrum by the existence of some existent groups in the molecule. By comparison λ max of an
unknown carotenoid with the λ max values of a known carotenoid, control, the number of
double bonds in the chromophore can be estimated shown in figure 4.3[14].

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 Extraction of carotenoid

Fig4.3 .HPLC-UV chromatogram of carrot extract using PFE, λ = 450.

Table 1. Carotenoids Content in Major Plant

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Table 2. Types and Analysis

Table 3: Qualitative comparison of extraction methods

Extraction Advantage Disadvantage

Method

Maceration No additional energy needed Very long extraction

times.

Long extraction
Solvent Easy industrial scaleup. times.Some solvents
extraction Well known technique not valid for food
industry
Higher efficiency (less extraction
Ultrasound time and solvent consumption
Expensive scale-up
assisted requirements b).Safe extraction of
heat labile compounds
Quicker heating. Reduced Risk of burning the
Microwave equipment size. No added sample and
assisted solvent needed denaturalizing
compounds
SFE-CO2 extraction: CO2 no
Pressure assisted toxicity, extraction in absence of Expensive scale-up
air and light, very pure extracts

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Table 4: Physical methods used in establishing the chemical structure of carotenoids.

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Table 4.3: Energy values according to the spectral region

Spin Orientation Configuration Electronic

Distribution
Change

RMN RES Micorwaves IR UV– X rays

Vis

Wave number 1 cm–1 - 102 cm–1 104 cm–1 106 cm–1 -

Wavelength 100m 1m 1 cm 100 µm 1 µm 100A

Frequency (Hz) 3 × 105 3 × 108 3 × 1010 3 × 1012 3× 1014 3×1016

Energy(kcal/mol) 0,0003 0,03 3 3 × 102 3 × 104 3 × 106

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RESULT

The solubility of b-carotene in various pressures and temperatures is presented. It is observed

that the solubility of b-carotene generally increased with the increase in extraction pressure.
However, it is interesting to note that at all temperatures studied; the solubility of b-carotene
decreased with pressure from 45 to 80 bar. Further pressure increase to 100 bar resulted in a
solubility increase for b-carotene at 60 C, apparently to its highest level. It is observation may
be due to the competing effects of solubility at 80 bar between b-carotene and other more
soluble components present in the sample[6]. Thus, a high solubility of the co-extracted
components with carotenoids tended to reduce the solubility of b-carotene. However, further
increase in pressure to 100 bar at all temperatures had increased the b-carotene solubility. The
increase might be due to high solubility of b-carotene compared to other components coexisting
in carotenoids. noted that the presence of other components being co-extracted with carotene
could result in low solubility of b-carotene. In terms of temperature effect, it was found that the
solubility of b-carotene increased with temperature from 40 to 80 C at pressure below 80 bar.
The analysis on b-carotene was performed during the final extraction yield to investigate the
total extractability of the component. It was observed that the concentration of b-carotene
increased with increase in extraction pressure. however, it is interesting to note that at all
pressures studied, the concentration of b-carotene decreased markedly at 80 bar, with
temperature of 60 C giving the lowest b-carotene concentration. This happened possibly due to
the high solubility of undesired components which were co-extracted with carotenoids at 80
bar, thereby resulting in lower b-carotene concentration. However, the b-carotene concentration
tended to rise again after the pressure was increased to 100 bar at all the temperatures
studied[10].

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CONCLUSION

The extraction of carotenoids from natural products is usually performed using organic
solvents. On the other hand, carotenoids are soluble in edible oils, which are called “green”
solvents. The present study indicated the higher effectiveness of SCT and MCT in carotenoid
extraction from edible brown seaweeds and vegetable leaves. Both TAGs are oxidatively more
stable than other types of edible oils containing polyunsaturated fatty acids. Additionally, MCT
is known to have several types of beneficial health effects; therefore, the oil extract of MCT
can be applied as functional food material. Soxhlet extraction is a conventional technique
providing the highest recovery of carotenoids. Nevertheless, it’s a time consuming process, and
also uses significant amounts of solvents, thereby increasing the cost of extraction. SFE using
CO2 as a solvent and ethanol as a co-solvent, has been documented as a superior “green” and
non-thermal method for the efficient and selective extraction of carotenoids[14]. However, the
process requires improvement to obtain a higher recovery of xanthophylls (polar carotenoids).
The ultrasound assisted extraction (UAE), pressurized liquid extraction, pulsed electric field
(PEF) extraction and enzyme assisted extraction (EAE), are the other evolving non-thermal
methods for the rapid extraction of carotenoids. However, detailed analysis of cost,
environmental safety, efficiency and reproducibility of these methods need to be further
evaluated. Additionally, automation of these methods can help for large scale industrial
applications. The carotenoid extraction using nontoxic solvents offers several advantages over
conventional solvents, in terms of environmental safety and also its application in food and
cosmetics. Thus, methodological and technological advancements in these fields are
desirable[3].

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