Ultrasound-Assisted Extraction of Lycopene from Tomatoes: A Report

This report explores the extraction of lycopene, a pigment responsible for the red color in tomatoes, using ultrasound-assisted extraction (UAE). It contrasts UAE with conventional organic solvent extraction (COSE), highlighting the advantages of UAE in terms of reduced extraction time, solvent consumption, and improved yield. The report discusses the structure, properties, and benefits of lycopene, emphasizing its antioxidant capabilities and role in preventing chronic diseases. It delves into the methodology of UAE, including materials, experimental design, and statistical analysis, and presents the results of comparative studies between UAE and COSE. The findings indicate that ultrasound-assisted extraction can significantly enhance lycopene recovery from tomatoes, making it a more efficient and environmentally friendly alternative to traditional methods. The report also touches on the optimization of UAE parameters such as temperature and solvent ratio to maximize lycopene yield.

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LYCOPENE EXTRACTION FROM TOMATO USING ULTRASOUND ASSISTED
EXTRACTION
Abstract
Lycopene is the pigment that is mainly responsible for the characteristics deep-red colour that is
observed in ripe fruits of tomatoes which makes up to about 80 per cent to 90 per cent of the
pigments that are found in the fruits. The previous studies gave a lot of attention to other pro-
vitamin A carotenoids among them α -carotene and β-carotene which in cases was negligible. In
the recent past, there has been an upsurge in the studies illustrated that lycopene and its acyclic
structure and an enormous array of conjugated double bonds is composed of numerous distinct
physiochemical and biological properties. Lycopene has been found to be one of the most singlet
oxygen quenchers of high efficiency among the natural carotenoids that do not exhibit pro-
vitamin A activity. It is used as a natural antioxidant in the human serum among other tissues so
as to protect the proteins, DNA, and lipids from oxidant damage. Other pieces of evidence
extracted from animal and epidemiological studies have concurred with the chemoprevention
capability of lycopene and a reduction in the chronic diseases among them cancer of the
lycopene metabolites.
Lycopene, in the form of a natural food additive, has received significantly large attention owing
to its extensive and elaborate use alongside the deep understanding of the natural ingredients that
are found in food. It is for this reason that the addition of lycopene to functional foods leads to an

increase in the concerns regarding green as well as efficient extraction technology. Conventional
solvent extraction techniques which have for decades been used as the method of solvent
extraction of products derived from nature has been associated with a number of limitations that
have seen a significant reduction in its efficiency. Among the limitations include longer times
spent in the extraction process as well as the use of large amounts of the organic solvent in the
extraction process. In countering the above limitations, ultrasound-assisted extraction can be
applied in the improvement of the rate of extraction through increasing the rates of mass transfer
as well as rupturing of the cell walls as a result of the ultrasonic cavitation impacts. In
comparison with the other methods of material pre-treatment, lyophilisation initiates
improvements in the retention of lycopene activity from tomatoes that are in raw forms.
Superfine grindings which are related to cellular level pulverizing methods and strategies are
usable in getting vegetable and fruits powder using small sizes of particles alongside a large
specific surface area. However, there have not been reports made on the extraction of lycopene
regarding the studies on the freeze-drying application in conjunction with superfine pulverizing
technology.

Contents
Abstract...................................................................................................................................................................... 1
Introduction.............................................................................................................................................................. 5
Lycopene................................................................................................................................................................ 7
Structure and Physical Properties of Lycopene................................................................................8
Biosynthesis......................................................................................................................................................... 9
Staining and removal..................................................................................................................................... 10
Benefits of Lycopene.................................................................................................................................. 10
Ultrasonic Assisted Extraction................................................................................................................... 11
Ultrasonic Assisted Extraction and features.................................................................................... 12
Impacts of the Involved Variable of UAE in the Production and Yield.......................................15
Impacts of Ultrasound System characteristics.................................................................................... 15
Ultrasound System and Amplitude...................................................................................................... 15
Mode and Frequency of Operation Equipment...............................................................................16
Impacts of pre-treatment of sample.................................................................................................... 17
Effect of Temperature............................................................................................................................... 17
Effect of the solute/solvent ratio.......................................................................................................... 18
Extraction time............................................................................................................................................. 18
Optimization of Ultrasound assisted Extraction............................................................................. 19
Comparison between Ultrasound assisted Extraction and other Extraction Methods.......20
Comparison against Conventional Methods..................................................................................... 20
CHAPTER 2: METHODOLOGY......................................................................................................................... 21
Research methodology.................................................................................................................................. 21
Introduction....................................................................................................................................................... 21
1) Use of questionnaires............................................................................................................................. 22
2) Interviews..................................................................................................................................................... 23
3) Secondary sources................................................................................................................................... 23
4) Use of mathematical models.............................................................................................................. 24
5) Computational methods and simulation..................................................................................... 24
The rationale for research methodology............................................................................................... 25
Materials and Methods.................................................................................................................................. 26

Materials......................................................................................................................................................... 26
Ultrasound-Assisted Extraction Method........................................................................................... 26
Conventional Organic Solvent Extraction, COSE, Method...........................................................27
Determination of lycopene and β-carotene...................................................................................... 28
Statistical analysis....................................................................................................................................... 29
CHAPTER 3: RESULTS AND DISCUSSION................................................................................................... 30
Conventional Organic Solvent Extraction, COSE................................................................................. 30
Ultrasound-assisted extraction UAE........................................................................................................ 33
Materials and Methods.................................................................................................................................. 38
Evaluation of lycopene.................................................................................................................................. 39
Experimental design....................................................................................................................................... 40
CHAPTER 4: RESULTS AND DISCUSSION................................................................................................... 42
Analysis of Variance results........................................................................................................................ 42
Contour plots and response surfaces...................................................................................................... 43
Ultrasonic-enzymatic extraction optimum conditions.....................................................................46
Conclusion............................................................................................................................................................... 46
References............................................................................................................................................................... 48

Introduction
Tomatoes and products related to tomatoes are heavily consumed across the globe both in the
raw and processed forms. Following their composition with regard to health, such products have
gained popularity and the trend is ever on the rise1. Among the health implications of
consumption of tomatoes and tomato products include prevention or reduction in certain kinds of
cancer among them prostate cancer and epithelial cancer. These products as well play a major
role in the minimization or prevention of cataract and cardiovascular diseases upon consumption
following their high lycopene content.
Different quantities and types of wastes at different stages are observed during the processing of
tomatoes. Dry pomade is composed of 44% by volume of seed and 56% by volume of the pulp
and skin. A greater portion of the waste is made up of the seed and skin that has been found to
contain at least five times more lycopene that tomato pulp. Owing to the fact that the skin acts as
the major source of lycopene, there is need to separate it from the other parts of the tomato in
order to achieve better extraction2. The total amount of lycopene in tomato pulp and tomato skin
is not constant and instead changes between 90 to 190 mg/kg and approximately 120 mg/ kg in
fresh weight in that order.
On the other hand, the quantity of β-carotene has been established to be approximately 3 mg/kg.
other than having high antioxidant activity, lycopene has been found to degrade and isomerize
when subjected to light irradiation and treatments of high temperature. In order to achieve a
complete isolation of all-trans-lycopene and prevent isomerization, conditions that are suitable
and relatively controlled should be achieved and maintained. This has seen the classic solvent
extraction methods used in the extraction of lycopene from tomato wastes since the ancient
times.

Lycopene forms fundamental components of cosmetic formulation and pharmaceutical due to its
protective properties in which it protects the human body against cancer and oxidants.
Furthermore, owing to its high solubility in fat and oil media, lycopene serves as one of the
agents colour in the food industry3. Lycopene has a characteristic red colour of the vegetables
and fruits and is composed of 2 non-conjugated and 11 conjugated double bonds which allow it
to act as an antioxidant two-fold relative to β-carotene and 100 folds as compared to
α −tocopherol.
Traditionally, the extraction of lycopene had been done using the COSE method. This is despite
the fact that the traditional extraction methods, there were need of lengthy times and high
quantities of solvents. New methods for the extraction of lycopene thus needed to be established
which could reduce the time of extraction, lower the consumption of the solvent, increase the
quality of the extract and improve the yield from extraction. Among the new methods that were
established and found to be in a position to meet part of or the whole of the obligations included
ultrasound assisted, supercritical extraction and microwave-assisted4.
Ultrasonic applications were found to be ideal in reducing the time of extraction with lower
ratios of the liquids to the solids by increasing the rate of mass transfer. Sound waves have the
capability of changing the materials in both chemical and physical aspects when setting at
frequencies that are higher that cavitation and sponge effect. Such effects improve the
penetration of the solvent in the solids and eliminate the release of extracts through the damage
of the solid matrix. High pressure and extremely high temperatures are generated by the
implosions of the cavitation bubbles which are high enough to attain the required energy for
reaction5. Findings from scientific experiment and research established that ultrasound aided in
the acceleration of the extraction rate and improve on the yield by approximately 10%.

Another impact of ultrasonic power is the generation of cavitation bubbles of very high energy
which are composed of solvent vapour. The bubbles have the capability to implode adjacent to
the cell walls resulting in very high local temperatures, a rise in the pressure as a destruction of
the cell wall6. These occurrences make it easy for mass transfer to occur from the check to a
solvent and the overall impact is a facilitated micro-streaming. A combination of such impacts
increases the penetration of solvent and meets the required mixing for the extraction of large
amounts of active components. In addition to easing extraction, ultrasound is also applicable in
the production of free radicals that are found within the cavitation's phenomenon like hydroxyl
radicals that are highly reactive in solutions that contain water6. Ultrasonic assistance has as
well found its applications in certain process food industry such as emulsification, separation,
extrusion, crystallization, fermentation, de-foaming, filtration, and microbial inhibition.
Lycopene
Lycopene is a chemical that occurs naturally and gives the vegetables and fruits their red colour.
It forms one of the pigments that are called carotenoids. The chemical substance is found in
apricots, pink grapefruits, watermelons as well as pink guavas. The highest concentration is
found in tomatoes and tomato products. Up to about 85% of the dietary lycopene in North
America is got from tomato products among them tomato juice or paste.
Research has established that a cup of tomato juice offers approximately 23 mg of lycopene.
Raw tomato processing through the use of heat which is applied in the making of tomato juice
alters the lycopene in the raw product to a form that can easily be used by the human body.
Lycopene is supplements are almost as for use by the body as the lycopene that is found in
food7.

In photosynthetic organisms among them algae and plants, lycopene is used as an intermediate
during the biosynthesis of numerous carotenoids such as beta-carotene which gives the yellow,
orange or even red pigmentation as well as photoprotection. Just like all the other carotenoids,
lycopene is a terpene and insoluble in water8. The deep red colour observed in lycopene is
attributed to the eleven conjugated double bonds. Due to the strong colour, lycopene is used as
an element in food colouring which has been approved for use in the USA, New Zealand and
Australia among other countries that are members of the European Union.
Structure and Physical Properties of Lycopene
Figure 1 (a): the Skeletal formula of all-trans lycopene
Figure 1(b): Ball-and-stick model of all-trans lycopene
Lycopene is a tetraterprene that is symmetrical in nature and assembled using eight isoprene
units. It belongs to the carotenoid family of compounds and since it is purely made up of carbon
and hydrogen, it is as well referred to as a carotene9. The procedures for isolation of lycopene
were first developed and reported in 1910 with the determination of the structure of the molecule
being done in 1931. In its natural form, which is all-trans form, the molecule is in long and
straight shape that is constrained by its systems that is of eleven conjugated double bonds.

The energy needed for transition for the transition of electrons to higher states of energy is
reduced by each of the extensions in the conjugated system. This permits the absorption of
visible light that are having progressively longer wavelengths. Lycopene has the capability of
absorbing all but the longest wavelengths of the visible light and thus having the red appearance.
All-trans lycopene is naturally produced by plants and photosynthetic bacteria. Lycopene
undergoes isomerization upon being exposed to light or heat to form any of the numbers of cis-
isomers. Cis-isomers are more of bent that linear in shape10. Owing to their molecular energy,
various isomers have varied stabilities and in the human blood, different cis-isomers make up to
more than 60% of the total concentration of lycopene even though there has not been an
investigation over the biological impacts of each of individual isomers.
Carotenoids such as lycopene are mainly found in the photosynthetic complexes of pigment and
protein in plants, fungi, algae and photosynthetic bacteria. They are in charge of the bright
orange-red colours that are observed in vegetables and fruits and engage in different functions in
photosynthesis as well as protection of the photosynthetic organisms from being damaged by
light. Lycopene is a major intermediate in the biosynthesis of carotenoids among them
xanthophylls and beta-carotene11.
Biosynthesis
The unconditioned biosynthesis is similar in eukaryotic plants and prokaryotic bacteria as is the
similarity in the enzymes that are involved. The process of the synthesis starts with mevalonic
acid, which is changed to dimethylallyl pyrophosphate which is further condensed using three
molecules of isopentenyl pyrophosphate which is an isomer of dimethylallyl pyrophosphate that
generates the twenty-carbon geranylgeranyl pyrophosphate12.

Two molecules derived from this product are then condensed to form a tail-to-tail configuration
to yield the forty-carbon phytoene. This forms the first step in the biosynthesis of carotenoid.
From numerous desaturation steps, conversion of phytoene to lycopene occurs3. Cyclization of
the two terminal isoprene groups can be done to generate beta-carotene which can then be
changed into numerous varieties of xanthophylls.
Staining and removal
Lycopene is the pigment that is found in sauces that contain tomato, having the capability of
changing plastic cookware to orange and also not soluble in water. The compound is only soluble
in organic solvents and oils12. Owing to its non-polarity, lycopene has the capability to stain any
materials that are sufficiency porous among them more plastics. Soaking the plastics is solutions
that contain the very little amount of bleaches that are used in the household can serve as a
mechanism for removing the stain.
Benefits of Lycopene
There are no major benefits that have been accrued as far as the treatment of prostate cancer
using supplemental lycopene is concerned. Still, another study has established that patients who
are on an intake of 15 mg of supplemental lycopene about 3 weeks to the surgery aimed at
removing the prostate recorded significant improvements in the cancerous lesions and a
reduction in the score of PSA which is a blood test used in the monitoring of prostate cancer.
A study on the different vitamins among them A and E, lutein, beta-carotene, and lycopene
illustrated that there are higher concentrations of such nutrients in the human body, more
specifically beta-carotene and lycopene illustrated a significant protection against breast cancer.
Protection against the development of tumors in the breast has also been illustrated in mice.
There are also benefits to lung cancer in animal research13.

Lycopene has also illustrated benefits for the high blood vessels that are around the heart
illustrating protection to the vessels of blood in the neck much better than vitamins A, E or
CoQ10. This is attributed to the fact that lycopene is considered a powerful antioxidant.
Protection against heart attack has also been shown by higher levels of lycopene. Tomato paste,
which is a great source of the nutrients, has illustrated some protection against the growth of the
cardiovascular disease. There is a greater protection as compared to the other carotenoid
nutrients in the protection of the women against heart disease13.
Lycopene acts as a supplement as part of a nutritional program even though most of the studies
propose that the food acts as the optimal way of delivering such an antioxidant to the tissues of
the human body. This is applicable to most of the vitamins. Numerous sources of information
have demonstrated that greater consumption of tomato products generation higher concentrations
of lycopene. The absorption of lycopene is enhanced through heating of the tomato with olive oil
or even taking the products such as tomato paste or sauce14.
Ultrasonic Assisted Extraction
Ultrasonic Assisted Extraction, UAE, has turned out to be one of the most sustainable extraction
alternatives that need a medium invest in energy and solvent as compared to the conventional
methods of extraction. Still, the method is easy to handle, can be reproduced, economical and
safe owing to the fact that it is technology which permits its development under controlled
conditions of atmospheric pressure alongside an ambient temperature. Ultrasonic Assisted
Extraction works on the principle of acoustic cavitation which has the ability to damage the cell
walls of the matrix of a plant and hence facilitating the release of the bioactive compounds from
a substance. The technology is applicable to the extraction of various phytochemicals from
various compounds of which lycopene has remained to stand out15. These have been

acknowledged and appreciated by different fields of industry, most particularly the food and
pharmaceutical industries, which is attributed to its antimicrobial, anti-cancer and anti-
inflammatory properties and most importantly their capability of being antioxidant.
The information and application of the biological properties of lycopene have led to the range of
studies which have focused on the recovery of such substances from the various plant sources
mostly tomatoes under the principle of the implementation of sustainable technologies of which
Ultrasonic Assisted Extraction is part.
Ultrasonic Assisted Extraction and features
Ultrasonic is mainly based on the principle of propagation of mechanical waves which are
generated by a set of cycles that are described by the combination of both low and high pressures
known as compressions and rarefactions16. Below are the main characteristics of Ultrasonic
waves:
 The frequency which defines the number of cycles made per unit time
 Length which is the distance between any two points on the rarefaction to compression
 Intensity is the power unit ration across the unit areas
 Speed whose SI unit is m/s is the product of the wavelength and the frequency
 Amplitude is the maximum height attainable by the wave.
The spectrum of the waves that are involved in the ultrasound is known as ultrasound waves and
the frequencies of such waves are in most cases those that go beyond the human audible range
which is > 20kHz bit below the frequencies of the microwave which go up to 10kHz. The
frequency magnitude that is assisted with the effects of ultrasound, which means those
frequencies that are lower than 20 to 100 kHz, are in most cases dominated by physical impacts