Optimization of some factors influencing lycopene extraction from tomato processing waste using response surface methodology

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE NGUYEN THI KIM THANH OPTIMIZATION OF SOME FACTORS INFLUENCING LYCOPENE EXTRACTION FROM TOMATO PROCESSING WASTE USING RESPONSE SURFACE METHOD

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

NGUYEN THI KIM THANH

OPTIMIZATION OF SOME FACTORS INFLUENCING

LYCOPENE EXTRACTION FROM TOMATO PROCESSING WASTE USING

RESPONSE SURFACE METHODOLOGY

Suppevisors: 1 Assoc Prof Tran Thi Dinh

2 Prof Marie-Louise Scippo

AGRICULTURAL UNIVERSITY PRESS - 2017

DECLARATION

I hereby declare that the data and results of research in my thesis are honest There

is no material that has been accepted for the award of any other degrees or diploma in any educational institution and, to the best of my knowledge and belief, it contains no material previously published or written by another person, except where due reference

is made in the text of the thesis

I hereby declare that, all the help to carry out of my thesis was thanked and the cited information in this thesis has been written clearly the source

Hanoi, May 10th, 2017 Master candidate

Nguyen Thi Kim Thanh

ACKNOWLEDGEMENTS

This thesis was realized at Department of Food Processing Technology and Central laboratories of Food technology-Vietnam national university of Agriculture under the supervisor of Assoc Prof Tran Thi Dinh and Prof Marie-Louise Scippo To complete this thesis, besides the effort of myself, I have received encouragement and great help of many individuals and groups

Foremost, I would like express my deep gratitude to my supervisor Assoc Prof Tran Thi Dinh and Prof Marie-Louise Scippo for their valuable advices and continuous guidance, encouragement and time sharing during my study I would like to express my sincere thanks to Msc Nguyen Thi Hoang Lan and Dr Hoang Hai Ha for enthusiasm, insightful comments, teaching me on the HPLC analytical technique and useful laboratory skills

I am grateful to Research and Teaching Higher Education Academy – Committee

on Development Cooperation (ARES – CCD) for awarding the scholarship grant I give

my thanks to Dr Nguyen Thi Thanh Thuy for her great support during my study

My sincere thanks are also sent to my friends especially, special thanks to my juniors Than Thi Huong, Nguyen Thi Hien and Pham Thi Bich for their assistance in the experimental work of this thesis

Last but not least, I owe more than thanks to my family, my parents, my elder sister and my younger brother for their love, support, patience and inspiration

Hanoi, May 10th, 2017

Master candidate

Nguyen Thi Kim Thanh

TABLE OF CONTENTS

Declaration i

Acknowledgements ii

Table of contents iii

List of abbreviations v

List of tables vi

List of figures vii

List of figures vii

Thesis abstract viii

Chapter 1 Introduction 1

1.1 Introduction 1

1.2 AIM 2

1.2.1 General objective 2

1.2.2 Specific objectives 2

Chapter 2 Literature review 3

2.1 Tomato 3

2.1.1 Origin and distribution of tomato 3

2.1.2 Tomato composition 5

2.1.3 Tomato processing waste 6

2.2 Lycopene 7

2.2.1 Source of lycopene 7

2.2.2 Role of lycopene in the human health 10

2.2.3 Physical and chemical properties of lycopene 11

2.3 Lycopene extraction 14

2.3.1 Solvent extraction method 15

2.3.2 Other methods of lycopene extraction 17

Chapter 3 Materials and methods 20

3.1 Materials 20

3.1.1 Sample collection and preparation 20

3.1.2 Equipment 20

3.1.3 Chemical 21

3.2 Research contents 21

3.3 Methodology 21

3.3.1 Experimental design 21

3.3.2 Analytical methods 25

3.3.3 Data analysis 28

Chapter 4 Results and discussion 29

4.1 Selection of the suitable organic solvent for lycopene extraction 29

4.2 Selection of treatment regime of tomato waste for lycopene extraction 31

4.3 Response surface methodology for optimization of lycopene extraction 36

Chapter 5 Conclusions and recommendations 42

5.1 Conclusions 42

5.2 Recommendations 42

References 43

Appendix 48

LIST OF TABLES

Table 2.1 World tomato area, production and productivity, 2013 3

Table 2.2 World leading tomato producing countries in the world 4

Table 2.3 Tomato area, production and productivity of some region in Viet Nam, 2009 4

Table 2.4 Typical composition in 100 gram of a ripe tomato fruit 5

Table 2.5 Carotenoid composition of tomato fruit, tomato processing wastes and tomato paste (mg/100g wet sample) 7

Table 2.6 Lycopene content of common fruit and vegetables 9

Table 2.7 Lycopene content in common tomato –based food 10

Table 2.8 Physical properties of lycopene 11

Table 2.9 Total lycopene and Cis-isomer content in the dehydrated tomato 14

Table 3.1 Effect of solvent system on lycopene extraction from tomato waste 21

Table 3.2 Experimental design for drying of tomato waste 22

Table 3.3 Box- Behnken experimental design for lycopene extraction 23

Table 4.1 Results of optimization treatment regimens for tomato waste 31

Table 4.2 Summary of effect of independent factors to the output variables 32

Table 4.3 Results of the analysis of variance on lycopene content 32

Table 4.4 Result of the analysis of variance antioxidant capacity of lycopene extract 34

Table 4.5 Results of optimization condition for lycopene extraction 37

Table 4.6 Summary of effect of independent factors to the output variables 38

Table 4.7 Results of the analysis of variance of lycopene content 38

Table 4.8 Result of the analysis of variance of antioxidant capacity of lycopene extract 39

extraction 40

THESIS ABSTRACT

Master candidate: Nguyen Thi Kim Thanh

Thesis title: Optimization of some factors influencing lycopene extraction from tomato processing waste using response surface methodology

Major: Food technology Code: 24180557

Educational organization: Vietnam National University of Agriculture (VNUA) Research Objectives: The aim of this research is to optimize some factor (solvent/material ratio, temperature and time) influencing lycopene extraction process from tomato waste which could be used to produce functional foods

Materials and Methods:

- Materials: The red ripe tomato cv Chanoka F1 was harvested in Bac Ninh province Tomato waste was obtained by removing the juice Tomato paste was passed through a fruit pulper to obtain waste Tomato waste was dried by a convective oven after that they were ground to use as material for lycopene extraction

- Methods: Suitable organic solvent for lycopene extraction from tomato waste was studied ranging from single solvent (acetone, ethanol, ethyl acetate), double solvent (acetone: ethanol) and triple solvent system (acetone: ethanol: ethyl acetate) The treatment regime (moisture content and drying temperature) of tomato waste for lycopene extract also investigated Tomato waste, which was dried in the oven at the optimal temperature and moisture content, was used to optimize of several factors (ratio

of solvent/dried tomato waste, temperature, time) influencing extraction of lycopene with the most suitable solvent by response surface methodology

- Analytical methods: Moisture content of tomato waste (%) was measured using fast moisture detector (MA37, Germany) Lycopene content was quantified by HPLC Antioxidant capacity of lycopene content was quantified by DPPH radical scavenging test

Main findings and conclusions

The results of the present study indicated that ethyl acetate solvent proved to be the most efficient compared to other solvents for lycopene extraction The optimal conditions for drying of tomato waste is temperature of 65oC until the moisture content

of the material reached 23% The optimal extraction conditions for lycopene were:

+ Ratio of solvent/waste 40/1 (v/w),

+ Temperature 55oC and

+ Extraction time 120 min

Under this optimization condition lycopene content in the extract was 7.391 mg/g DW and antioxidant capacity of extract was 10.384 µmol TE/g DW

CHAPTER 1 INTRODUCTION

1.1 INTRODUCTION

Lycopene is one of 750 carotenoids found in nature and is responsible for the red color of fruits It is present in high concentration in red fruit and vegetable, such as tomato, gac, carrot, watermelon… (Britton, 2004) In the food industry, lycopene is used as a natural pigment in the dyeing of food product Besides, lycopene is also known as a potential antioxidant which is believed to be responsible for protecting cell against oxidative damage and thereby decreasing the risk of chronic diseases (Rao et al., 2006) Thus, lycopene demands on using

in pharmaceutical, food, feed and cosmetic industries calls more attention nowadays

Tomato, Lycoperisicon esculentum, is one of the most widely cultivated vegetable in worldwide and known as one of fruits which are rich in lycopene World tomato production in 2013 was about 163 million tons of fresh fruit from

an estimated 4.7 million hectares (Faostat, 2014) Tomato contains a wide variety

of antioxidants including vitamin E, ascorbic acid, carotenoids, flavonoids, phenolic compounds (Sathish et al., 2009) Lycopene represents about 80-90% of total carotenoids in tomato Lycopene is located in different fractions of tomato such as tomato skin, water insoluble fraction, and fibrous fraction including fiber and soluble solids Tomato processing industry produces large amounts of solid waste It is about 10–40% of the total tomato processed in the facility and includes 33% seeds, 27% skin and 40% pulp (Topal et al., 2006) Toor and Savage (2005) indicated that 70–90% of the lycopene was associated with the water insoluble fraction and the skin In Vietnam and other countries, the waste is usually used for animal feed or for organic fertilizer but it is not used for human consumption (Knoblich et al., 2005) Therefore, large quantity of carotenoids is lost as waste In addition, this waste has a high moisture content that makes it susceptible to microbial proliferation and spoilage Therefore, it can be preserved

by drying or other methods and then for lycopene extraction

Tomato carotenoids are liposoluble Recently, there are several methods used for lycopene extraction Sabio et al (2003) studied a lycopene extraction process based on supercritical CO2, which allows the extraction of over 60% of

the lycopene from tomato waste Xi (2006) reported that the lycopene yield from high pressure processing treatment of tomato paste waste for 1 min was much higher than from solvent extraction for 30 min However, lycopene is commonly extracted with organic solvents due to the cheap cost of technology and better recovery as compared to other methods There are a lot of organic solvents, which are usually used in several studies to non-polar carotenoid extraction such

as ethyl acetate, ethanol, acetone, etc However, their parameters (solvent/material ratio, temperature and time) are largely influence on lycopene extraction Therefore in the current study, we conduct research entitled

“Optimization of some factors influencing lycopene extraction from tomato processing waste using response surface methodology”

1.2 AIM

1.2.1 General objective

The aim of this research is to optimize some factors (solvent/material ratio, temperature and time) influencing lycopene extraction process from tomato waste which could be used to produce functional foods

CHAPTER 2 LITERATURE REVIEW 2.1 TOMATO

2.1.1 Origin and distribution of tomato

Tomato (Lycopersicon esculentum Mill.) is one of the most widely cultivated vegetable worldwide Tomatoes are members of the family Solanaceae (also known as the nightshade family), genus Lycopersicon, subfamily Solanoideae and tribe Solanceae (Taylor, 1986) It was originated in the coastal highlands of Andean region that includes parts of Chile, Colombia, Ecuador, Bolivia and Peru (Sims, 1979) The Spanish introduced tomato into Europe in the early 16th century (Harvey et al., 2002) European acceptance of tomato as a cultivated crop and its inclusion in the cuisine were relatively slow Tomatoes were initially grown only as ornamental plants: the fruits were considered to be poisonous, because of the closely related deadly nightshade (Solanum dulcamara) Since the mid-16th century tomatoes have been cultivated and consumed in southern Europe, though they only became widespread in north-western Europe by the end of the 18th century (Harvey et al., 2002) In 17thcentury, European took the tomato to South, Southeast Asia and China In the

18th century, tomato came to Japan and the USA The production and consumption of tomato expanded rapidly in the USA in the 19th century, and by the end of that century, processed products such as soups, sauces and ketchup were regularly consumed (Harvey et al., 2002)

Table 2.1 World tomato area, production and productivity, 2013

(1000 ha)

Production (1000 tons)

Productivity (tons/ha)

Nowadays, tomatoes become the most important vegetable in the world According to Faostat (2014) tomato grows more than 175 countries around the world World tomato production in 2013 was about 163 million tons of fresh fruit from an estimated 4.7 million hectares (Faostat, 2014) (Table 2.1) China leads world tomato production with about 50 million tons with 30.1% of world production followed by India with 18.2 million tons (11.1% global production) (Table 2.2)

Table 2.2 World leading tomato producing countries in the world

(tons)

Share of world production (%)

Table 2.3 Tomato area, production and productivity of some region

in Viet Nam, 2009

Regions Area (ha) Production (tons) Productivity (tons/ha)

Tomatoes are mainly cultivated in the winter season Besides, it is also grown in the summer- autumn, autumn-winter, spring-summer season on the rice land in order to bring high profit to farmers Tomatoes are grown popularly in Red Delta region (Table 2.3), concentrated in Ha Noi, Hai Duong, Hung Yen, Bac Ninh… also in the Southern such as An Giang, Tien Giang, Lam Dong provinces…) (Dang, 2014)

2.1.2 Tomato composition

Tomato is a kind of vegetable which had high nutrition value to human diet and subsequent importance in human health They are rich in minerals, vitamines, essential amino acids, sugars and dietary fibers Tomato contains much vitamin B and C, iron and phosphorus (Ayandiji et al., 2011) The Table 2.4 gives the main nutrients and their quantities that can be derived from consuming 100 gram of ripened tomatoes

Table 2.4 Typical composition in 100 gram of a ripe tomato fruit

Tomatoes are widely known for their outstanding antioxidant content, including their high concentration of lycopene and excellent amounts of other conventional antioxidants like vitamin C and tocopherols, additional carotenoid (β- carotene, lutein, and zeaxanthin) (Arab and Steck, 2000) According to Naika

et al (2005), yellow tomatoes have higher vitamin A content than red tomato, but red tomato contain lycopene, an anti-oxidant that may contribute to protection against carcinogenic substances

2.1.3 Tomato processing waste

According to FAOSTAT (2014), world tomato productions are huge about

163 million tons of fresh fruit in 2012 and on the rise in recent years More than a third of these were used for processing industry such as juice, soup, concentrate, dry- concentrate, sauce, salsa, puree, dry-tomato, ketchup or paste (Kaur et al., 2008) Commercial processing of tomato produces large amounts of solid waste

or by-products, namely tomato seeds and peels, representing 10-40% of total processed tomato (Al-Wandawi et al., 1985; Topal et al., 2006)

The importance of utilization of the waste is no revenue from the sale and dumping of this processing waste at the nearest landfill site will add to the processing cost On the other hand, if this waste remains unutilized, they not only add to the disposal problem but also aggravate environmental pollution (Al-Wandawi et al., 1985) These wastes can be used for animal feed Wet tomato processing wastes can be ensiled with corn plants and the resulting silage supported good milk production (Weiss, 1997)

One way of avoiding this problem would be to re-use the tomato processing wastes to take advantage of the large quantity of potentially beneficial compounds they contain Tomato processing waste contain a variety of biologically active substances being a promising source of dietary fibers, proteins, carotenoids, tocopherols, polyphenols and other compounds (Vagi et al., 2007) Among these bioactive compound polyphenol, carotenoid and vitamins have a lot of physiological properties such as anti-inflammatory, anti-allergenic, antimicrobial, anti-thrombotic, cardio-protective anti antioxidant effects (Yang et al., 2008) The results of Al-Wandawi‘s (1985) research showed that tomato skins yield about 71% of the lycopene found in tomato pastes Finally it is clear that a large quantity of nature color is normally disposed in tomato processing “as waste” (Al-Wandawi et al., 1985) The Table 2.5 gives

carotenoid composition of tomato fruit, tomato processing waste and tomato paste that can be derived from consuming a 100 gram of wet sample (Al-Wandawi et al., 1985)

Table 2.5 Carotenoid composition of tomato fruit, tomato processing wastes

and tomato paste (mg/100g wet sample)

Carotenoid Whole

mature fruit

Tomato processing wastes Tomato

paste Tomato skin Tomato seed

Source: Al-Wandawi et al (1985)

In recent years, a number of food scientists proposed that utilization of tomato processing waste can be as source of lycopene for food, in order to increase the intake of this carotenoid in the diet Calvo et al (2008) indicated that tomato peel could be added to dry fermented sausages to produce a meat product enriched in lycopene Dry tomato peel was added to the meat mixture used in sausage manufacture with ratio from 0.6 to 1.2% (w/w) After 21 days ripening, lycopene level remained between 0.26 and 0.58 milligram per 100 gram of sausage (Calvo et al, 2008) Garcia et al (2009) also indicated that the direct adding of dry tomato peel to hamburger could be useful both to obtain a new product enriched in lycopene and to provide a use for this by-product from the tomato industry The addition of dry tomato peel to 4.5% w/w in hamburgers with good overall acceptability and a lycopene content of 4.9 mg/100 g of cook hamburger (Garcia et al 2009) In addition, dry tomato waste is not only added

to meat product (sausage, hamburger, minced meat, dry-cured, beef patties and frankfurters) but also add to bread to enrich in lycopene and other bioactive compounds (Nour et al., 2015)

2.2 LYCOPENE

2.2.1 Source of lycopene

Lycopene belongs to carotenoid family that is a natural pigment synthesized exclusively by plant and microorganisms Lycopene is a natural pigment widely used in the food industry as a food additive due to its strong color and non-

toxicity It is approved for food use with registering as 160d (i) - synthetic lycopene, 160d (ii) – lycopene extract from tomato, 160d (iii) – lycopene Blakeslea trispora (tracuuphugia.vfa.gov.vn)

 Chemical synthesis

Synthetic lycopene is the final product of a series of reactions, starting from synthetic reagents and using chemical solvents The final product often contains trace of chemical solvents, impurities and by-products, which could be toxic and very dangerous for health This industrial production of synthetic lycopene has a negative environmental impact due to high amount of chemical solvents utilized (Lycopene, 2015)

Synthetic lycopene is highly concentrated, it has a purity of 90-95% and it could not be directly used for human consumption It is used for soaps, creams and cosmetics In fact, it has a very low bio-availability and is very labile to air and light The dietary supplements including this kind of lycopene are obtained

by diluting lycopene up to 1-10% with vegetable oils, preservatives and other exogenous chemicals (Lycopene, 2015)

 Biological sources

Lycopene belongs to a group of naturally-occurring pigments known as carotenoids Lycopene is a natural constituent of red fruits and vegetables and of certain algae and fungi (Olempska – Beer, 2005)

Microbial source:

Blakeslea trispora is a fungal plant pathogen It is known as a source to produce lycopene and is also the microbe used for producing commercial β-carotene for dietary supplements and food additives (Wikipedia: Blakeslea trispora, 2016)

Lycopene from B trispora is produced by mating and co-fermentation of two non-pathogenic and non-toxigenic strains Lycopene is extracted by isopropanol and isobutyl acetate from the biomass and purified by filtration and crystallization Pure lycopene crystals are unstable when exposed to oxygen and light so lycopene is stored under nitrogen or other inert gases in light-proof containers Olempska-Beer (2005) indicated that lycopene from B trispora contains at least 90% of all-trans-lycopene and minor quantities of 13-cis-lycopene and β- and γ-carotene

Plant sources:

Lycopene and other carotenoid are responsible for red color of many kinds

of fruit and vegetable in nature Many fruit and vegetables are known to contain lycopene such as tomato, watermelon, pink guava, pink grapefruit, papaya, apricot and so on (Table 2.6)

Table 2.6 Lycopene content of common fruit and vegetables

Fruit/ vegetables Lycopene (µg/g of weight)

Source: Rao and Agarwal (1999)

Tomatoes, especially deep-red fresh tomato fruits, are considered the most important source of lycopene in many human diets (Schwartz et al., 2002) Lycopene can represent about 80-90% of total carotenoids in tomatoes The amount of lycopene in tomato fruits depend on variety, maturity, and the environmental conditions under which the fruit matured (George, 2004) Most of lycopene compound (70-90%) is located in the insoluble fraction of the tomato such as peel According to Al- Wandawi et al (1985) tomato skin contains 12 mg lycopene per 100 gram skin (wet basis), while whole mature tomato contain only 3.4 mg lycopene per 100 gram (wet basis) Skin tomato is a rich source of lycopene, as they contain about five times more lycopene than the whole tomato pulp (Sharma and Le Maguer, 1996)

Besides, the product was made from in tomato also contain large amount of lycopene (Table 2.7) They are also mainly source of lycopene in human diets

Table 2.7 Lycopene content in common tomato –based food

Source: Rao and Agarwal (1999)

2.2.2 Role of lycopene in the human health

Human and animals cannot synthesize lycopene, and thus amount of lycopene in their body depends on dietary sources (Shi and Magure, 2000) Of more than 700 carotenoids in nature, there are 50 type may be absorbed and metabolized by the human body Only 14 carotenoids have been identified in human serum, and lycopene is the most abundant (Xianquan et al., 2005) Lycopene is particularly high concentration in the prostate gland, adrenal glands, skin, liver and kidneys (Shi and Magure, 2000) Unlike other carotenoids, lycopene cannot be converted into vitamin A (Rao and Agrawal, 1999) The ability of lycopene to act as a potent antioxidant is thought to be responsible for protecting cells against oxidative damage and thereby decreasing the risk of chronic diseases (Rao and Agrawal, 1999)

Several recent studies have been shown that dietary intake of tomatoes and tomato products containing lycopene associated with a decreased risk of chronic diseases (Agrawal and Rao, 2000) Dorgan et al (1998) also indicated that serum and tissue lycopene levels have been found to be inversely related to the incidence of several types of cancer, including breast cancer and bladder cancer Lower serum lycopene levels were found associated with increased risk and mortality from coronary heart disease (Kristenson et al., 1997) Similarly, Coodle et al (1995) and Periquet et al (1995) also reported that lower serum lycopene levels were in human immunodeficiency virus (HIV) positive women and also in children infected with HIV

Lycopene is not only reducing the risk of chronic diseases but also used in cosmetic products It is a common ingredient in anti-aging creams due to its potent antioxidant Lycopene can decrease inflammation and help to protect the skin from damage resulting from UV sun exposure (Stahl et al., 2001)

2.2.3 Physical and chemical properties of lycopene

The molecular formula of lycopene is C40H56, and is an acyclic open-chain polyene with 13 double bonds There are 11 conjugated double bonds arranged in

a linear array, making it longer than any other carotenoid The acyclic structure

of lycopene causes symmetrical planarity, and therefore lycopene has no vitamin

A activity Lycopene is more soluble in chloroform, benzene, carbon disulfide, acetone, ethyl acetate, and other organic solvents than in water (Shi and Maguer, 2000) The solubility of lycopene in vegetable oil is about 0.2 g/L at room temperature (Borel et al., 1996) In aqueous systems, lycopene tends to aggregate and to precipitate as crystals This behavior is suspected to lower lycopene bioavailability in humans In ripe tomato fruit, lycopene takes the form of elongated, needle like crystals that are responsible for the typical bright-red color

of ripe tomato fruits (Shi and Maguer, 2000)

The physical properties of lycopene are shown in Table 2.8

Table 2.8 Physical properties of lycopene

Solubility Soluble in chloroform, hexane, benzene, carbon disulfide,

acetone, petroleum ether and ethyl acetate Insoluble in water, ethanol, methanol Sensitivity Light, oxygen, high temperature, acids

Source: Shi and Maguer (2000)

The chemical name of lycopene is

include Ψ, Ψ-carotene, all-trans-lycopene, and (all-E)-lycopene Lycopene

occurs in all-trans-lycopene and various cis configuration In literature source, all-trans-lycopene is referred to as (all-E)-lycopene and cis isomers are referred

to as Z isomers (Olempska-Beer, 2005)

The all-trans isomer of lycopene is most predominant geometrical isomer in fruits and vegetables, and is the most thermodynamically stable form In red tomato fruit, lycopene presents about 94-96% of all-trans-lycopene (Schierle et al., 1997) In nature, lycopene exists in all-trans form and may be expected to undergo changes into mono-cis or poly-cis forms under the influence of heat, light, or certain chemical reactions (Cole and Kapur, 1957) The 5-cis, 9- cis, and 15-cis isomers of lycopene have presented in various tomato –based foods In human serum and tissue, cis isomers of lycopene contribute more than 50% of total lycopene (Shi and Maguer, 2000) Cis isomers of lycopene have physical characteristics and chemical behaviors distinct from those of their all-trans counterparts, including decreased color intensity and lower melting points; they are more polar than their all-trans counterpart, less prone to crystallization, and more soluble in oil and hydrocarbon solvents Experimental results revealed that cis isomers of lycopene are better absorbed by human than the all-trans form (Boileau et al., 2002) On the other hand, the conversion of cis- isomer to trans- form is another reaction that can occur during the product storage Cis-isomers are in the unstable, wherease trans-isomers are in the stable The chemical structure of lycopene isometrics in tomatoes are shown in Figure 2.1

Figure 2.1 Structure of trans and cis isomeric forms of lycopene

Source: Rao et al (2006)

Lycopene has acyclic structure, large array of conjugated double and extreme hydrophobicity Therefore, lycopene exhibits many unique and distinct biological properties Although it has no pro-vitamin A activity, lycopene is able

to function as an antioxidant Di Mascio et al (1991) indicated that antioxidant capacity of lycopene was more than double that of β-carotene and 10 times more than that of α-tocopherol

2.3 LYCOPENE EXTRACTION

Lycopene is a pigment principally responsible for dark-red color of ripe tomato fruit and tomato products (Montesano et al., 2008) Fresh tomato industrial waste, which can be material for lycopene extraction, has high moisture content about 82.9 % (Montesano et al., 2008) Drying process of the waste may be affected lycopene content due to exposure of temperature, light and oxygen Favati et al (2003) reported that the lycopene content of dried sample was lower than that of fresh tomato industrial waste sample The dried methods

of tomatoes make significantly increase in cis- isomers and simultaneously made decrease in all-trans isomers (Table 2.8) Cis- isomers were not detected in the fresh tomato sample after HPLC analysis (Rodriguez-Amaya and Tavares, 1992) The highest amount of cis-isomers was found in air-dried tomato samples (Shi et al., 1999)

Table 2.9 Total lycopene and Cis-isomer content in the dehydrated tomato

Sample Total lycopene

(µg/g dry basis)

Lycopene loss (%)

All- trans isomers (%)

Cis- isomers (%)

Source: Shi and Le Mague (1999)

Lycopene is sensitive compound to light, oxygen, heat and acid Therefore, lycopene extraction from tomato waste has to be carried out under controlled environmental factors to minimize lycopene degradation through oxidation or isomerization Actually, in the world, there are a lot of studies about various methods, which are used for lycopene extraction from tomatoes such as solvent extraction, supercritical fluid extraction using CO2, enzymatic aided treatment… However, preferred method, which is used popularly, is solvent extraction The main reason for the adoption of solvent extraction method is the cheaper cost of technology and better recovery as compared to other methods

2.3.1 Solvent extraction method

Lycopene is a fat-soluble substance therefore it is more commonly extracted with single organic solvents, such as ethanol, acetone, ethyl acetate, petroleum ether, hexane, benzene and so on or mixture of various solvents During extraction process, whole extracted systems should be guaranteed safety to avoid detonating combustion because the solvent has low boiling point, and easy to volatile Some main factor influencing lycopene extraction by solvent extraction includes kinds of solvent, solvent/material ratio, temperature, extraction time, particle size and number of extraction

2.3.1.1 Solvent

The lycopene is used in production of healthy food, food additives, medicines and cosmetics One of the problems is the elimination of the residual solvents to obtain a safe extract Solvents should have not only low toxicity and considered to be safe for foods but also low cost, high affinity toward the target compound, stability during the extraction process, low detrimental effect on the environment, low flammability (for safety) United Stated Department of Health and Human Services (2012) has classified solvents into three categories Among

of them, solvents belong to Class 3 such as acetone, ethanol, ethyl acetate, propanol and propyl acetate are acceptable in small residual amount, and are used

in the food and pharmaceutical industries

Acetone: is the organic compound with the formula (CH3)2CO It is a colorless, volatile, flammable liquid, and is the simplest ketone Acetone can be soluble in water and boils at 56.05oC (Allen et al., 1952)

Ethanol: also called alcohol, ethyl alcohol and drinking alcohol, is the principal type of alcohol found in alcoholic beverages It is a volatile flammable, colorless liquid with a slight characteristic odor Its chemical formula is C2H6O

or C2H5-OH It boiling point is at 78.24oC

Ethyl Acetate: is the organic compound with the formula C4H8O2 Ethyl acetate is the ester of ethanol and acetic acid; it is manufactured on a large scale for use as a solvent Ethyl acetate is used primarily as a solvent and diluent, being favored because of its low cost, low toxicity, and agreeable odor In perfumes, it evaporates quickly, leaving only the scent of the perfume on the skin Ethyl acetate is fairly volatile at room temperature and has a boiling point of 77oC

2.3.1.2 Solvent/material ratio

The ratio of solvent/material affects to extraction process due to influence

of diffusion capacity of soluble compound into solvent Hence, depending on the form of material extraction, the appropriate material/ solvent ratio improve extraction yield

2.3.1.3 Extraction temperature

Temperature of extraction makes speed and yield of extraction process to increase due to the coefficient of diffusion is increased and viscosity is reduced However, if extraction temperature is too high, it will be influenced lycopene yield

Calvo et al (2007) also indicated that lycopene concentration in ethanol extraction increased with the increase in the extraction temperature from 25 to 50

oC However, at 60 °C, the obtained concentration was approximately half than that at 50 °C at all the assayed time; this may be due to a lower stability of the lycopene at 60 °C than at 50 °C In addition, lycopene is a sensitive compound to temperature; high temperature can break down lycopene In hexane and light petroleum solution, 26.1% of the lycopene was lost when heated for 3 hours at 65

oC, and 35% was lost when heated for 3h at 100 °C (Shi and Maguer, 2000) Therefore, in the extraction process, defining suitable temperature is necessary to keep lycopene stable

2.3.1.4 Extraction time

The longer the extraction time, the more extraction yield of compound is made However at a certain time, when extraction time is increase, the lycopene recover will be insignificantly increased Furthermore, lycopene also is sensitive

to oxygen and light If extraction time is too long, while the conditional extraction contains oxygen with high temperature, lycopene will be significantly lost More than 30% of the lycopene was degraded when heated at 100°C in the presence of oxygen (Monselise and Berk, 1954) In hexane and light petroleum solution, lycopene loss is 15% in 1 hour and 26.1% in 3 hours at 65°C

2.3.1.5 Particle size

Particle size affect to extraction yield The smaller the particle size, the larger the lycopene recovery obtained This can be explained that the small particle size makes contact surface between material and solvent to increase In

addition, many broken cells help release compounds to outside Therefore, the efficiency of extraction was increased However, lycopene is sensitive with to light and oxygen The small dried tomato waste size may be lost lycopene content due to exposure with light oxygen

2.3.1.6 Number of extraction

In theory, the higher the extracted time, the higher the lycopene content obtained However, when extracted to a certain threshold, the amount of lycopene recovery is not significantly increased, while it takes time and solvent

to extract Therefore, it is necessary to select number of extraction, which is the most appropriate and highest economic efficiency

 In previous work, Kaur et al (2008) also studied the effect of extraction condition on lycopene extraction from tomato processing waste skin using response surface methodology The tomato skin was dried in cabinet dryer and contained 5.74% moisture The experiment was run at the optimum condition of solvent/meal ratio 30:1 v/w, four times of extraction, at 50oC and particle size of 0.15 mm and extraction solvent was hexane: acetone: ethanol 2:1:1

Calvo et al (2007) evaluated the extraction yield of the food grade solvents ethanol and ethyl acetate by extracting lycopene, β-carotente, phytoene and phytofluen from tomato peel powder at varying heating intensities, and the influence of the solvent and heating intensity on carotenoids isomerization and degradation during extraction The carotenoid yield using the ethanol solvent was higher than that using the ethyl acetate solvent This research also concluded that the temperature increase caused an increase in the carotenoid concentration However in the extractions performed with ethanol at 60oC, the yield of trans-lycopene and their isomer was low than that at 50 oC

Pandya et al (2015) was undertaken to find out the effective solvent system for efficiently extracting lycopene from the fresh tomato pomace The polar and non-polar solvents such as acetone, ethanol, ethyl acetate, hexane and petroleum ether were investigated Acetone- ethyl acetate was identified as the suitable solvent system for efficient extraction of lycopene from tomato pomace

2.3.2 Other methods of lycopene extraction

 Supercritical fluid extraction with CO2

Supercritical fluid extraction using CO is an ideal solvent for extracting

food components and has been proposed to obtain a high purity lycopene extract (Gomez-Prieto et al., 2003) However, the use of supercritical fluids extraction is more laborious than the solvent (Calvo et al., 2007)

Choksi and Joshi (2007) reported that supercritical fluid extraction of lycopene with CO2 gives better results than other methods In this method, experiment condition such as pressure and temperature also influence extraction yield and amounts of lycopene The highest concentration of carotenoid with 90.1% of lycopene was obtained by supercritical CO2 extraction at 460 bars and 80°C (Vagi et al., 2006)

Beside, Shi et al (2009) investigated effect of the addition of non-toxic modifiers (ethanol, water, and canola oil) in supercritical carbon dioxide (SC-CO2) extractions from tomato skins The extraction was carried out with various temperatures (45°C, 60°C, and 75°C) and pressures (25, 30, and 35 MPa) The suitable operating condition for highest lycopene yields was 75°C and 35 MPa and the highest lycopene yields were obtained with 15% olive oil at 45°C and 10% olive oil at 75°C

 Enzymatic aided treatment

Enzymatic cell wall lysis employing hydrolytic enzymes, which can degrade the cell wall constituents, thus aiding in the release of intracellular contents is a widely reported method for the extraction of various kinds of natural compound (Choudhari and Ananthanarayan, 2007) Pre-treatments with cell wall hydrolases have proved effective in enhancing the extraction yield from tomato

by organic solvent based methods (Choudhari and Ananthanarayan, 2007) or supercritical carbon dioxide (Lenucci et al., 2015) Since the plant cell wall comprises of cellulose and pectins, most of the work in the field utilize cellulases and pectinases

Choudhari and Ananthanarayan (2007) also studied using cellulase and pectinase enzymes for assisting lycopene extraction from tomato tissue by petroleum ether and acetone (1:1) The results showed that enzyme aided extraction of lycopene from whole tomatoes under optimized condition resulted

in an increase in the lycopene yield by 132µg/g (198%) in cellulose treated sample and 108µg/g (224%) in case of pectinase treated sample

 Ultrasonic assisted extraction

Ultrasonic assisted extraction (UAE) of lycopene promotes extraction yield of lycopene and minimizes degradation and isomerization of lycopene Ultrasonic assisted extraction of lycopene was at lower extraction temperature and shorter total extraction time (enhancement of 26% extraction yield of lycopene in 6 replications at 40.0 min, 40.0 °C and 70.0% v/w in the presence of ultrasound) (Eh and Teoh, 2012)

 Hydrostatic pressure processing

Xi (2006) studied the effect of high pressure processing (HPP) on the extraction of lycopene in tomato paste waste HPP without heating can increase the yield of lycopene The result of study showed that HPP do not destroy the molecular structure of lycopene The extraction yield of lycopene obtained by high pressure processing for 1 min was higher than that obtained with solvent extraction for 30 min

CHAPTER 3 MATERIALS AND METHODS 3.1 MATERIALS

3.1.1 Sample collection and preparation

The red ripe tomato cv.Chanoka F1 was harvested at Bac Ninh province in the winter season Tomato waste was obtained by removing the juice Briefly, whole tomatoes were first washed and the seeds were removed Tomatoe paste was passed through a fruit pulper to obtain waste, in the form of flesh pulp and peel residues with moisture content about 85% Afterward, the paste was dried and stored in the tightly closed falcon tubes which had been covered with aluminum foil

Figure 3.1 A Tomato ‘Chanoka F1’ fruit, B Fresh tomato waste,

C Dried tomato waste 3.1.2 Equipment

- Fruit pulper (HR1823, Philips, China)

- Convective drying equipment (ED 115, Binder, Germany)

- Moisture analysis equipment (MA37, Sartorius, Germany)

- Centrifugal equipment ( Centrifuge 5810R, Eppendorf, Germany)

- Vortex (Vortex 4 basic, IKA, Germany)

- HPLC system (LC - 10Ai, Shimadzu, Japan)

- Spectrophotometer ( Shimadzu UV 1800, Japan)

3.1.3 Chemical

* Solvents (Merck, Germany): Acetone (C3H6O, 100%), ethyl acetate

C4H8O (99.9%), ethanol (C2H5OH, 99.7%), methanol (CH4O) (99.9%)

* Analytical standards and reagents

- Lycopene (C40H56, >90% from tomato, Sigma- Aldrich, China)

- Trolox (C14H18O4, Sigma- Aldrich, USA)

- DPPH (C18H12N5O6, Sigma- Aldrich, USA)

Table 3.1 Effect of solvent system on lycopene extraction from tomato waste

T7 acetone: ethanol: ethyl acetate = 1:1:1

Each treatments had 3 replicates (n=3)