OPTIMIZATION OF MICROWAVE-ASSISTED EXTRACTION OF

ANTIOXIDANTS FROM POTATO PEELS

ASHUTOSH SINGH

Department of Bioresource Engineering

Faculty of Agricultural and Environmental Sciences

McGill University

Ste Anne De Bellevue, Quebec, Canada

August 2010

A thesis submitted to the McGill University in partial fulfillment of the

requirements of the degree of

Master of Science

in

Bioresource Engineering

2010 Ashutosh Singh

ii

OPTIMIZATION OF MICROWAVE-ASSISTED EXTRACTION OF ANTIOXIDANTS

FROM POTATO PEELS

Ashutosh Singh

ABSTRACT

Potato processing by industries generates large volumes of potato peels which are a low-

value by-product. They have been shown to contain significant levels of phenolic antioxidants

with high antioxidant capacity. In the present study, effect of microwave-assisted extraction

(MAE) on recovery of phenolic compounds from potato peels was investigated. Operational

parameters of MAE were optimized using response surface methodology individually based on

total phenolics, ascorbic acid, chlorogenic acid, caffeic acid, ferulic acid, DPPH (1,1-diphenyl-2-

picrylhydrazyl) radical scavenging activity.

The independent extraction parameters for MAE such as extraction time, solvent

concentration and microwave-power level were evaluated using a central composite design to

access their effects and interactions on the extraction of phenolics from potato peels. Optimal

predicted contents for total phenolics [3.94 mg g-1

dry weight (DW)] was obtained with a

solvent concentration of 67.3% (v/v), an extraction time of 15 min and a microwave power level

of 14.7% (102 W). Maximum ascorbic acid (1.44 mg g-1

DW), caffeic acid (1.33 mg g-1

DW)

and ferulic acid (0.50 mg g-1

DW) contents were obtained with a solvent concentration of 100%

(v/v), extraction time of 15 min, and power level of 10% (63W), while the maximum chlorogenic

acid content (1.35 mg g-1

DW) was obtained at a solvent concentration of 100% (v/v), an

extraction time of 5 min, and a power level of 10% (63W). The radical scavenging activity of the

extract was evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and optimum antioxidant

activity (74%) was obtained at a solvent concentration of 100% (v/v), an extraction time of 5

min, and a power level of 10% (63W). MAE was able to extract higher level of antioxidants

compared to traditional methods of extraction in less time and with lower solvent consumption.

iii

In the second part of the study effect of dielectric properties of solvent-water mixture

used in MAE on extraction of antioxidants from potato peel was investigated. Linear regression

models were obtained for establishing the effect of solvent concentration and temperature on

dielectric properties of solvent-water mixture with and without introduction of potato peel.

Solvent concentration significantly affected the dielectric properties of the solvent-water mixture

and the total phenolics content of the extract obtained from the MAE process.

iv

Rsum

Le grand volume de pelures de pomme de terre issues de la transformation des pommes de terre

par lindustrie alimentaire, reprsente un sous-produit de faible valeur. Cependant, ces pelures

contiennent une importante quantit de composs phnoliques haute activit antioxydante. Lors

de la prsente tude, leffet dune extraction assiste par micro-ondes (EAM) sur la rcupration

de composs phnoliques des pelures de pomme de terre fut tudi. Les paramtres oprationnels

de lEAM furent optimiss par une mthode de surface rponse pour les phnoliques totaux,

lacide ascorbique, lacide chlorognique, lacide cafique, lacide frulique, ainsi que lactivit

dintercepteur radical valu avec DPPH [hydrazyle(diphnyl picryl)].

Limportance des paramtres de transformation de la EAM, tels la dure dextraction, la

concentration du solvant (mthanol), et le niveau de puissance des micro-ondes, sur lextraction

de composs phnoliques des pelures de pomme de terre, fut value grce un plan central

compos. Un contenu optimal en composs phnoliques totaux [3.94 mg g-1

poids sec (p.s.)] fut

obtenu avec une concentration du solvant de 67.3% (v/v), une dure dextraction de 15 min et un

niveau de puissance des micro-ondes de 14.7% (102 W). Les niveaux maximum dacide

ascorbique acid (1.44 mg g-1

p.s.), dacide cafique acid (1.33 mg g-1

p.s) et dacide frulique

furent obtenus avec une concentration du solvant de 100% (v/v), une dure dextraction de 15

min et un niveau de puissance des micro-ondes de 10% (63W), tandis que le niveau maximum

dacide chlorognique (1.35 mg g-1

p.s.) fut obtenu avec une concentration du solvant de 100%

(v/v), une dure dextraction de 5 min et un niveau de puissance des micro-ondes de 10%

(63 W). Lactivit dintercepteur radical de lextrait fut value avec un dosage par DPPH, et

atteignit un maximum de 74% pour une concentration du solvant de 100% (v/v), une dure

dextraction de 5 min et un niveau de puissance des micro-ondes de 10% (63W). LEAM

permetta dextraire des niveaux dantioxydants plus levs que les mthodes traditionnelles

dextraction, en moins de temps et avec une moindre quantit de solvant.

Le seconde volet de ltude porta sur les proprits dilectriques du mlange solvant-eau

utilis dans lEAM des antioxydants des pelures de pomme de terre. Des modles de rgression

linaire furent dvelopps reliant les effets de la concentration du solvant et de la temprature sur

les proprits dilectriques du mlange solvant-eau, avec ou sans pelures. La concentration du

v

solvant montra un effet significatif sur les proprits dilectriques du mlange solvant-eau et sur

la teneur totale en composs phnoliques de lextrait obtenu par EAM.

vi

ACKNOWLEDGEMENTS

I would like to thank my supervisor Dr. G.S.V. Raghavan, for his guidance and support

throughout my Masters Thesis; above all I am grateful to him for his encouragement and belief

in me. It has been and will always be a matter of great pride to work for Dr. Raghavan, his

perpetual energy and enthusiasm in research had motivated all his advisees including me.

I would like to thank Dr. Stan Kubow for serving on my advisory committee and for the

guidance that he provided. I would also like to thank Dr. Valrie Orsat and Dr. Danielle

Donnelly for their scientific advice and support for this project.

Special thanks to Mr. Yvan Garipy for his patience, knowledge and his willingness to

help me with all technical problems that I faced during my masters program. I am also

appreciative of the technical help extended by Dr. Kebba Sabally for HPLCwith all the

leakages and problems we faced with the instrument!!

I would like to thank the faculty and staff in the Dept. of Bioresource Engineering.

Special thanks to Mrs. Susan Gregus, Mrs. Abida Subhan and Ms. Patricia Singleton for their

help in administrative affairs.

I would like to express my appreciation to the people with whom Ive worked with in Dr.

Raghavans lab including Gopu, Abid, Pansa, Simona, Satya, Baishali, Valquria, Kirupa,

Ramesh, Winny, Yanti, Juliette and Kartheek. I would like to extend my special thanks to my

friends Meera, Shireen, Archi, Jamshid and Kumaran for their love, support and home-cooked

meals!. Thanks for lending me your ears on countless occasions to vent my frustration. Life at

Mac campus would have been a misery without you all by my side.

I am grateful to my parents for their love and support. I am also thankful to almighty God

for 819,936,000 seconds (and still counting) of life on this beautiful planet Earth.

vii

CONTRIBUTIONS OF THE AUTHORS

The work reported here was performed by Ashutosh Singh and supervised by Dr.

G.S.V Raghavan of the Department of Bioresource Engineering, Macdonald Campus of McGill

University, Montreal. The entire research work was carried out at the Postharvest Technology

laboratory, Macdonald Campus of McGill University, Montreal.

The authorship of the first paper (chapter 3) includes Ashutosh Singh, Dr. Kebba Sabally,

Dr. Stan Kubow, Dr. Danielle Donnelly, Dr. Valrie Orsat, Yvan Garipy and Dr. G. S. V.

Raghavan. For the second paper (chapter 4) the authorship is Ashutosh Singh, Pansa Liplap,

Yvan Garipy, Dr. Stan Kubow, Dr. Valrie Orsat and Dr. G. S. V. Raghavan.

Co-authors, Dr. Danielle Donnelly from Department of Plant Science, Dr. Stan Kubow

and Dr. Kebba Sabally from School of Dietetics and Human Nutrition and Pansa Liplap, Yvan

Garipy, Dr. Valrie Orsat from Department of Bioresource Engineering were involved in the

development, implementation and data analysis. Dr. Stan Kubow was also the member of

advisory Committee, Yvan Garipy, Dr. Valrie Orsat provided additional guidance and support

in the development of the manuscripts.

viii

TABLE OF CONTENTS

Title Page.... i

Abstract.................................................................................................................................. ii

Rsum ......................................................................................................................................... iv

Acknowledgements ................................................................................................. vi

Contributions of the authors ............................................................................vii

Table of contents .................................................................................................viii

List of Figures ......................................................................................................xii

List of Tables .............................................................................................................................. xv

CHAPTER 1

INTRODUCTION ....................................................................................................................... 1

CHAPTER 2

LITERATURE REVIEW................................6

Abstract.................................................................................. 6

2.1 Lipid oxidation in food and food products............................................................................. 7

2.1.1 Mechanisms of lipid oxidation.7

2.1.2 Deleterious activity of free radicals and oxidants on human health. .....10

2.2 Antioxidants ..10

2.2.1 Synthetic Antioxidants. .12

2.2.2 Natural Antioxidants.. 13

2.2.2.1 Polyphenols .... 15

2.2.2.2 Carotenoids.... 18

2.2.2.3 Agricultural residues as sources of natural antioxidants... 19

2.3 Extraction of phytochemicals from plant materials.. 20

2.3.1 Solid-liquid extraction process 21

2.3.2 Accelerated solvent extraction 23

ix

2.3.3 Supercritical fluid extraction.. .. 24

2.3.4 Microwave-Assisted Extraction 25

2.4 Conclusion 30

References 30

CONNECTING TEXT.. 40

CHAPTER 3

OPTIMIZATION OF MICROWAVE-ASSISTED EXTRACTION OF PHENOLIC

ANTIOXIDANTS FROM POTATO PEELS BY RESPONSE SURFACE METHOD..41

Abstract. 42

3.1 Introduction. 43

3.2 Materials and Methods 44

3.2.1 Materials 44

3.2.2 Equipment and apparatus... 45

3.2.3 Experimental Design. 45

3.2.4 Preparation of potato peel extracts.. 46

3.2.5 Conventional extraction methods 46

3.2.6 Determination of total phenolic compounds 48

3.2.7 Scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DDPH) radicals.. 48

3.2.8 HPLC Analysis.. 48

3.3 Results and discussion... 50

3.3.1 Model Fitting. 50

3.3.1.1 Total Phenolics...... 50

3.3.1.2 Ascorbic acid.. . 52

3.3.1.3 Chlorogenic acid... 53

3.3.1.4 Caffeic acid... 54

3.3.1.5 Ferulic acid. 54

3.3.1.6 Radical scavenging activity .... 54

x

3.3.2 Response surface Analysis.. 54

3.4 Comparison of MAE and conventional extraction techniques. 60

3.5 Conclusion..... 63

3.6 Acknowledgements.. 63

References.. 64

CONNECTING TEXT 68

CHAPTER 4

EFFECT OF DIELECTRIC PROPERTIES OF A SOLVENT-WATER MIXTURE ON

MICROWAVE-ASSISTED EXTRACTION OF ANTIOXIDANTS FROM POTATO

PEELS ................................................................................................................................... 69

Abstract. 70

4.1 Introduction. 71

4.2 Materials and Methods. 73

4.2.1 Materials. 73

4.2.2 Equipment and apparatus.. 73

4.2.3 MAE extraction . 74

4.2.4 Preparation of potato peel extracts.. 75

4.2.5 Determination of total phenolic compounds.. 75

4.2.6 Scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DDPH) radicals.. 75

4.2.7 HPLC Analysis.. 76

4.2.8 Statistical analysis . 76

4.3 Results and discussion 76

4.3.1 Effect of temperature on dielectric properties of methanol-water mixtures 76

4.3.2 Effect of temperature, methanol concentration and presence of plant material on

dissipation factor (tan) 83

4.3.3 Effect of dielectric properties of methanol fraction on MAE extraction of

antioxidant 86

4.4 Conclusion.. . 89

References. 90

xi

CHAPTER 5

SUMMARY AND CONCLUSIONS.. 92

xii

LIST OF FIGURES

Figure 2.1 Free-radical chain mechanism of autoxidative reactions ...............................8

Figure 2.2 Mechanism of photosensitized oxidation...............................................................9

Figure 2.3 Deleterious activity of free radicals and oxidants on human health......................10

Figure 2.4 Antioxidant (AH) reaction with several types of free radicals generated during lipid

oxidation...................................................................11

Figure 2.5 Interfacial phenomenon of hydrophilic and lipophilic antioxidants in bulk oil and

oil-in-water emulsion system............................................................................................12

Figure 2.6 Chemical structure of synthetic antioxidants.........................................................13

Figure 2.7 Chemical structure of a) hyroxybenzoic b) and hydroxycinnamic acids...........16

Figure 2.8 Basic structure of a flavonoid molecule.........................................................17

Figure 2.9 Structure of major classes of flavonoids............................................18

Figure 2.10 Schematic representation of individual steps in process of extraction.....................22

Figure 2.11 Schematic of Accelerated Solvent Extraction instrument........................................23

Figure 2.12 A typical phase diagram...........................................................................................24

Figure 2.13 Schematic of different MAE systems.......................................................................27

Figure 2.14 Heating principle of conventional and microwave-assisted extraction processes28

Figure 3.1 Chromatogram of standards: ascorbic acid (peak 1), chlorogenic acid (peak 2), caffeic

acid (peak 3), ferulic acid (peak 4) and rutin (peak 5)..................................................................49

Figure 3.2 HPLC Chromatogram of a potato peel polyphenolic extract. Ascorbic acid (peak 1),

chlorogenic acid (peak 2), caffeic acid (peak 3), ferulic acid (peak 4) and rutin (peak 5)50

Figure 3.3 Predicted (mg g-1

) vs. measured (mg g-1

) total phenolics............................................52

Figure 3.4 Predicted vs. actual ascorbic acid content (mg g-1

).....................................................53

Figure 3.5 Response surface plot of the effect of Methanol (MeOH) concentration (% v/v) and

extraction time (min) on total phenolics content of potato peel extract........................................56

xiii

Figure 3.6 Response surface plot of the effect of Methanol (MeOH) concentration and effect of

microwave power on ascorbic acid content of Potato peel extract................................................57

Figure 3.7 Effect of solvent concentration on concentration of chlorogenic acid and caffeic

acid.................................................................................................................................................58

Figure 3.8 Effect of solvent concentration on % inhibition of oxidation by potato peel

extract.............................................................................................................................................59

Figure 3.9 Measured total phenolic and individual phenolic content in selected potato

cultivars..........................................................................................................................................62

Figure 3.10 Comparison of MAE and conventional extraction techniques..................................63

Figure 4.1 Dipolar rotation in an electromagnetic field...............................................................72

Figure 4.2 Schematic of experimental setup for measurement of dielectric properties of

methanol fractions..........................................................................................................................74

Figure 4.3 Measured dielectric constant ( ') values for a binary mixture of methanol-water at

20C, 40C, 60C and 80C, in the presence or absence of a plant matrix, at different methanol

volume fractions and at frequency 2450 MHz...............................................................................78

Figure 4.4 Measured dielectric loss ( '') factor values for a binary mixture of methanol-water at

20C, 40C, 60C and 80C, in the presence or absence of a plant matrix, at different methanol

volume fractions and at frequency 2450 MHz...............................................................................79

Figure 4.5 Measured dielectric constant (') values for different binary mixture of methanol-

water (0, 35, 65, 100% MeOH) at 20C, 40C, 60C and 80C, with and without ground dried

potato skin at a, microwave frequency 915 MHz ........................................................................81

Figure 4.6 Measured dielectric loss factor ('') values for different binary mixture of methanol-

water (0, 35, 65, 100% MeOH) at 20C, 40C, 60C and 80C, with and without ground dried

potato skin at a, microwave frequency 915 MHz ........................................................................82

Figure 4.7 Effect of temperature, methanol concentration and potato peel on dissipation factor

(tan ) at 2450 MHz.......................................................................................................................84

Figure 4.8 Effect of temperature, methanol concentration and potato peel on dissipation factor

(tan ) at 915 MHz.........................................................................................................................85

Figure 4.9 Effect of solvent concentration on ' and ''................................................................87

Figure 4.10 Effect of dielectric constant on total phenolic content of the potato peel

extract.....87

xiv

Figure 4.11 The Effect of solvent concentration on tan..............................................................88

Figure 4.12 Effect of dissipation factor (tan) on total phenolic content of the potato peel

extract.............................................................................................................................................89

xv

LIST OF TABLES

Table 2.1 Sources of some natural antioxidants........................................................................14

Table 2.2 Different classes of phenolic acids in plants...............................................................16

Table 2.3 Phenolic content of agricultural by-products..............................................................20

Table 2.4 Dielectric constant and dissipation factors for organic solvents widely used in MAE

processes......................................................................................................................................26

Table 2.5 Comparison of MAE and other extraction processes.........................................29

Table 3.1 Coded (individually for each treatment factor) and corresponding actual values of the

independent variables analyzed in RSM.............................................................................46

Table 3.2 Central composite design for Response Surface Analysis of antioxidant extraction

from potato peel using methanol.................................................................................................47

Table 3.3 ANOVA for the effect of solvent concentration (S), and time (t) on total

phenolics..................................................................................................51

Table 3.4 Optimal conditions and predicted contents of ascorbic, chlorogenic, caffeic acid,

ferulic acid and total phenolic content of the extract ......................................................60

Table 3.5 Total phenolic and HPLC measured individual phenolic content in selected potato

cultivars extracted by MAE process .......................................................61

Table 3.6 Amount of Total phenolics (mg GAE/g dw) extracted by microwave-assisted

extraction (MAE), heat-reflux (HRE) and overnight extraction (ONE) techniques ..................62

Table 4.1 Equations obtained defining the relationship of ' and '' with temperature (T) and

methanol fraction concentration ( [MeOH] ) at 2450 MHz ...80

Table 4.2 Equations obtained defining the relationship of ' and '' with temperature (T) and

methanol fraction concentration ( [MeOH] ) at 915 MHz .....83

Table 4.3 Equation obtained defining the relationship tan with temperature (T) and methanol

fraction concentration ([MeOH] ) at 2450 MHz and 915 MHz .86

1

CHAPTER 1

INTRODUCTION

Lipid oxidation is one of the major phenomena that limit the shelf-life, nutritional and

organoleptic qualities of food products. Development of lipid peroxidation is influenced by

several factors such as storage, packaging and processing parameters (Adegoke et al., 1998).

The rate of deterioration caused by lipid oxidation can be reduced by adding antioxidants to food

products. Food industries extensively use synthetic antioxidants such as butylated

hydroxytoluene (BHT), butylated hydroxyl anisole (BHA) and tert-butyl hydroquinone (TBHQ).

However, several studies on animal models have suggested that synthetic antioxidants can be

associated with unfavourable effects such as increased mutagenic activity and formation of

tumours causing severe damage to the liver (Kahl, 1984; Lindenschmidt et al., 1986). Because of

safety concerns and consumer interest in all natural food products, there is currently much

interest in finding natural substitutes for synthetic antioxidants. Several natural antioxidants

(e.g., tocopherols) are being widely used as natural antioxidants in the food industry (Bruun-

Jensen et al., 1996).

There are thousands of naturally-occurring antioxidants belonging to several different

classes of compounds: e.g., carotenoids, polyphenolics, polyamines, tannins, catechins.

Polyphenols are the most abundant natural antioxidants present in plants and plant by-products.

Fresh fruits and vegetables are the greatest source of natural antioxidants and their consumption

has been positively related to a decrease in the risk of chronic conditions such as cancer,

atherosclerotic heart disease, neurological disorders, including Alzheimers and Parkinsons

diseases (Chu et al., 2002). Phenolic compounds with high antioxidant activity have been

identified in several agro-industrial by-products. Winery by-products can be used to extract

anthocyanins and flavanols which have been reported to possess antibacterial, anti-inflammatory,

anti-carcinogenic and antioxidant activities (Corrales et al., 2008). Ascorbic acid (Vitamin C)

and alpha-tocopherol (Vitamin E) obtained from agricultural residues are being synergistically

used in the food industry as substitutes to synthetic antioxidants or as synergists to enhance the

2

antioxidant activity of synthetic antioxidants in different food systems, such as emulsions

(Heinonen et al., 1997).

In several countries, potato (Solanum tuberosum L.) is amongst the most commonly

consumed vegetables. Potatoes have been reported to contain several phenolic antioxidants with

several health benefits. In Canada total 2009 potato production was approximately 4,6 Tg

(Statistics Canada 2009-2010 report on Canadian potato production), of which roughly 90% was

used in the food industry, consumed directly, fed to livestock, or kept as seed. Annually, over

50% of Canadas total potato production is processed, mostly for French fries and chips. (Agri-

Food Trade Service, Agriculture and Agri-Food Canada). The potato processing industries

generate a large quantity of potato peel, which is either used as cattle feed or as a source for

biofuel-production. Potato peels have been shown to contain several important and high market

value phenolic antioxidants, which can substitute for synthetic ones. Several researchers have

employed traditional solid-liquid extraction techniques to extract phenolic antioxidants from

potato peels and estimate the antioxidant activity of the extracts in increasing the shelf-life of

food products (Kanatt et al., 2005; Al-Weshahy and Venket Rao, 2009; Arora and Camire, 1994;

Lachman et al., 2008).

Microwave assisted-extraction is a recently developed technique which has been widely applied

to the extraction of organic compounds from environmental samples (Camel, 2000). This

techniques inherent advantages compared to traditional conventional soli-liquid extraction

techniques (reduction in extraction time, reduction in solvent consumption) have drawn interest

(Ballard et al., 2010; Dai et al., 1999; Venkatesh and Raghavan, 2004). Localized heating by

microwaves increases the temperature of the solvent above its boiling point and thus enhances

the extraction efficiency and reduces the extraction time. Recently studies have used MAE to

extract phenolic antioxidants from agricultural residues such as peanut (Arachis hypoga L.)

skin (Ballard et al., 2010), longan (Dimocarpus longan Lour.) peel (Pan et al., 2008) and citrus

peel (Hayat et al., 2009).

3

Hypothesis:

Our hypothesis was that microwave-assisted extraction could be used to extract phenolic

compounds of high antioxidant activity from potato peels with greater efficiency than

conventional techniques.

Study Rationale:

Consumer concerns and reported harmful effects of synthetic antioxidants have led the

food industry to reduce their reliance on such compounds to control oxidative rancidity caused

by lipid peroxidation. Extraction of polyphenols from several natural sources using traditional

extraction techniques is an expensive process and the yields obtained are not sufficient to meet

the huge demand. Thus, using agro-industrial residues, such as potato peel, as a source of natural

antioxidants requires efficient alternative extraction techniques. Extracting antioxidants from

potato peel waste confers added value on this material. MAE has proven its worth in extraction

of phenolic antioxidants from several agricultural residues. It not only maximizes the recovery of

the antioxidants but also reduces the extraction time and solvent consumption.

Objectives:

The overall objective of this research was to optimize the extraction of antioxidants from

potato peel.

Specific objectives of each study:

1) Determination of optimal conditions required for microwave-assisted extraction of

antioxidants from potato peels and comparison of results obtained with those of

conventional methods of extraction.

2) Investigate the effect of the dielectric properties of aqueous solvent mixtures on

microwave-assisted extraction of antioxidants from potato peels.

4

References:

Adegoke, G. O., Vijay Kumar, M., Gopala Krishna, A. G., Varadaraj, M. C., Sambaiah, K. &

Lokesh, B. R. (1998). Antioxidants and Lipid Oxidation in Foods - A Critical Appraisal.

Journal of Food Science and Technology 35(4): 283-298.

Al-Weshahy, A. & Venket Rao, A. (2009). Isolation and characterization of functional

components from peel samples of six potatoes varieties growing in Ontario. Food

Research International 42(8): 1062-1066.

Arora, A. & Camire, M. E. (1994). Performance of potato peels in muffins and cookies. Food

Research International 27(1): 15-22.

Ballard, T. S., Mallikarjunan, P., Zhou, K. &O'Keefe, S. (2010). Microwave-assisted extraction

of phenolic antioxidant compounds from peanut skins. Food Chemistry 120(4): 1185-

1192.

Bruun-Jensen, L., Skovgaard, I. M., Madsen, E. A., Skibsted, L. H. &Bertelsen, G. (1996). The

combined effect of tocopherols, L-ascorbyl palmitate and L-ascorbic acid on the

development of warmed-over flavour in cooked, minced turkey. Food Chemistry 55(1):

41-47.

Camel, V. (2000). Microwave-assisted solvent extraction of environmental samples. Trends in

Analytical Chemistry 19(4): 229-248.

Chu, Y. F., Sun, J., Wu, X. &Liu, R. H. (2002). Antioxidant and antiproliferative activities of

common vegetables. Journal of Agricultural and Food Chemistry 50(23): 6910-6916.

Corrales, M., Toepfl, S., Butz, P., Knorr, D. &Tauscher, B. (2008). Extraction of anthocyanins

from grape by-products assisted by ultrasonics, high hydrostatic pressure or pulsed

electric fields: A comparison. Innovative Food Science & Emerging Technologies 9(1):

85-91.

Dai, J., Yaylayan, V. A., Raghavan, G. S. V. &Pare, J. R. (1999). Extraction and Colorimetric

Determination of Azadirachtin-Related Limonoids in Neem Seed Kernel. Journal of

Agricultural and Food Chemistry 47(9): 3738-3742.

Hayat, K., Hussain, S., Abbas, S., Farooq, U., Ding, B., Xia, S., Jia, C., Zhang, X. &Xia, W.

(2009). Optimized microwave-assisted extraction of phenolic acids from citrus mandarin

5

peels and evaluation of antioxidant activity in vitro. Separation and Purification

Technology 70(1): 63-70.

Heinonen, M., Haila, K., Lampi, A. M. & Piironen, V. (1997). Inhibition of oxidation in 10% oil-

in-water emulsions by -carotene with and -tocopherols. Journal of the American Oil

Chemists' Society 74(9): 1047-1052.

Kahl, R. (1984). Synthetic antioxidants: Biochemical actions and interference with radiation,

toxic compounds, chemical mutagens and chemical carcinogens. Toxicology 33(3-4):

185-228.

Kanatt, S. R., Chander, R., Radhakrishna, P. & Sharma, A. (2005). Potato peel extract - A natural

antioxidant for retarding lipid peroxidation in radiation processed lamb meat. Journal of

Agricultural and Food Chemistry 53(5): 1499-1504.

Lachman, J., Hamouz, K., Orsk, M., Pivec, V. & Dvok, P. (2008). The influence of flesh

colour and growing locality on polyphenolic content and antioxidant activity in potatoes.

Scientia Horticulturae 117(2): 109-114.

Lindenschmidt, R. C., Tryka, A. F., Goad, M. E. & Witschi, H. P. (1986). The effects of dietary

butylated hydroxytoluene on liver and colon tumor development in mice. Toxicology

38(2): 151-160.

Pan, Y., Wang, K., Huang, S., Wang, H., Mu, X., He, C., Ji, X., Zhang, J. &Huang, F. (2008).

Antioxidant activity of microwave-assisted extract of longan (Dimocarpus longan Lour.)

peel. Food Chemistry 106(3): 1264-1270.

Venkatesh, M. S. &Raghavan, G. S. V. (2004). An Overview of Microwave Processing and

Dielectric Properties of Agri-food Materials. Biosystems Engineering 88(1): 1-18.

6

CHAPTER 2

LITERATURE REVIEW

ABSTRACT

Lipid oxidation is one of the major contributor to deterioration of food products,

particularly meat and meat products. Besides contributing to the deterioration of foods

organoleptic qualities, food oxidation also leads to a significant decline in their nutritional

quality. Lipid oxidation also reduces product shelf-life and can render foods inappropriate for

consumption. At present the food industry uses a variety of synthetic antioxidants to prevent

lipid oxidation in food products: butylated hydroxytoluene (BHT, 2,6-ditert-butyl-4-

methylphenol) and butylated hydroxyanisole (BHA, 2-tert-butyl-4-hydroxyanisole and 3-tert-

butyl-4-hydroxyanisole). Consumer interest in natural food additives and their beneficial effects

has increased tremendously, leading to extensive research into the extraction of naturally-derived

antioxidants, mainly from agricultural material (e.g., potato by-products, peanut skin, grape

products, etc.) as an alternative to synthetic antioxidants. This review summarizes recent

research related to agricultural waste materials being used as alternate sources of natural

antioxidants and their beneficial effect on human health. It also highlights the use of

conventional (solid-liquid extraction, heat-reflux and Soxhlet extraction) and novel (microwave-

assisted extraction and supercritical fluid extraction) methods used for extraction of antioxidants

(e.g., polyphenols, anthocyanins, etc.).

Key word: lipid oxidation, natural antioxidants, synthetic antioxidants, polyphenols,

anthocyanins, potato peels, microwave-assisted extraction

7

2.1 Lipid oxidation in food

Lipids are one of the primary components of several food products. Lipid oxidation is a

major concern for the food industry as it leads to generation of undesirable off-flavors

(rancidity), loss of colour, loss of nutrient value and formation of several toxic byproducts

detrimental to human health (Addis, 1986). Lipids can be classified as saturated or unsaturated

fatty acids. Saturated fatty acids has all bonding positions between carbon atoms occupied by

hydrogen atom, but unsaturated fatty acids have one or more double bonds between carbon

atoms making them susceptible to oxidation as oxygen can attack these double bonds forming

free radicals and oxidative by-products.

Oxidative rancidity caused by lipid oxidation is the most important factor that limits the

shelf-life of dairy, fish, oil, meat and meat products (Addis, 1986; Ryan et al., 2008). Due to

strong interactions between proteins and lipids in food, the oxidative reactions are transferred

from lipids to proteins. Several secondary products of lipid oxidation like 4-hydroxynoneral (4-

HN, (E)-4-Hydroxy-2-nonenal) and malondialdehyde (MDA, propanedial) have been known to

interact with proteins. These interactions impact on the functionality of proteins, causing changes

in texture and nutritional properties (Viljanen et al., 2004; Sarker et al., 1995).

2.1.1 Mechanisms of lipid oxidation

Oxidative deterioration of food lipids is mainly caused by auto-oxidative reactions which

are further accompanied by various secondary oxidative and non-oxidative reactions. Auto-

oxidation of unsaturated fatty acids proceeds through a free-radical chain mechanism (Fig. 2.1)

involving initiation (formation of free radicals), propagation (free-radical chain reaction), and

termination steps (formation of non-radical species) (Gray, 1978; Porter et al., 1995).

Initiation

The initiation step is marked with the abstraction of the most labile hydrogen atoms from

unsaturated fatty acids. Abstraction is caused by oxidizing agents like singlet oxygen, transition

metals and free radicals, leading to the generation of lipid free radicals (R). These R

immediately react with oxygen to form lipid peroxyl radicals (ROO).

8

Propagation

During the propagation step the lipid peroxyl radical abstracts a hydrogen atom from

another unsaturated fatty acid to form a lipid hydroperoxide (ROOH) and another R; thus

leading to rapid acceleration of free radical formation process begun in the initiation step.

Lipid hydroperoxides are very unstable and are further degraded into aldehydes, ketones,

acids and alcohols (Sherwin, 1972). These secondary reaction products are responsible for the

development of off-flavours and off-odours, as well as further reactions with other food

constituents like proteins and free amino acids (Gray, 1978; Kubow, 1992).

Termination

The termination reaction is marked by the formation of stable non-radical species. Free

radicals (autocatalysts) bind to each other to give rise to these non-radicals, thus completing the

lipid oxidation cycle.

Initiation: RH Initiator

R

Propagation: R + O2 ROO

ROO + RH ROOH + R

ROOH RO + OH

OH + RH R + H2O (etc.)

Termination: ROO + ROO ROOR + O2

R + R RR

Fig. 2.1 Free-radical chain mechanism of autoxidative reactions (Sherwin, 1972)

Lipid oxidation can also occur due to photosensitized oxidation and enzyme-catalyzed oxidation.

9

Photosensitized oxidation process

Photo-oxidation of lipids in food occurs due to photodynamic generation of singlet

oxygen via a photosensitizing agent like a food dye, chlorophyll, hmoproteins, and flavins

(Tejero et al., 2004). When a photosensitizer (S) is subjected to ultraviolet light (h ), it reaches

its first excited singlet state (1S). Subsequent exposure brings the singlet state to a more stable,

long-lived first excited triplet state (3S). The excited sensitizer can then transfer its excitation

energy to molecular oxygen forming highly reactive singlet oxygen species (1O2) (Fig 2.2). Since

these sensitizers are fat soluble they can generate reactive singlet oxygen species which can

directly attack the unsaturated acids in the vicinity.

S + h 1S

3S

3S +

3O2

1O2 +

1S (excitation energy transfer)

1O2 + RH ROOH

Fig 2.2 Mechanism of photosensitized oxidation

Enzyme-catalyzed oxidation

Enzyme-catalyzed oxidations of lipids occur when an endogenous enzyme catalyzes

reactions that lead to generation of singlet oxygen species. For example, the enzyme superoxide

dismutase (SOD) catalyzes dismutation of superoxide (O2-) into oxygen and hydrogen peroxide

(H2O2). Metal ions can react with hydrogen peroxide to form the reactive hydroxyl radical (OH).

Hydroxyl radical then can attack double bonds in lipids and cause lipid oxidation (St. Angelo,

1996).

O2- + SOD H2O2 + O2 (1)

Fe2+

+ H2O2 Fe3+

+ OH + OH-

(2)

10

2.1.2 Deleterious activity of free radicals and oxidants on human health

Generation of free radicals and oxidants not only impacts the food industry, reducing the

quality and shelf-life of food products, but also have a deleterious effect on human health. When

such compounds are produced in excess they generate a phenomenon commonly termed as

oxidative stress. Free radicals are deficient in electrons and get attracted to electron-rich sources

in the cell. Deoxyribonucleic acid (DNA), proteins and lipids have a high nucleophilic potential

to react with these free radicals to form stable bonds and cause oxidative and structural damage

(Fig 2.3). Oxidative damage to proteins leads to structural changes and loss of enzymatic

activity. Damage to DNA produces oxidative DNA lesions which can cause mutations (Halliwell

and Gutteridge, 1985). If this process is not regulated they can cause several chronic and

degenerative diseases, for example, several forms of cancer, cardiovascular (CVD), neurological

and pulmonary diseases which are induced by free-radical-generated oxidative stress (Halliwell,

1994).

R + DNA, RNA, Proteins, Lipids

Tissue Damage Cancer, CVDs, Aging, etc.

Fig 2.3 Deleterious activity of free radicals and oxidants on human health

2.2 Antioxidants

It is well known that the food industry employs several measures to combat lipid

oxidation of food products, including the use of food-grade synthetic antioxidants like butylated

hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate (PG, Propyl 3,4,5-

trihydroxybenzoate) and tert-butylhydroquinone (TBHQ, 2-(1,1-Dimethylethyl)-1,4-

benzenediol). Antioxidants are termed as any substance that prevents or delays oxidation of a

substrate when present in lower concentration than that of the oxidizable substrate (Shahidi,

2000a; Valenzuela and Nieto, 1996).

Reaction of an antioxidant (AH) and free radicals (Fig 2.4), generated during lipid

oxidation, is a complex process.

R +AH RH + A

RO + AH ROH + A

11

ROO + AH ROOH + A

RR + A RA

RO + A ROA

Fig 2.4 Antioxidant (AH) reaction with several types of free radicals generated during lipid

oxidation (Adegoke et al., 1998; Dziezak, 1986)

It might be expected that if these antioxidants were present at adequate levels, they could totally

prevent lipid oxidation; however, this is not possible as the reaction between free radicals and

antioxidants not only depends on the type of lipid under investigation but also on the type of

oxidative products generated during the process.

It is useful to understand the structure of the antioxidants as it governs their functionality.

The tertiary butyl groups of BHT and BHA molecules produce steric hindrance which suppresses

their free radical inhibitory property and electron release mechanism (Sherwin, 1972; Shahidi,

2000b). Varieties of free radicals are formed during the oxidation process and no evidence is

available to suggest that any one antioxidant can intercept and destroy all the radicals generated.

It would be wise to use a combination of antioxidants for retarding lipid oxidation in food as they

can act synergistically to enhance the oxidative stability of the product (Frankel, 1996).

In a series of experiments Frankel (1996) concluded that interfacial phenomena play an

important role in the action of antioxidants in food and biological systems. The great majority of

food products exist as emulsions, where the interaction of antioxidants depends not only on their

solubility but also on the structure of the antioxidants. Natural antioxidants when present in

adequate amounts have been shown to exhibit complex interfacial affinity between air-oil and

oil-water interfaces (Frankel, 1996). Frankel also observed that in food systems of low surface to

volume ratio (e.g. bulk vegetable oils) polar antioxidants (hydrophilic) are more effective than

non-polar lipid bound (lipophilic) antioxidants as they accumulate at the air-oil interface where

oxygen concentration and relative oxidation reaction rates are higher (Fig 2.5). Lipophilic

antioxidants are more effective in food systems of high surface to volume ratio (e.g. emulsified

oil) and tend to accumulate at oil-water interface (Fig 2.5) to protect oil against oxidation.

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Comparatively, in the same system hydrophilic antioxidants move into the water phase and get

too diluted to provide adequate protection from oxidation to the oil (Frankel, 1996; Frankel et al.,

1994).

Fig 2.5 Interfacial phenomenon of hydrophilic and lipophilic antioxidants in bulk oil and

oil-in-water emulsion system (Frankel, 1996)

2.2.1 Synthetic Antioxidants

Currently the food industry uses synthetic antioxidants widely to extend the shelf-life of

various food products. The most commonly used synthetic antioxidants are BHT, BHA, PG and

TBHQ (Fig 2.6). Due to their proven effectiveness and low cost these antioxidants are preferred

over natural antioxidants. Their efficiency and stability in several lipid systems is based on their

structure and ability to donate a hydrogen atom from their aromatic hydroxyl group to a free

radical and also on their ability to support an unpaired electron due to delocalization. Both BHT

and BHA are hydrophobic phenolic antioxidants which makes them suitable for oil-water

emulsion systems. They are commonly used in combination with PG which is more effective in

intercepting free radicals due to its polyhydroxyl structure (Sherwin, 1972). The polyhydroxyl

group of PG makes it water soluble and unfit for water-oil emulsion; a combination with BHT

and BHA provides synergistic effects and improves the overall free radical intercepting

efficiency.

13

Fig 2.6 Chemical structure of synthetic antioxidants (Shahidi, 2000)

Recently the use of TBHQ has increased in the food industry as it is less volatile and can

endure the high temperatures food products are subjected to during processing (Schmidt and

Pokorn, 2005). However current consumer interest is in natural products because of proven

harmful effect of synthetic antioxidants in food. Food processors are moving towards avoiding

addition of synthetic antioxidants to foods and are interested in supplying all natural products

for consumers (Zia Ur et al., 2004).

2.2.2 Natural Antioxidants

Stabilization of food products against oxidative rancidity using natural antioxidants has been

applied for decades. When synthetic antioxidants came on the market they soon replaced natural

antioxidants as they were cheap, consistent and had a greater antioxidant capacity. But gradual

change in consumer preference and food safety legislations has made the use of synthetic

antioxidants more complex, time consuming and expensive. Comparatively, natural antioxidants

are known to be safe by the perception of consumers and legislators as they are derived from

unadulterated plant materials. Natural antioxidants including tocopherol (Vitamin E),

14

tocotrienols, sesamol, phospholipids derived from oils and oilseeds, phenolic compounds,

ascorbic acid, flavonoids, extracts from herbs and

spices like rosemary (Rosmarinus officinalis L.), thyme (Thymus vulgaris L.) have been proven

to be equally effective in many food systems (Pokorn, 1991).

Natural antioxidants not only provide protection against lipid oxidation, but have potential health

benefits. They prevent a number of chronic and degenerative diseases such as CVDs, cancer,

Alzheimers and Parkinsons diseases (Chu et al., 2002; Chung et al., 1999). Most readily-

available antioxidants are common food ingredients (Table 1). Effectiveness of natural

antioxidants over synthetic ones has been a topic of debate for decades. Several researchers have

proved that natural antioxidants are as effective as synthetic ones when added to food products.

Their antioxidant activities depend very much on the food to which they are added, their

concentration, availability of oxygen and presence of heavy metals and various synergists like

citric acid and amino acids (Pokorn, 1991 , 2007). Therefore it is not possible to state

which type of antioxidant is most effective as they both act through similar mechanisms.

Table 2.1: Sources of some natural antioxidants (Adapted from Pokorn, 1991)

Sources Oxidation inhibitors

Oils and oilseeds Tocopherols and its derivatives; olive oil resins; phospholipids

Oats and rice brans Various lignin-derivatives

Fruits and vegetables Ascorbic acid; hydroxycarboxylic acids; flavonoids; carotenoids; phenolic compounds

Spices and herbs Phenolic compounds

Proteins and protein hydrolysates

Amino acids; dihydropyridines

Estvez et al. in 2006 studied the antioxidant effect of plant essential oils (sage (Salvia

officinalis L.) and rosemary (Rosmarinus officinalis L.)) and BHT on refrigerated stored porcine

liver pt. They analyzed the liver pt for protein oxidation and modification of hme and non-

hme components. They observed that the addition of rosemary essential oils significantly

reduced hardness of liver pts. They also reported that both rosemary and sage essential oils

exhibited similar antioxidant properties as BHT (Estevez et al., 2006). Freeze-dried extract from

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15

potato peels and other plant materials have been used as antioxidants for prevention of oxidation

in meat products (Mansour and Khalil, 2000; Kanatt et al., 2005) and soybean (Glycine max (L.))

oil (Zia Ur et al., 2004). Mansour and Khalil while investing the antioxidant activity of several

plant extracts observed a relation between pH and rancid odour and addition of plant extract.

Antioxidant activity of plant extracts decreased with increase in pH and products containing

plant extracts had a lower rancid score than products without any antioxidants. Extracts from

rapeseed (Brassica napus L.) and pine (Pinus spp.) bark have also proven to be a good sources of

phenolic compounds and suitable for application at industrial scale for prevention of lipid

peroxidation (Vuorela et al., 2005).

2.2.2.1 Polyphenols

It is generally assumed that an increased consumption of fruits and vegetables are

associated with a lower risk of chronic and degenerative diseases such as cancer, CVDs, cataract

and several immunological disorders (Ames et al., 1993; Vinson et al., 1998). Several

researchers reported a negative association between intake of total fresh fruits and vegetables and

ischemic heart diseases and cerebrovascular disease mortality (Armstrong et al., 1975;

Verlangieri et al., 1985; Acheson and Williams, 1983). The protection that fruits and vegetables

provide against diseases has been attributed to the various antioxidants contained in them. A

major portion of these antioxidants are phenolics and polyphenols (Ames, 1983; Steinberg et al.,

1989; Steinberg, 1991). These compounds are also abundant in agricultural by-products such as

potato peels, hulls, roots, leaves, grape seeds, peanut skins and in number of herbs and spices.

Polyphenols are secondary plant metabolites and are made up of several classes of compounds

including phenolic acids, anthocyanins, catechins and flavonoids (flavones, isoflavones,

flavanones); these compounds confer both desirable and undesirable food qualities. The

antioxidant activities of these plant metabolites is mainly a result of their redox properties, which

allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers (Kaur and

Kapoor, 2001; Evans, 1997). Phenolic acids (Table 2) consist of two subgroups, i.e. the

hydroxybenzoic and hydroxycinnamic acids (Fig 2.7). Hydroxybenzoic acids include gallic acid,

p-hydroxybenzoic acid, protocatechuic acid, syringic acid and vanillic acids, which have a

common C6-C1 structure. Hydroxycinnamic acids include caffeic, ferulic and p-coumaric acid

http://en.wikipedia.org/wiki/Carolus_Linnaeushttp://en.wikipedia.org/wiki/Carolus_Linnaeus

16

and sinapic acids, these are aromatic compounds with a three carbon side chain (C6-C3)

(Balasundram et al., 2006; Moyer et al., 2002).

Table 2.2: Different classes of phenolic acids in plants (Balasundram et al., 2006)

Phenolic acid compounds in plants

Classes Structure

Simple phenolics C6

Hydroxybenzoic acids C6-C1

Phenylacetic acids C6-C2

Hydroxycinnamic acids C6-C3

Napthoquinones C6-C4

Xanthones C6-C1-C6

Anthraquinones C6-C2-C6

Flvanoids, isoflavonoids C6-C3-C6

Lignans (C6-C3)2

Biflavonoids (C6-C3-C6)2

Lignins (C6-C3)n

Tannins (proanthocyanidins) (C6-C3-C6)n

Fig 2.7 Chemical structure of a) hyroxybenzoic b) and hydroxycinnamic acids

(Balasundram et al., 2006)

17

Flavonoids are the largest group of plant phenolics. Their structure consists of fifteen carbon

atoms arranged in a C6-C3-C6 configuration (diphenylpropane skeleton). The structure consists of

two benzene rings (A and B) connected to a third heterocyclic ring (C) by a 3-carbon bridge (Fig

2.8)(Havsteen, 1983; Balasundram et al., 2006).

Fig 2.8 Basic structure of a flavonoid molecule (Balasundram et al., 2006)

Variation in the substitution on ring C results into major classes of flavonoids such as

flavonols, flavones, flavanones, isoflavones and anthocyanidin (Fig 2.9). Isoflavones have been

shown to provide protection against estrogen-related cancers (e.g., breast, ovarian, prostate and

colon), while other flavonoids have been implicated in antiproliferative effects in human

intestinal and lung cancer cells (Arai et al., 2000; Wattenberg, 1992; Dziedzic and Hudson,

1983). Flavonols (catechin and catechin gallate esters) are among the most potential polyphenol

used to combat carcinogenesis. Green tea extract contains high concentration of catechin and has

been found to act as anti-carcinogenetic antioxidants at all stages of carcinogenesis (Dreosti,

2000; Blot et al., 1996; Chen et al., 1998). Polyphenols have also been shown to decrease

formation of atherosclerotic plaques, reduce arterial stiffness and block low density lipoprotein

(LDL) oxidation, which helps in prevention of heart strokes and related cardiovascular diseases

(Arai et al., 2000).

Polyphenols are ubiquitous within the plant kingdom. Their concentration in plants can

vary by many orders of magnitude under the influence of factors such as season, age, climate and

postharvest storage practices. This suggests that better processing and storage can improve the

levels of these compounds and also the marketability of the produce.

18

Fig 2.9 Structure of major classes of flavonoids (Balasundram et al., 2006)

2.2.2.2 Carotenoids

Carotenoids are another set of phytochemicals found in abundance in fruits and

vegetables. Carotenoids like polyphenols exhibit activity in protecting from oxidative damage,

increasing metabolic detoxification and inhibiting cancer initiation (Khachik et al., 1995). These

compounds have a polyene structure which allows them to absorb light and intercept free

radicals (Britton, 1995). This class of phytochemicals includes lycopene, -, -, and -carotene,

lutein, zeaxnthin, neoxanthin and viloxanthin. Their antioxidant activity in plants and several

food products depends on the stability of intermediate formed during the interception of free

radicals produced from the interaction of light and photosensitizers (for example, chlorophyll)

(Ames, 1983). Their importance in human health is significant, they have been proved to provide

protection against tumour initiation and also against proliferation of already initiated cells. In

human eyes lutein and zeaxanthin are present in the centre of the retina and have are involved in

the prevention of retinal damage due to aging and also prevention against cataract formation

(Seddon et al., 1994).

19

2.2.2.3 Agricultural residues as sources of natural antioxidants

Growing interest in finding natural substitutes to synthetic food antioxidants has led

many investigators to look for appropriate sources of these antioxidants (Peschel et al., 2006).

Until now most attention has been paid to oral administration of natural antioxidants like green

tea extract as health supplements or salvia and rosemary extracts as food additives

., 2001; Karpiska et al., 2000). Fruits and vegetables have been used to extract

these phytochemicals for commercialisation. The number of studies on the use of agricultural

residues as a source of antioxidants has risen considerably, given the interest in deriving added

value from recyclable agro- and food industry wastes (Peschel et al., 2006). Recycling of several

agricultural and industrial byproducts such as peels and agro-industry waste water containing

high concentrations of antioxidants supports the idea that agro-food industry wastes can be used

as alternative sources for the extraction of phytochemicals.

Phenolic compounds with antioxidant activity have been identified in several agricultural

by-products, such as grape (Vitis vinifera L.) and apple (Malus domestica Borkh.) peels, rice

(Oryza sativa L.) hulls, almond (Prunus dulcis (Mill.)) hulls, banana (Musa sapientum L.) peels

etc.. Grape by-products consists mainly of peels and seeds, containing a high amount of

secondary metabolites including phenolic acids, flavanols and anthocyanins (Corrales et al.,

2006). The abundance of these phytochemicals has been documented by a number of studies

(Bonilla et al., 1999; Corrales et al., 2008; Kanner et al., 1994). Grape seeds and skin contain

high concentrations of proanthocyanadins (Koga et al., 1999). It is estimated that the phenolic

content of grape skin ranges from 285-550 mg kg-1

skin depending on the grape variety and type

of extraction technique used (Pinelo et al., 2005a; Pinelo et al., 2005b). Researchers have also

reported that total phenolics in peels of lemon (Citrus limon (L.)), oranges (Citrus sinensis

(L.)) and grapefruits (Citrus paradisi) were 15% higher than in peeled fruits (Gorinstein et al.,

2001). Peels of apple, peaches (Prunus persica (L.)) and pears (Pyrus spp. L.) have been shown

to contain twice the amount of phenolics than the peeled fruits (Gorinstein et al., 2002). Olive

(Olea europaea L.) mills waste consists mainly of aqueous phase wastewater or solid phase

pomace (Rodis et al., 2002). Visioli and Galli reported that depending on the variety and

processing, olive oil mill waste contains 1.0% to 1.8% of total phenols by weight. Peels and

seeds of tomatoes (Solanum lycopersicum L.) have also been reported to be a valuable source of

20

antioxidant phenolics. They have been found to be a richer source of phenolic compounds then

the fleshy pulp. Phenolic content (expressed in terms of mg catechin/100g, fresh weight) of

tomato pulp ranges from 9.2 to 27 mg/100g and that of peels ranges from 10.4-40 mg/100g.

Phenolic content of several other agricultural by-products are listed in Table 2.3.

Table 2.3: Phenolic content of agricultural by-products

By-product phytochemicals Concentration (mg/g dry weight

(dw) or mg/g fresh weight (fw)

References

Apple peals flavonoids ,

anthocyanin

2299 52 mg CEa/ 100g dw,

169.7 1.6 mg CGEb/ 100g dw

(Wolfe and Liu,

2003)

Grape seeds flavanols 199mg/ 100g dw (Gonzlez-Params

et al., 2003)

Peanut roots resveratrol 130mg/ 100g dw (Chen et al., 2002)

Grape

cane

trans-resveratrol

trans-viniferin

345 mg/ 100g dw

130 mg/ 100g dw

(Rayne et al., 2008)

Potato peel chlorogenic acid,

caffeic acids

279 mg/ 100g dw,

52mg/100g dw

(Al-Weshahy and

Venket Rao, 2009)

Almond hulls chlorogenic acid,

4-O-caffeoylquinic acid, 3-O-

caffeoylquinic acid

42.52 4.50 mg/100g fw,

7.90 mg/ 100g fw,

3.04 mg/ 100g fw

(Takeoka and Dao,

2003)

sweet potato

leaves

flavonoids 185.01 mg/ kg (green leaves),

426.82 mg/kg (purple leaves)

(Chu et al., 2000)

a CE: catechin equivalents,

b CGE: cyanidin 3-glucoside equivalent

2.3 Extraction of phytochemicals from plant materials

The search of natural sources of antioxidants has led many research groups to place their

focus on the extraction of antioxidants from plant and agro-industrial by-products. The extraction

process is one of the most important unit operations in the agro-food industry. Compounds

obtained from the process may be used as food additives or as nutraceuticals (Ames, 1983;

Moure et al., 2001).

21

At present extraction is carried out using traditional methods including solid-liquid

extraction, Soxhlet extraction and liquid-liquid extraction. Disadvantages associated with these

methods are high solvent consumption, risk of thermal degradation of heat-labile components,

and longer extraction times. Soxhlet is the most widely used conventional method of extraction

at the lab scale; however, industrial application of this process show low compound throughput.

Several novel extraction techniques have been sought to completely remove or reduce the

disadvantages associated with conventional methods. Some of these novel methods include

microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), ultrasound-assisted

extraction (UAE) and accelerated solvent extraction (ASE).

Extraction of phytochemicals from agricultural wastes requires the process to be cost-

effective and efficient. Since the yield of antioxidant compounds from plant materials is

influenced mainly by the conditions under which the process is carried out, one must optimize

the extraction process. A few of the most commonly used conventional and novel extraction

techniques are discussed below.

2.3.1 Solid-liquid extraction process

Solid-liquid extraction (SLE) or leaching is an un-steady mass transfer operation which

involves removal of the desired component from a solid matrix using an organic solvent capable

of dissolving the solute (Pinelo et al., 2006). A series of steps are required for the mass transfer

of solutes from the solid matrix to the solvent: (a) penetration of solvent into the solid matrix, (b)

solubilization of components, (c) transfer of solute to the exterior of the solid matrix, (d)

migration of the solute from the solid surface into the bulk of the solvent (Fig 2.10)

(Giergielewicz-Moajska et al., 2001).

22

Fig 2.10 Schematic representation of individual steps in process of extraction

Since the extraction process depends on physical and chemical characteristics (e.g.,

viscosity) of the solvent as well as the structure (surface area) and composition of the solid

matrix; the interaction between these factors is unpredictable (Pinelo et al., 2004). Temperature

also plays an important role in the extraction process. Increase in temperature increases the

solubility of the solutes, but most heat-labile phytochemicals are lost due to this process.

The SLE process has been used in several studies for the extraction of phytochemicals

from plant materials. The most commonly-used organic solvents in this process include

methanol, ethanol, water or combinations of these. Choosing the solvent is one of the most

important steps in this operation, and depends on several factors previously mentioned, including

extraction temperature and how fast the compound is dissolved and its equilibrium in the liquid

is reached. The SLE process suffers from several disadvantages including high solvent

consumption, increased extraction time and poor solute yield.

23

2.3.2 Accelerated solvent extraction

Accelerated solvent extraction (ASE) also known as pressurized fluid extraction (PFE)

uses high temperatures (40-200C) and pressures (3.3-20 MPa). High temperatures weaken the

molecular interactions between solute and solid matrix and thus increase the mass transfer of

solute into the bulk of the solvent; it also reduces the viscosity and surface tension of the solvent

improving its penetration into the solid matrix. High pressure is maintained during the process to

retain the boiling solvent in a liquid state and push the solvent into matrixs micro-pores,

increasing the accessibility of the solute to the solvent (Fig 2.11)(Giergielewicz-Moajska et al.,

2001).

ASE has been utilized in several studies for the extraction of phytonutrients from plant matrices;

lipids from corn (Zea mays L.), oat (Avena sativa L.) and egg-containing foods, carotenoids from

processed foods, xanthones and flavanones from root bark, tocopherols from seeds and nuts,

antioxidants from microalgae and anthocyanins from dried red grape skin (Giergielewicz-

Moajska et al., 2001; Ibaez et al., 2006; Kawamura et al., 1999; 2006).

Fig 2.11 Schematic of Accelerated Solvent Extraction instrument (Turner, 2006)

24

2.3.3 Supercritical fluid extraction

A liquid or gas becomes supercritical when the temperature and pressure are increased

beyond their critical points (Tc, Pc) (Fig 2.12). At the critical point the liquid and gas phase of a

substance are indistinguishable and it exhibits properties of both liquid and gas (Perretti, 2006).

Several supercritical fluids can be used for extraction process, but CO2 is the most commonly

utilized because of its low toxicity, low critical temperature and pressure (31C and 7.3 MPa). It

is also an attractive extraction medium due to its high diffusivity into solid matrices, and given

that it is gaseous at room temperature and ambient pressure, it is easily recycled after the solute

dissolved in it is recovered by precipitation upon depressurization (Turner, 2006; Perretti, 2006).

In the supercritical fluid extraction (SFE) process, the selectivity of the solvent towards a

specific solute can be adjusted by making small adjustments in pressure. Variation in pressure

alters the density of the fluid. A compounds solubility increases with the fluids increasing

density (Turner, 2006; Cavero et al., 2006; Zougagh et al., 2004).

Fig 2.12 A typical phase diagram (Turner, 2006; D. Steytler, 1996)

25

Given SFEs ability to control the solubility and selectivity of solutes for extraction of

phytochemicals from several natural resources, this process has been widely studied. The SFE

process is conducted in the absence of oxygen and light, which provides an added advantage

over conventional methods. SFE has been used for extraction of vitamin E from wheat [Triticum

stivum L.] germ (Ge et al., 2002), phenolics from number of products like grape seeds (Palma

et al., 2000), cranberry [Vaccinium macrocarpon Ait.] seeds (Bhagdeo et al., 2006), lycopene

from tomato by-products (Rozzi et al., 2002), rosemary and sage (Djarmati et al., 1991).

Methanol has been used as co-solvent by several authors to extract phenolics as supercritical

fluids are not suitable for polyphenolic extraction. Methanol enhances the solubility of polar

compounds into the supercritical solvent by introducing stronger molecular interactions between

them (Palma et al., 2000). The yield obtained by SFE process is considerably higher than by

conventional methods, but equipment setup and initial investment costs are very high, making

the process undesirable for industrial use at present.

2.3.4 Microwave-Assisted extraction

Microwaves are a form of non-ionizing electromagnetic radiation with frequencies

ranging from 300 300 000 MHz. Microwaves are made up of two oscillating perpendicular

fields, one magnetic and one electric (Vivekananda Mandal, 2007). The oscillation fields cause

molecular motion (ionic conduction) and migration of ions and dipole rotations. Ionic conduction

is the electrophoretic migration of ions such as salts under an alternating electric field. The

resistance provided by the solution to the migration of ions generates friction, which eventually

leads to heating of the solution (Vivekananda Mandal, 2007). Many molecules exist as electric

dipoles i.e. they have a negatively and positively charged ends. When placed in an

electromagnetic field these dipoles attempt to orient themselves according to the polarity of the

field. At an electromagnetic frequency of 2450 MHz, that which is used in commercial systems,

the electric component of the wave changes at a rate of 4.9 104 sec

-1. At this speed a dipole

fails to realign itself and starts vibrating, which generates heat due to friction (Eskilsson et al.,

2006; Camel, 2000, 2001).

The ability of a material to interact with microwaves is dependent on the dielectric

properties of the material: the dielectric constant (') and dielectric loss (''). The ' is the measure

26

of the materials ability to absorb microwave energy and the '' represents the efficiency of

converting microwave energy into heat. The relationship between ' and '' is given by:

'' = ' tan (2.1)

where tan is the dissipation factor or loss tangent, which represents the ability of the material to

absorb microwave energy and pass it on in the form of heat to other molecules. Thus, both '' and

tan determine the amount of heat that will be generated when a solvent is subjected to

microwave. A list of the organic solvents most widely used in microwave-assisted extraction

process (Table 2.4), shows that both ethanol and methanol will absorb fewer microwaves as

compared to water but will dissipate more energy into heat. Non-polar solvents like acetone and

hexane have no dissipation factor value as they are transparent to microwaves.

Table 2.4: Dielectric constant and dissipation factors for organic solvents widely used in

MAE processes (Vivekananda Mandal, 2007)

Solvent Dielectric Constant at 20C Dissipation factor (tan )

Acetone 20.7

Acetonitrile 37.5

Ethanol 24.3 0.25

Hexane 1.89

Methanol 32.6 0.64

2-propanol 19.9 0.67

Water 78.3 0.16

In MAE the samples being processed are subjected to microwave energy using two

technologies (Fig 2.13): closed vessels (under controlled pressure and temperature) and open

vessels (at atmospheric pressure) (Camel, 2000; Letellier and Budzinski, 1999). Both systems

have been shown to have similar efficiencies in extraction of several analytes from plant and soil

samples (Saim et al., 1997). In open system the maximum temperature that can be reached is

determined by the boiling point of the solvent or solvent combination used, whereas in a closed

system temperature it can be elevated by applying correct pressure, similar to the case of a

pressurized fluid extraction system (Richter et al., 2001; Venkatesh and Raghavan, 2004).

27

Fig 2.13 Schematic of different MAE systems (Camel, 2001)

Conventional solvent extraction methods rely on conductive and convective processes to heat the

sample, where as microwave heating occurs by direct energy transfer to the sample (Fig 2.14)

(Venkatesh and Raghavan, 2004; Nemes and Orsat, 2010). Microwave heating is known to be

volumetric in nature so microwave irradiation produces efficient internal heating by coupling

microwaves with polar components inside the solvent and the sample.

28

Fig 2.14 Heating principle of conventional and microwave-assisted extraction processes

(Kaufmann and Christen, 2002)

Ganzler et al. (1986) used a microwave-assisted extraction process for the extraction of vicine

and convivine from faba (Vicia faba L. beans, and showed this process to outyield the

conventional Soxhlet extraction process by 20% , 1987; Ganzler et al., 1986).

After its initial success, MAE process has been widely used for extraction of phytonutrients from

natural sources and pollutants from environmental samples. Recently MAE has been widely used

in extraction of phenolics from agro-industrial waste samples such as pectin from apple pomace

(Wang et al., 2007), phenolics from citrus mandarin (Citrus reticulata) peels (Hayat et al., 2009),

peanut skin (Ballard et al., 2010), longan peel (Pan et al., 2008), solanesol from potato leaves and

stems (Chen et al., 2001), flavonoids from the Chinese herb Radix puerariae (Wang et al., 2010),

Capsaicinoids from capsicum (WILLIAMS et al., 2004). Almost all the researchers reported that

MAE process provided significantly higher yield than conventional methods of extraction.

Microwave heating also allows reduction in the volume of solvent and sample needed for the

extraction, thus being cost effective and efficient (Table 5).

http://en.wikipedia.org/wiki/Carolus_Linnaeus

29

Table 2.5 Comparison of MAE and other extraction processes (Eskilsson and Bjorklund, 2000)

30

2.4 Conclusion

Consumer interest and health concerns over use of synthetic antioxidants in food industry

to combat lipid oxidation, has led demand for natural antioxidants to sky-rocket in recent

years. Phenolic antioxidants derived from agricultural wastes, such as potato peels and

other by-products can be viable alternative to synthetic antioxidants. A novel cost-

effective and energy-efficient method needs to be developed to improve upon the

conventional methods and make these agricultural by-products an attractive source with

potential commercial appeal.

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