i

Characterization of Hass and Fuerte Avocado (Persea americana) Varieties Grown

in Wondogenet and Optimization of Some Oil Extraction Parameters

By Kenbon Beyene

Advisor: Dr.Eng. Shimelis Admassu (Assoc. Prof.)

A Thesis Submitted to

School of Chemical and Bioengineering

Presented in Partial Fulfillment of the Requirements for the Degree of Master of

Science in Chemical Engineering (Food Engineering Stream)

Addis Ababa University

Addis Ababa, Ethiopia

November, 2017

ii

Addis Ababa University

Addis Ababa Institute of Technology

School of Chemical and Bioengineering

This is to certify that the thesis prepared by Kenbon Beyene, entitled “Characterization of Hass

and Fuerte Avocado (Persea americana) Varieties Grown in Wondogenet and Optimization of its

Oil Extraction Conditions” and submitted in partial fulfilments of requirement for the degree of

masters of Science (Food Engineering) complies with the regulation of the university and meet

the accepted standard with respect to originality and quality.

Kenbon Beyene

Submitted by Signature Date

Approved and Signed by the Examining Committee

Dr. Eng. Shimelis Admassu (Assoc. Prof.)

Advisor Signature Date

Dr. Solomon Kiros

Internal Examiner Signature Date

Eng. Gizachew Shiferaw

External Examiner Signature Date

Dr. Eng. Abubeker Yimam (Assoc. Prof.)

Chairperson, School’s graduate committee Signature Date

iii

Acknowledgements

First and foremost I am pleased to thank the “Almighty God”, the source of all knowledge and

wisdom, for his guidance and blessing, for helping me start, pursue and successfully complete

my studies. “In whom are hid all the treasures of wisdom and knowledge”, Col 2:3. Next, I

would like to thank all the people and organizations who contributed to this thesis.

I am very grateful to my advisor, Dr. Eng. Shimellis Admassu, for his constant support,

encouragement, unreserved guidance and constructive suggestions and comments from the

stage of proposal to this end. He made sprit of research enthusiasm and zeal in me.

Also, I express my gratitude thanks to Birhane G. for having given me an opportunity to

work on interesting projects and for his support and encouragement.

All the technical help especially Debebe, Yemane and Woineshet, thank you for helping me

during laboratory experiments. And also all my colleagues’ thank you for all the fun we had

while struggling through all the two years life.

Wondogenet agricultural research center, thank you for providing/allowing me the raw

material/ avocado (especially Basaznew and Wondimu).

Food Science and Nutrition laboratory of Addis Ababa University, Bless agri-food

laboratory, Human nutrition laboratory of Ethiopian Public Health Institute, for providing me

conducting the laboratory experiments.

Also, I would like to acknowledge School of Chemical and Bio-Engineering of Addis Ababa

University for acceptance and training me in the program, and to all the staff members of the

school for helping me in my effort to be fruitful.

Finally, I extend my heartfelt gratitude to my parents and siblings for all the love and

support, especially my great brother, “Yadeta” you are a star for me. Also my darling fiancée

J.G thanks for all your love and endless support, putting me and the completion of this thesis

first.

iv

Abstract

The purpose of this research was to determine the physicochemical characteristics, proximate,

mineral and the phytochemical composition of pulp and seed of two avocado cultivars, namely

Fuerte and Hass, produced in Wondogenet, Ethiopia with respect to the unripe and ripe.

Furthermore, extraction of oil from Hass avocado pulp using soxhlet and ultrasonic extraction

and some process parameter optimization were carried out. The percentage oil yield, analysis of

chemical properties and fatty acid profile of extracted oil were done. The basic characteristics

evaluated were weight (of the fruit, pulp, seed, peel and seed coat), Dry matter (DM), Total

soluble solid (TSS), pH, Titratable acidity (TA), Moisture content (MC), ash, total lipid, fatty

acid composition, protein and minerals. The concentrations of phytochemicals (total phenolics,

flavonoids, tannin, and phytate) were also analysed. Hass avocado cultivar showed greater flesh

to seed ratio (13.57±10.43) and had lower fruit weight (183.92±47.58g) than Fuerte cultivar. The

highest pulp MC (77.82±0.19%), protein (2.92±0.04%), ash (0.42±0.01%), fat (22.80±0.00%)

and crude fiber (4.50±0.09%) corresponds to UHP except MC which is to UFP. The analyses of

the nutritional compositions demonstrated that the pulp of the Hass proved to be rich in ash,

fiber, total lipid, protein and potassium content than fuerte pulp. The seed, in turn, had higher

DM (48.4±1.02%), TA (2.88±0.02%), Mg (2.01 ±0.02%), Na (12.67±0.01%), and Ca (24.77±0.02)

mg/100g, wet basis contents than its pulp. With regard to the contents of phytochemicals, the

seed was superior to the pulp. The effects of three factors (particle size, solvent to sample ratio

and extraction time) on the oil yield were considered. The optimum extraction conditions for

soxhlet extraction were found to be at 8 h reaction time, particle size of 1.4mm and solvent to

solid ratio of 20:1 with a maximum yield of 69.7%, while the optimum extraction conditions for

ultrasonic extraction were found at 1.5 h reaction time, particle size of 2mm and solvent to solid

ratio of 15:1 with a maximum percentage yield of 67.2%. The analyses of the oil characteristics

demonstrated that the oil extracted by soxhlet extraction shows lower PV, FFA, MVM and

Alkalinity than oil extracted using Ultrasonic extraction. The results of fatty acid profile

displayed that the oleic and linoleic acid contents of the soxhlet extracted oil were higher than

that of the Ultrasonic extracted oil while palmitic and butanoic acid were higher in ultrasound

extracted oil. Generally, Hass avocado variety was found to be better interms of pulp to seed

ratio, proximate composition and minerals. Ultrasound assisted extraction showed a promising

oil yield regarding short extraction time and low solvent consumption; but its oil quality is lower

than that of soxhlet extracted oil.

Keywords: avocado, cultivar, fatty acid, peroxide value, ripening, soxhlet, ultrasonic

v

Table of Contents

Page

Acknowledgements iii

Abstract iv

List of Figures ix

Acronyms x

CHAPTER ONE 1

1. Introduction 1

1.1. Background 1

1.2. Statement of the problem 3

1.3. Objective of the research 4

1.3.1. General objective 4

1.3.2. Specific objectives 4

1.4. Significance and scope of the study 5

CHAPTER TWO 6

2. Literature Review 6

2.1. Overview of Avocado 6

2.1.1. The Avocado Fruit and its seeds 6

2.2. Global Avocado Production 9

2.2.1. World avocado production 9

2.2.2. Avocado production in Africa 10

2.2.3. Avocado production in Ethiopia 11

2.3. Avocado quality characteristics 13

2.3.1. Physical properties 14

2.3.2. Sensory properties 16

2.3.3. Chemical properties 16

2.4. Nutritional value, health benefits and uses of avocado 19

2.5. Phytochemicals composition 21

2.6. Avocado oil processing methods and oil characteristics 25

2.6.1. Avocado oil processing methods 25

2.6.2. Factors that influence avocado oil yields 31

vi

2.6.3. Avocado oil characteristics 32

2.7. Concluding remarks 33

CHAPTER THREE 35

3. Materials and Methods 35

3.1 Materials 35

3.1.1 Raw Material collection, transportation, storage and sample preparation 35

3.2. Framework of the thesis 36

3.3. Processing Methods 37

3.3.1. Extraction of oil using soxhlet Extraction 37

3.3.2. Extraction of oil using Ultrasound Assisted Extraction 37

3.4. Analytical Methods 38

3.4.1. Physicochemical analyses 38

3.4.2. Proximate Composition Analyses 39

3.4.3. Mineral Composition Analyses 42

3.4.4. Phytochemical Analyses 43

3.4.5. Quality characterization of extracted avocado oil 45

3.5. Experimental Design and Statistical Data Analysis 48

CHAPTER FOUR 50

4. Results and Discussion 50

4.1. Physical properties of avocado 50

4.2. Chemical properties of avocado pulp and seed 50

4.3. Proximate composition of avocado pulp and seed 53

4.4. Mineral composition of avocado pulp and seed 56

4.5. Phytochemical composition of avocado pulp and seed 59

4.6. Yield of avocado oil extracted using soxhlet and ultrasound assisted extraction 61

4.6.1. Effect of raw material particle size on oil yield 65

4.6.2. Effect of solvent to solid ratio on oil yield 66

4.6.3. Effect of extraction time on oil yield 67

4.6.4. Interaction effect of solvent to sample ratio and extraction time on oil yield 68

4.7. Quality characterization of the extracted avocado oil 70

CHAPTER FIVE 75

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5. Conclusions and Recommendations 75

5.1. Conclusions 75

5.2. Recommendations 76

References 77

Appendixes 90

viii

List of Tables

Page

Table 2.1 Fruit characteristics of different avocado cultivars 8

Table 2.2 Composition of Avocado seed 9

Table 2.3 Africa top 10 avocado producing countries (2008-2012, in mt) 11

Table 2.4 Summary of major fruit crops produced in Ethiopia in 2012/2013 cropping season 13

Table 2.5 Nutritional profile of US-grown avocados and avocado oil (per 100 g) 21

Table 4.1 Physical properties of avocado 50

Table 4.2 Chemical properties of avocado pulp and seed 53

Table 4.3 Proximate composition of avocado pulp and seed 56

Table 4.4 Mineral composition of pulp and seed of avocado variety in wet basis 58

Table 4.5 Phytochemical composition of pulp and seed of avocado variety in wet basis 61

Table 4.6 Actual and predicted oil Yield value by soxhlet extraction 63

Table 4.7 Actual and predicted oil Yield value by ultrasound extraction 64

Table 4.8. Quality characteristics of oil extracted by UAE and Soxhlet Extraction 72

List of Tables under Appendixes

Appendix A1 ANOVA for Response Surface Quadratic model of oil yield from ultrasound 90

Appendix A2 ANOVA for Response Surface Quadratic Model of oil yield from soxhlet 91

ix

List of Figures

Page

Figure 2.1 World avocado production trend (2000 - 2012) in '000mt 10

Figure 2.2: Proposed structures of phytic acid. Source: Reddy (2002) 23

Figure 3.1 Frameworks of Experiments 36

Figure 4.1 One Factor Plot of effect of Particle Size on oil yield for (a) ultrasonic extraction (b)

soxhlet extraction 66

Figure 4.2 One Factor Plot of effect of solvent to sample ratio on oil yield for (a) ultrasonic

extraction and (b) soxhlet extraction 67

Figure 4.3 One Factor Plot of effect of extraction time on oil yield for (a) ultrasonic extraction

and (b) soxhlet extraction 68

Figure 4.4 graph of effect of solvent to sample ratio and extraction time on oil yield for soxhlet

extraction (a) Interaction graph (b) contour plot (c) 3D surface 69

Figure 4.5 GC-MS result for ultrasound extracted oil. 73

Figure 4.6 GC-MS result for soxhlet extracted oil. 74

List of Figures under Appendixes

Appendix B1 Standard calibration curve of D-catechin for the determination of tannin

content 92

Appendix B2 Standard calibration curve of phytic acid for the determination of phytate content

of pulp 92

Appendix B3 Standard calibration curve of phytic acid for the determination of phytate content

of seed 93

Appendix B4 Standard calibration curve of gallic acid for the determination of total phenolic

content 93

Appendix B5 Standard calibration curve of quercetin for the determination of total flavonoid

content 94

Appendix C Some of the pictures and photos taken during conducting research 94

x

Acronyms

Acronyms Nomenclature

AAS Atomic Absorption Spectrophotometry

CRFG California Rare Fruit Growers

CSA Central Statistical Authority

DM Dry Matter

FAO Food and Agricultural Organizations

FAOSTAT Food and agricultural Organization statistics

HCl Hydrochloric Acid

HDL High Density Lipoprotein

LDL Low Density Lipoprotein

MC Moisture Content

M-T Magness-Taylor

R & D Research and Development

RFP Ripe Fuerte Pulp

RFS Ripe Fuerte Seed

RHP Ripe Hass Pulp

RHS Ripe Hass Seed

RH Relative Humidity

SAS Statistical Analysis System

SEO Soxhlet Extracted Oil

SNNP Southern Nations Nationalities and peoples

TSS Total Soluble Solids

TTA Total Titratable Acidity

USDA United States Department of Agriculture

UEO Ultrasound Extracted Oil

UFP Unripe Fuerte Pulp

UFS Unripe Fuerte Seed

UHP Unripe Hass Pulp

UHS Unripe Hass Seed

USAE Ultrasound Assisted Extraction

WARC Wondogenet Agricultural Research Center

1

CHAPTER ONE

1. Introduction

1.1. Background

Avocado (Percea americana) is a tropical and mediterrenian trees and shrubs which belongs to

the Lauraceae family. Lauraceae family trees are trees those are ever green and soft leafs which

includes avocado, laurel, cinnamon, saffras and green-heart (a timber of the Guianas). The

English name is derived from the Spanish word ‘abogado’, which was avocet in French. Its tree

is a fruit plant originated in the Americas, especially Mexico and Central and South America,

belonging to the Lauraceae family and Perseal genus (Maranca, 1980).

Avocado is one of the climacteric fruits of an excellent nutritional quality with low sugar content

which makes avocado very recommendable source of high energy food for those who are

diabetic. It is highly consumed in the world due to the presence of unsaturated lipids and its

relevance in improving and maintaining healthy heart and circulatory system. Its fat contents

make it a valuable source of energy as well as a potential raw material for the manufacture of

pleasantly tasting spreads for breads and biscuits. Besides, the lipids contain linoleic acid, a

polyunsaturated fatty acid which together with alpha linoleic acid (Omega-3 fatty acid) form

vital parts of body structures, perform important roles in immune system and vision, help form

cell membranes and produce hormone-like compounds called eicocasnoids (Wardlaw & Kessel,

2002). The oil content of the fruit depends upon its ecological origin and on the cultivar, as for

example, in Guatemalan and Mexican cultivars, the oil content varies from 10 to 13% and from

15 to 25%, respectively (Biale & Young, 1971).

From a nutritional point of view, avocado is an important and high caloric fruit. Indeed its high

content of unsaturated fatty acids is one of its distinguishing characteristics. Moreover, avocado

is rich in vitamin E, ascorbic acid, vitamin B6, b-carotene, and potassium (Bergh, 1992).

Avocados are divided into three horticultural races or species according to the areas of origin and

distinctive features: Guatemalan (Persea nubigena var. guatamalensis L. Wms.), Mexican (P.

americana var. drymifolia Blake), West Indian (P. americana Mill. var. americana). Hybrid forms

2

exist between all three types which are more cultivated now (CRFG, 1998). In Ethiopia there are

about six types of avocado cultivars registered for production which are hybrids of original races.

These are Hass, Fuerte, Pinkerton, Nabal, Bacon and Ettinger (FAO, 2010). These cultivars have

different chemical composition, phytochemical composition, physicochemical properties and

ripening time. But, their composition (pulp and seed), physicochemical properties and oil

extraction conditions are not studied and characterized yet which were investigated in this study.

Annual avocado production in Ethiopia is 25633.16 tons. The crop is now produced by

1,149,074.00 farmers countrywide who collectively farm more than 8938.24 ha of land (CSA,

2012/13). According to Garedew (2010) even though avocado has economically and socially

play a significant role its production is confronted by a number of constraints:- this are

degeneration of fruits, disease problem and absence of agronomic practices.

The avocado cultivars ‘Fuerte’ and ‘Hass’ are the most commercially valuable varieties and

account for up to two-thirds of the avocado production around the world. Hence, most studies of

avocado quality characteristics use these two cultivars (Ashton et al., 2006; Rodríguez-Carpena

et al., 2011; Villa-Rodríguez et al., 2011). However, similar studies on avocado varieties grown

in Ethiopia have been limited.

Avocado pulp is enriched with oil which can be extracted by different extraction methods. A

number of new methods for extracting oils have been investigated in recent years, including

mechanical compression (Karaj and Müller, 2011), ultrasonic extraction (Ozkan et al., 2007),

microwave extraction (Kumaran and Karunakaran, 2007), and supercritical fluid extraction

(Louli et al., 2004). Compared with traditional Soxhlet extraction, ultrasonic extraction provides

higher selectivity, is less time-consuming, has lower energy consumption and reduced emissions

(Ward et al., 1985). It is also environmentally friendly because most of the extraction solvent can

be recovered, and the equipment is inexpensive. So, application of ultrasonic extraction for

extraction of oil from avocado and optimization of some extraction parameters should be done.

3

1.2. Statement of the problem

Horticulture can be an important factor for economic development and contribute to increased

food security and improve the populations’ nutrition intake (Weinberger & Lumpkin, 2007). The

growing population and changing dietary habits in Ethiopia has increased the demand for fruit

(ILRI, 2011). Especially the demand for local fruits with higher quality for example mango,

papaya, apple and avocado are emerging. Two examples of fruits were the production have

increased with over 60 percent during the last 10 years in Ethiopia is avocado and mango (www,

faostat, 2, 2014; www, faostat, 3, 2014).

In order to increase food availability, diversification and consume nutritious fruit it is therefore

not enough to increase the productivity in agriculture, there is also a need to identify which

variety is more nutritious by characterizing the variety of each fruit specifically avocado. This

fruit can be consumed either fresh or processed in to different forms like juice, salads,

guacamole, oil etc. In this case avocado is useful to reduce hunger and malnutrition and promote

agricultural growth. In the future, this fruit could represent the raw material for oil extraction and

other processed products. Even if avocado production in Ethiopia is increasing, the inter-cultivar

variety of this fruit is not characterized yet interms of its Physical property and chemical

composition including oil content and phytochemical composition since it may differ due to

location, cultivar (variety), harvesting season, ripening, maturity level etc. which may cause a

general loss. The lipid content in avocados varies greatly with the cultivar, and the same is

observed for fatty acid composition, which depends on growth rate and variety (Tango et al,

2004). Concerning the region of cultivation, differences may reside not only between countries,

as suggested by Landahl, Meyer, and Terry (2009), but also between different geographical

locations within the same country, due to variation in climate, soil composition, and other

environmental factors. Therefore, the choice of an avocado cultivar for oil extraction might be

based on lipid content as well as on fatty acid composition (which is related to the intended use

of the oil) Isabelle Santana1 et al. (2015).

Also at present only the flesh (pulp) part is commonly utilized in Ethiopia and its seed part is

rejected due to its poor taste, lack of information on its nutritional composition. So this study

helps to characterize the composition of avocado seed and recommend development of some

4

products from it. Utilization of it may also helpful in reducing the cost for its disposal and

minimize environmental waste.

However, it is rich in oil content but in Ethiopia its oil is not commonly extracted and its

extraction condition is not optimized. Furthermore, the fatty acid content and characteristics of

Ethiopian avocado oil have, however, not been determined. Different extraction methods (using

soxhlet and Ultrasound assisted extraction) have influence on quantity (yield) and quality of oil.

So, extraction of oil from avocado using different extraction methods can improve oil yield and

minimize economic loss. Therefore, Ethiopia needs further understanding of the fruit and its

main components; its variation in composition among different cultivars and also produce oil

from it.

1.3. Objective of the research

1.3.1. General objective

The main objectives of this research were characterization of Hass and Fuerte avocado variety

grown in Ethiopia (Wondogenet) and optimization of some oil extraction parameters for Hass variety.

1.3.2. Specific objectives

The Specific Objectives of the research were to:

determine the physicochemical properties of selected unripe and ripe avocado varieties

(Hass and Fuerte).

analyse mineral content, proximate and phytochemical composition of the avocado pulp

and seed of selected unripe and ripe varieties.

optimize some extraction parameters (particle size, solvent to sample ratio and extraction

time) of soxhlet and ultrasound assisted extraction and evaluate their effects on the yield

of oil.

characterize the extracted avocado oil at optimum condition and compare their results.

perform analyses of fatty acid composition of avocado oil.

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1.4. Significance and scope of the study

In recent years, avocado production and consumption is increasing in Ethiopia due to its high

nutritional composition and health benefit as well as good economic aspect. Despite its

importance, no work has so far been carried out on characterization and seeing inter-cultivar

variations of avocado grown in Ethiopia. Also, extraction of oil using different extraction

methods and its condition optimization was not carried out. So, characterization of some avocado

cultivars grown in Ethiopia and extraction of oil from it is necessary in order to minimize loss

(economic and nutritional), select cultivars which is mostly nutritious, select cultivar which

contains high oil content and produce it, select cultivars which contains much of pulp (flesh)

rather than seed and skin. Since Ethiopia has a large agricultural sector and there is increasing

interest in the growth of small and developing farmers, if a new market for avocado fruit can be

created by production of high quality avocado oil at a premium price, more small and developing

farmers would be interested in cultivating avocado fruit. This will in turn benefit the agricultural

and economic sectors in the long term. Oil production can also create an alternative market for

the commercial farmer, which has the benefit of less risk, compared to the fresh fruit market

where visual appearance of the fruit is very important. From this study many individuals and

organizations will be benefited which may include: Individual avocado producer (farmer) and

consumer, food security sector, avocado producing farms, industry sector, ministry of agriculture

and horticulture, research institute, avocado suppliers, traders and exporters.

In this research physicochemical properties of avocado fruit varieties (Hass and Fuerte) were

studied. Also proximate, mineral and phytochemical composition of avocado pulp and seed of

selected varieties were determined. Furthermore, effects of different oil extraction methods on

the yield of oil were investigated and their extraction conditions were optimized. The extracted

oil were characterized and analyzed including interms of its fatty acid profile.

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CHAPTER TWO

2. Literature Review

2.1. Overview of Avocado

2.1.1. The Avocado Fruit and its seeds

The avocado fruit is botanically described as a berry with a thick, fleshy mesocarp surrounding a

single large seed. Fruits are globose, have a yellow-green to maroon or purple skin, which can be

smooth or warty, and range in weight from 50g to 1 kg. The avocado fruit consists of an exocarp

or rind, a fleshy mesocarp, a thin fleshy endocarp (collectively the flesh or pulp) and the seed.

The edible flesh or mesocarp contributes to 50 to 80% of total fruit while the large seed of the

avocado comprises 10 to 25% of the total fruit weight (Lewis, 1978). The seed consists of two

fleshy cotyledons covered by two thin seed coats adhering to each other. The cotyledons consist

of parenchyma tissue with scattered oil drops and contain starch as the main storage material

(Biale and Young, 1971).

Avocados are divided into three horticultural races or species according to the areas of origin and

distinctive features: Guatemalan (Persea nubigena var. guatamalensis L. Wms.), Mexican (P.

americana var. drymifolia Blake-), West Indian (P. americana Mill. var. americana). Hybrid

forms exist between all three types (CRFG, 1998).

The Mexican race originated from the highlands in Mexico. Mexican avocados mature relatively

quickly within six to eight months and are the richest of all avocados with as much as 30% lipid

content. Fruit from the Guatemalan race also originated from the highlands in Central America

and have thick woody skins and are generally the latest maturing of the three races (Schaffer and

Andersen, 1994), taking a year or longer before they are ready for harvest. Guatemalan avocados

are generally small (the size of a tennis ball) and typically have a lipid content around 8 to 15

percent (Smith et al., 1992). The West Indian race occurs in lowland forests of Central America

and northwestern parts of South America. The West Indian avocados are large, have a leathery

skin, mature in 6 to 9 months, weight up to 1.5 kilograms and have the lowest lipid content (only

3 to 10 percent) of all avocados (Smith et al., 1992).

7

West Indian avocados are the most cold sensitive and well adapted to high temperatures. Due to

their origin in the cool highlands, fruit of the Mexican race are the most cold tolerant and

because of this, they do not show normal development in high temperatures either. On the other

hand avocados of the Guatemalan race fall in between the other two showing more sensitivity to

high temperatures (Whitmore, 1986).

Most avocado trees grown in commercial plantations are a mix of Guatemala and Mexican

ancestors or have Guatemala and West Indian parents. Avocado cultivars found in the market are

the result of selection among the three avocado races with advantages for commercialization and

cross-breeding work done basically in California, US (CAC, 1998).

Systematic studies have classified more than 500 varieties; however, most of them have been

discarded in order to "create" commercial varieties, which adapt to production in commercial

scale (Smith et al., 1992). From this large number of varieties, most have had productivity

problems (production time, amount of fruit), quality (protein and fat content), and commercial

handling problems (resistance to transportation, etc.). Commercial varieties are developed from

the selection and improvement of these types, or by hybridation. For example, from the Antillean

type the Pollock, Peterson, and Waldin varieties are obtained; from the Guatemalan, MacArthur,

Orotava, Nabal, Anaheim, Hass, Booth 7, Booth 8; and from the Mexican: Puebla, Mayapán,

Zutano, Topa-Topa, and Bacon. Some hybrid varieties are: Mexican-Guatemalan: Fuerte,

Ettinger, Rincón, Robusto, Lula. Antillean-Guatemalan: Gema, Choquette (Rodríguez-Suppo,

1992).

Once the edible pulp is separated, the skin and seed are left as residues. The seed has lower lipid

content than pulp; therefore, seeds are not considered important in processes such as the

obtaining of oil. However, Lee (1981) found that the fatty acids in the seed have higher levels of

polyunsaturated acids than in the pulp.

8

Table 2.1 Fruit characteristics of different avocado cultivars.

Cultivar Fruit shape Skin color Flesh color Fruit weight

Hass Ovate Purplish black Creamy yellow 140 t0 400 g

Fuerte Pyriform Green Pale yellow 170 t0 500 g

Edranol Pyriform Dark green Buttery yellow 255 to 500 g

Ryan Pyriform Green Golden yellow 255 to 420 g

Pinkerton Prominantly

pebbled

Dark green Cream-colored 230 to 425 g

Ettinger Pyriform Bright green Light cream to yellow 170 to 570 g

Bacon Ovate Green Very pale yellow to green 170 to 510 g

Source: Whiley et al. (1996b)

Proteins, carbohydrates, vitamin C and phytochemicals are present in both the dry and fresh seed

samples. Carbohydrates exist in varying amounts in the avocado seeds depending on the

different activities that took place in the fruit during storage process before the analysis. During

storage, fruits lose weight, shrivel and change colour, lose acidity and ascorbic acid but gain

sweetness (Pareek et al., 2009). Also during this period, the enzyme activity, sugar and

carotenoid contents increase with corresponding decrease in acidity, pectin and tannin content.

To compare the carbohydrates in both fresh and dry seeds it needs a test that is specific for a

specific type of carbohydrates. The study by Flitsch & Rein (2003), explains the vital functions

of carbohydrate found in avocado seeds as supplying energy for the body process. Some of the

carbohydrates are immediately utilized by the tissues and the remaining is stored as glycogen in

the liver and muscles and some are stored as adipose tissues for future energy needs (Flitsch &

Rein, 2003).

Avocado seed also contains a diverse number of nutrients and phytochemical compounds of

nutritional value. Some of the phytochemical compounds found in avocado seeds are tannins,

saponins and flavonoids which are used as antioxidants, lower bad cholesterol level etc. The

phytochemical compounds in avocado seeds are responsible for color and organoleptic properties

and also for prevention and treatment of many health conditions, including cancer, heart disease,

diabetes, and high blood pressure (Kushi et al, 2006). There is some evidence that certain

phytochemicals may help prevent the formation of potential carcinogens (substances that cause

9

cancer), block the action of carcinogens on their target organs or tissues, or act on cells to

suppress cancer development (Kushi et al., 2006).

Table 2.2 Composition of Avocado seed

Composition Percentage

Water 50.4%

Wet Basis (%) Dry Basis (%)

Ash 1.3 2.7

Protein 2.5 5.0

Reducing sugars 1.6 3.2

Common sugars 0.6 1.2

Starch 29.6 60.0

Pentosans 1.6 3.3

Arabinos 2.0 4.1

Ether Extract 1.0 2.0

Fiber 3.7 7.2

Undetermined 5.6 11.3

Source: California Avocado Association (1934)

2.2.Global Avocado Production

2.2.1. World avocado production

World production of avocados has increased more than fourfold over the past four decades,

according to FAO. World production in 2010 production was about 3,581,711 metric tons (mt) it

increased to 4,188,912mt in 2012. Mexico is the largest producer of avocados in the world,

followed by Chile, Indonesia, Dominican Republic and USA. According to FAO data, Mexico

produced 1.1mln mt, Indonesia 224,000mt, Chile 330,000mt, Dominican Republic 275,000mt

and USA 149,000mt. The growth in production has been fuelled by increasing public awareness

on healthy eating habits as well as economic growths coupled with disposable income. It is

important to note that globally and indeed it has to be the case in Tanzania that the major driver

for avocado production is domestic consumption. Statistics show that exports accounts for less

than 20% of the production in major producing countries. For example in Mexico, the largest

producer consumes 35 per cent of the World avocado production, per capita consumption since

the 2000s had reached 9 kilos per annum, however, imports are competing with domestic

market as a result Mexicans now eat about 7 kilos/annum2.

10

Source FAOSTAT

Figure 2.1. World avocado production trend (2000 - 2012) in '000mt

2.2.2. Avocado production in Africa

Over a period of 2005 – 2012 avocado production in Africa has grown but unevenly from

497,339mt in 2005 to 751,881mt in 2012. The leading producers with their volumes in mt in

2012 were Kenya (186,292mt), Rwanda (145,000mt), South Africa (91,603mt), Cameroon

(72,000mt) and DRC (70,000mt). With an exception of South Africa all the other four top

ranking producers lie closer to the equator with tropical conditions. Africa’s annual production

growth has been closer to the global rate that between 2005 and 2012 averaged at 6.4%. Year-on-

year growths have been the highest in Morocco (23%), Tanzania (20%), Rwanda (18%) and

Kenya (10%). In absolute terms Kenya and Rwanda carry more weight due to the larger base

while a country like Tanzania had a smaller base of 22,000mt.

It is promising to know that the crop has market foothold in both export and domestic markets,

data from the South African Subtropical Growers Association indicate that 45 % of total

production is exported, 15 % is processed, 25 % is consumed in local markets and 15 % is sold

on the informal markets. While South Africa ranks low in terms of volume of fruit produced, it

has the most advanced subsector and is among the top five of exporters. Other Africa exporters

are Ethiopia, Cameroon, Rwanda and Kenya. It also has a well advanced R&D base.

11

Table 2.3 Africa top 10 avocado producing countries (2008-2012, in mt)

Country 2008 2009 2010 2011 2012 Percent in

2012

Kenya 103,523 145,204 202,294 201,478 186,292 12.17%

Rwanda 79,291 141,130 129,732 143,281 145,000 9.47%

South Africa 83,534 76,726 83,204 75,748 91,603 5.98%

Cameroon 55,000 54,000 56,000 69,532 72,000 4.70%

RDC 65,220 66,112 67,016 67,933 70,000 4.57%

Morocco 19,253 26,000 33,645 33,519 54,340 3.55

Cote d’Ivore 27,700 28,614 31,700 31,000 32,000 2.09%

Madagascar 24,000 25,762 26,080 25,632 26,000 1.70%

Ethiopia 32,452 37,651 57,299 73,097 25,633 1.67%

Tanzania 7,500 10,420 15,540 19,660 22,000 1.44%

Others 23,133 23,966 24,386 25,714 27,013 1.76

Total 520,606 635,585 726,896 766,594 751,881

Source: FAOSTAT

2.2.3. Avocado production in Ethiopia

Fruits have significant importance with a potential for domestic and export markets and

industrial processing in Ethiopia. The main fruits produced and exported are banana, citrus fruits,

mango, avocado, papaya and grape fruits (Zeberga, 2010). Owing to its shortest introduction to

Ethiopia, these days the crop is produced in several countries where Ethiopia stands the 10th

leading producer and 6th most important consumer in the world (FAOSTAT, 2010).

Avocado was first introduced to Ethiopia in 1938 by private orchardists in Hirna and Wondo-

genet and production gradually spread into the countryside where the crop was adapted to

different agro-ecologies (Edossa, 1997). Annual avocado production in Ethiopia is 25633.16

tons. The crop is now produced by 1,149,074.00 farmers countrywide who collectively farm

more than 8938.24 ha of land (CSA, 2012/13). According to Garedew (2010) even though

avocado has economically and socially play a significant role its production is confronted by a

12

number of constraints;- this are degeneration of fruits, disease problem and absence of

agronomic practices.

According to FAO, (2010) description of some varieties introduced in Ethiopia and presently

available includes:

Hass: high yielding, resistant to main pests and diseases. not presenting a marked biennial

fruiting behavior. Fruit size variable; oil % in the fruit: medium, month to ripen: 9, seed size:

small; cold tolerance: medium

Pinkerton: high yielding; fruit size; medium; oil % in the fruit: high; month to ripen: 6-8; seed

size: big; cold tolerance: medium

Fuerte: a Mexican Guatemalan cross; medium yielding, fruit size , small to medium; oil % is

high; month to ripen 5-6; seed size : tolerant to frost.

Bacon: high yielding; medium size fruit; oil % high; tolerant to cold -5oC

Ettinger: a Mexican Guatemalan cross, resistant to cold

Nabal: Guatemalan type, big size fruit; suitable for warm climate.

In general, fruit production is still backward, the business is underdeveloped and the private

sector is not much attracted. In connection with this lack of access to improved varieties,

production is exclusively based on distribution of mixed materials; consequently the local seed

system has come out as best-bet arena and is now a common route for seedling dissemination

(Ayelech, 2011 ). CSA (2013) indicated Avocado as one of the second potential fruit crop

produced in Ethiopia.

Ethiopian Farmers produce avocado without knowing its variety. Knowing their variety is useful

in selecting variety which give high yield, high oil content and also in determining time of

harvesting. In Ethiopia avocado is consumed mostly as a juice either alone or mixed with other

fruit juice and also as a salad. Its juice is consumed round a year especially during fasting days

and mostly utilized by urban dwellers rather than rural dwellers due unavailability of it

everywhere since avocado is not produced all over Ethiopia. Eating small amount of avocado

feels stomach full which incase useful in reducing weight due to its high fat content. Its fat

content among different varieties in Ethiopia is not yet investigated which was determined in this

study.

13

Even if its consumption trend is high, avocados grown in Ethiopia is highly perishable and

easily deteriorated due to various reasons which may include:

lack of good hygienic practice during harvesting, transporting, packing, distribution and

storage

lack of cold storage during transportation and storage

fluctuation of temperature and relative humidity from area to area

sunburn and direct contact with dusts, soils and mud during selling which may cause

physical and fungal damage.

Poor hygienic environment around marketing etc.

Therefore, in order to reduce postharvest loss all value chains from farmers up to consumers

should know the behaviour of each cultivars and as much as possible facilitate appropriate

harvesting, holding, transportation and storage conditions.

Table 2.4 Summary of major fruit crops produced in Ethiopia in 2012/2013 cropping season

Crop

Fruits

Area in Ha Production in quintal Yield (Qt/ha)

61,972.60 4,793,360.64 77.35

Avocados 8,938.24 256,331.64 28.68

Bananas 36,012.19 3,025,022.32 84.00

Guavas 1,492.32 11,730.03 7.86

Lemons 754.23 55,167.50 73.14

Mangoes 8,808.64 697,507.30 79.18

Oranges 2,999.21 357,458.39 119.18

Papaya 2,752.08 386,943.15 140.60

Pineapples 215.69 * *

Source: CSA, 2012/2013, Agricultural Sample Survey Result

2.3. Avocado quality characteristics

The quality of horticultural produce are composed of sensory attributes, nutritive attributes,

chemical constituents, mechanical properties, functional properties and defects (Abbott, 1999).

Colour, texture, flavour and aroma have been found to be essential in determining the eating

quality of avocados and are the main characteristics to which consumers refer to when

14

purchasing (Lee et al., 1983). In order to investigate and maintain the quality of avocados it is

essential to be aware of the quality related attributes which are outlined here.

2.3.1. Physical properties

Physical properties are mainly related to the appearance and aesthetic appeal of avocados to

which consumers are initially exposed influencing their decision to purchase. The physical

quality parameters of avocados include skin colour, firmness, texture, and physiological

disorders.

a. Skin colour

Avocado skin colour is an important indication of the stage of ripening for industry and

consumers (Arzate-Vázquez et al., 2011). Skin colour can be measured either objectively,

commonly using a chroma meter or colorimeter or alternatively using subjective means by

experienced sensory panellists using eye colour rating. Skin colour is found to vary among

different avocado cultivars. The ‘Hass’ variety is characteristic of a colour change from green to

purple and eventually black (Arzate-Vazquez et al., 2011). However, Chen et al. (2009) revealed

that the skin colour of the ‘Sharwil’ variety does not darken with maturity. Therefore, other

methods must be utilised to distinguish the various stages of maturity.

b. Firmness

The firmness of avocados is a vital determinant in assessing the degree of ripening (Mizrach and

Flitsanov, 1999; Arzate Vazquez et al., 2011). Firmness can be described as the resistance to

penetration (Mizrach and Flitsanov, 1999) determined by employing invasive, such as hand

tactile methods, destructive methods such as the Magness-Taylor puncture test (M-T), or non-

destructive methods such as impulse response and ultrasonic methods. Destructive techniques do

not allow for continuity in monitoring on a commercial basis but is, rather, well suited for

laboratory analysis. Mizrach and Flitsanov (1999) employed ultrasonic techniques to evaluate the

firmness in a non-destructive manner which rendered comparable results to that of destructive

methods.

c. Texture

Texture is a significant indicator of avocado quality and of concern to the consumer

(Maftoonazad and Ramaswamy, 2008; Landahl et al., 2009). Avocados undergo drastic changes

15

in texture (Landahl et al., 2009; Li et al., 2010). Chen et al. (2009) stated that the oil content is a

key component in the texture of avocados and which Hofman et al. (2002b) identified as

contributing to the ‘smoothness’. Despite the relation between texture and oil content, it was

discovered by Chen et al. (2009) that an increase in the oil content over the harvest period did

not manifest into any change in the texture. Storage temperature, oxygen and carbon dioxide

concentrations and wounding directly affect the texture (Maftoonazad and Ramaswamy, 2008).

The relationship between texture and firmness can be extended to the strength of avocados and

the ability of the fruit to withstand loading during storage. Firmness can be described as the force

necessary to attain a previously defined deformation during textural evaluation (Landahl et al.,

2009). It was found that as the avocados ripened the texture, firmness and strength were reduced.

d. Physiological disorders

Every biological system operates optimally within specific limits. If these limits are significantly

reduced or increased, physiological disorders are likely to ensue. Storage at low temperature is

commonly used to extend the shelf life of fresh commodities, however, these results in chilling

injury of avocados (Woolf et al., 2003).

The main symptoms associated with chilling injury are black spots on the peel or gray or dark-

brown discolouration of the mesocarp and Hofman et al. (2002b) found that employing hot water

treatments prior to storage were effective in reducing the effects of chilling injury. Exposing

avocados to low temperature storage conditions just above those at which chilling injury is likely

to occur prior to storage have been proven to alleviate the effects of chilling injury (Woolf et al.,

2003). The optimum low temperature was found to be 6 or 8°C for three to five days. However,

Woolf et al. (2003) found the minimum temperature to be 4°C.

Other disorders include sunburn leading to development of symptoms which emerge as

yellowing or bleaching or a roughened skin. On the other hand controlled or modified

atmospheres that expose avocados to too low of oxygen or too high carbon dioxide

concentrations can lead to disorders (Ferguson et al., 1999).

16

2.3.2. Sensory properties

One of the main sensory properties of avocados is flavour which encompasses both aroma and

taste and forms an important component of the eating quality of the fruit (Abbott, 1999).

a. Flavour

Workneh and Osthoff (2010) and Paull and Duarte (2011) defined flavor as the ratio of sugar to

acid influenced by temperature as in the case of grapefruit held at 8ºC resulting in a sugar and

acid decline as compared to those stored at 12ºC. Premature harvesting can lead to an

undesirable taste (Wu et al., 2011). The off flavour can be ascribed to increased levels of ethanol

and acetaldehyde (Paull and Duarte, 2011). Burdon et al. (2007) observed that exposure of

‘Hass’ to oxygen and carbon dioxide concentrations of less than 0.5% and up to 20%,

respectively, resulted in increased levels of acetaldehyde and ethanol. As with texture, the oil

content also forms a key component of flavour (Chen et al., 2009) and, hence, it can be deduced

that a positive correlation exists between texture and taste.

2.3.3. Chemical properties

Identification of horticultural maturity is often difficult to determine in avocados as changes in

external appearance are sometimes not easily distinguishable (Lee et al., 1983). Other techniques

of determining maturity and that employ chemical properties are therefore required. The

chemical properties of avocados discussed here are total soluble sugar, pH, total titratable acid,

moisture content, dry matter and oil content.

a. Total soluble sugars

Carbohydrates are an essential source of energy for growth, development and maintenance in

avocados (Liu et al., 1999b). Five major soluble sugars were identified within the avocado

include the rare seven carbon (C7) reducing sugar mannoheptulose, its reduced polyol form,

perseitol, the common disaccharide sucrose, and its component hexoses, fructose and glucose.

These constituted approximately 98% of the total soluble sugars (TSS). Liu et al. (1999b)

demonstrated that ripening of avocados at 20°C resulted in a considerable decline in the TSS in

the peel and flesh, particularly the C7 sugars, and that a decrease in the TSS was concomitant

with an increase in the oil content. During storage at 1 and 5°C a decrease in the TSS was

observed but at a slower rate. Similarly Liu et al. (2002) found a decrease in the C7 sugars

17

during the progression of the ripening process. During avocado growth, carbohydrates are stored;

however, once the fruit is harvested these carbohydrates are consumed for postharvest

physiological processes such as respiration via enzymatic mechanisms that metabolize the C7

sugars (Liu et al., 1999b). This suggests that the C7 sugars play an important role in the

respiration of the avocado during the ripening process.

b. pH

Avocado pH lies in the range of 6 to 6.5 (Soliva-Fortuny et al., 2004). Maftoonazad and

Ramaswamy (2008) observed that the pH decreased with time during storage. Avocados treated

with pectin based coatings illustrated a slower rate of decrease in pH values compared to

untreated fruit and those exposed to higher temperatures. Exposure to low oxygen and/or high

carbon dioxide for short periods of time has been used as a pretreatment to alleviate

physiological disorders and for enhanced storage atmospheres. These conditions can also lead to

a decrease in the intracellular pH thereby altering the various physiological processes that are

dependent upon pH (Ke et al., 1995). Avocados subjected to (a) 0.25% oxygen, (b) 20% oxygen

in combination with 80% carbon dioxide or (c) 0.25% oxygen and 80% carbon dioxide reduced

the pH value from 6.9 to 6.7, 6.3, and 6.3, respectively, at 20°C (Ke et al., 1995). Similarly

Lange and Kader (1997) stored avocados at 20°C in atmospheres of varying concentrations of

oxygen and carbon dioxide and found that concentrations of (a) 21% oxygen, (b) 20% carbon

dioxide (17% oxygen and the remainder Nitrogen) and (c) 40% carbon dioxide (13% oxygen and

the remainder Nitrogen) produced pH values of 7.0, 6.6 and 6.4 respectively. These show that the

lowest concentration of oxygen and highest concentration of carbon dioxide results in a

reduction of pH to form an acidic medium.

c. Total titratable acid

Acidity is associated with both sweetness and sourness of fruit. The method used to measure

acidity is titratable acidity. Maftoonazad and Ramaswamy (2008) observed an increase in the

titratable acid at higher storage temperatures in both pectin-based coated and non-coated

avocados. Holcroft and Kader (1999) showed that strawberries exposed to higher concentrations

of carbon dioxide at 5°C exhibited increased pH and decreased levels of titratable acidity.

Mangoes subjected to postharvest treatments, packaging and storage for 28 days resulted in a

decrease in the titratable acidity from 3.42 to 0.2% (Tefera et al., 2007). It can thus be deduced

18

that the postharvest changes in strawberries and mangos differ to that of avocados in terms of

total titratable acidity.

d. Moisture content

Moisture content is the preferred indicator of maturity in South Africa with the recommended

moisture content in the range of 69 to 75% depending on the cultivar (Mans et al., 1995). Export

of early season ‘Fuerte’ commences once the moisture content has reached 78 to 80% equivalent

to oil content of 9 to 11 standards of moisture percent (Dodd et al., 2008).

e. Oil content

Avocado is considered to be an important oil fruit and oil content serves as a significant indicator

of fruit maturity (Hofman et al., 2002a; Ozdemir and Topuz, 2004). As the fruit matures, the

concentration of oil within the mesocarp increases as described by Hofman et al. (2002a),

Ozdemir and Topuz (2004) and Chen et al. (2009). This increase in oil results in a reduction in

the water by the same amount within the fruit implying that the percentage of total water plus oil

remains constant throughout the avocado life (Hofman et al., 2002a; Ozdemir and Topuz, 2004).

Lee et al. (1983) and Chen et al. (2009) observed a close correlation between the percentage oil

content and percentage dry matter. The minimum oil content necessary for marketing avocado

fruit is 8%. After maturation, values greater than 20% can occur. These values occur in the

period between harvesting, when commercial maturity is reached and full maturation, when the

oil content increases and change occurs in the oil composition. Concentrations of unsaturated

fatty acids increase and those of saturated fatty acids decrease (Martinez & Moreno, 1995).

The oil content of the fruit depends upon its ecological origin and on the cultivar, as for example,

in Guatemalan and Mexican cultivars, the oil content varies from 10 to 13% and from 15 to 25%,

respectively (Biale and Young, 1971), while in the fruits from Carrebean, a low fat (2.5 to 5%)

has been reported (Hatton et al., 1964).

f. Dry matter

Hofman et al. (2002a) referred to percentage dry matter determination as an alternative to oil

content determination in assessing the maturity. Extending the maturation stage of the avocado

allows for more oil accumulation and dry matter however the risk of increased disease is

introduced. Maturity standards are being used by avocado producing countries to avoid

19

marketing of low quality immature fruit. The standards adopted are the Californian minimum dry

matter of 20.8% for ‘Hass’ or a slightly higher minimum dry matter content of approximately

25% to decrease disorders during storage. An oil content of 8% has been reported by Ozdemir

and Topuz (2004) to be acceptable for marketing of avocados. Villa-Rodriguez et al. (2011)

found that the dry matter had increased from 31.65 to 36.52% over eight days at 15°C and

thereafter decreased to 32.91 on the day 12.

Hofman et al. (2000) suggested that the percentage oil content and dry matter are not suitable

indicators of avocado maturity in late-harvested ‘Hass’ due to late harvested fruit having

inconsistent changes. No distinct correlation could be drawn between the effect of varying

temperature and relative humidity on the percentage dry matter and oil content of avocados

during storage, thus, motivating research to be conducted in this field.

2.4. Nutritional value, health benefits and uses of avocado

Composition of nutrients of any fruit may vary depending on the variety, grade of ripening,

climate and composition of soil, and fertilizers. Avocados have significantly higher lipid content

(8–25%) as compared to other fruits. The avocados have diverse fats yet they are one of the best

foods one can eat. They are full of nutrients and heart-healthy compounds. According to USDA

report in 2004, each 100g (3.5oz) of avocado pulp gives 670KJ (160Kcal) of energy; 75% of

which is from its fat. It contains 2.13g saturated fatty acid, 9.80g monounsaturated and 1.82g

polyunsaturated fatty acids. 2g of that amount was protein while water was 73.23g. The avocado

also contains many vitamins and minerals; especially it contains 35% more potassium than

banana which has 358mg per 100g. 75% of the high fibre content is insoluble while 25% is

soluble (Naveh et al., 2002). The health benefits of avocados are quite obvious, from their

nutritional components and hence cannot be overemphasized. High intake of avocados lowers

blood cholesterol levels (low density lipoprotein, harmful cholesterol) due to its high content of

High density lipoproteins (HDL), helpful cholesterol (Naveh et al., 2002).

The avocados are a great source of liteine, a carotenoid that works as an antioxidant and helps

protect against eye disease. Avocado helps in weight loss because the monounsaturated fats

make one feel full and resist temptation to eat. It contains good amount of fiber both soluble and

insoluble. Fiber is needed by the digestive system to run smoothly. Oleic acid is a fat that

20

activates the part of the brain that makes one feel full, and it is also present in avocado. Oleic

acid in avocado has been shown to produce greater level of satiety than less healthy saturated fats

and Trans fats contained in processed food. The processed products of avocado pulp include the

paste, puree, and guacamole. Guacamole is a fruit pulp seasoned with salt, onion, lemon, pepper

and tomato, being produced not only in an artisanal way but also marketed by some US

companies (Daiuto et al., 2011).

The sensory quality of guacamole of Hass variety made without chemical additives and stored

under refrigeration was evaluated according to the type of packaging used. A greater consumers’

acceptance was observed for the product stored in container with gas barrier when compared to

that stored in polyethylene package (Daiuto et al., 2011). Although these authors have also

considered that the heat treatment may have been effective on the polyphenol oxidase

inactivation, it can result in the development of bitterness and off-flavors in avocado, which

changes the guacamole texture, negatively contributing to a mashed appearance.

Chaves et al. (2013) studied avocado pulp Margarida variety dehydrated and defatted by cold

pressing and avocado oil to partially replace wheat flour and butter, respectively, in whole grain

crackers. The authors reported that the flour from avocado pulp, in general, showed

characteristics similar to those of conventional flour and whole wheat flour. The biscuits had

higher minerals and fiber levels, with good sensory acceptance.

Meat derivatives can also be supplemented with avocado pulp, since most of these processed

foods contain relatively high levels of saturated fats in the formulation whose consumption is

restricted by health issues. Thus, an alternative to reduce and enhance fatty acids balance is the

incorporation of fats or vegetable oils in emulsified meat products. The replacement of animal

fats by vegetable oils in meat products has been studied with positive effects on the chemical,

physical and sensory characteristics of the products, but with negative effects on water activity

and texture (Lugo, 2006).

21

Table 2.5 Nutritional profile of US-grown avocados and avocado oil (per 100 g).

Nutrient Unit Raw Avocado (average, all

US varieties)

Raw Avocado

(California grown)

Avocado Oil

Proximate

Water

Energy

Protein

Total lipid (fat)

Ash

Carbohydrate

Fiber, total

dietary

Sugars, total

g

kcal

g

g

g

g

g

g

73.23

160

2

14.66

1.58

8.53

6.7

0.66

72.33

167

1.96

15.41

1.66

8.64

6.8

0.3

0

884

0

100

0

0

0

-2

Minerals

Calcium

Iron

Magnesium

Phosphorus

Potassium

Sodium

Zinc

mg

mg

mg

mg

mg

mg

mg

12

0.55

29

52

485

7

0.64

13

0.61

29

54

507

8

0.68

0

0

0

0

0

0

0

Vitamins

Vit C, as

ascorbic acid

Niacin

Pantothenic

acid

Vitamin B- 6

Folate, total

Choline, total

Carotene, beta

Carotene, alpha

Cryptoxanthin,

beta

Vitamin A, IU

Lutein +

zeaxanthin

Vitamin E (α-

tocopherol)

mg

mg

mg

mg

µg

mg

µg

µg

µg

IU

µg

mg

10

1.738

1.389

0.257

81

14.2

62

24

28

146

271

2.07

8.8

1.912

1.463

0.287

89

14.2

63

24

27

147

271

1.97

0

0

0

0

0

0

-

-

-

-

-

-

1By difference; 2not reported. Source: USDA (2010a)

2.5. Phytochemicals composition

Phytochemical comes from the Greek word “Phyto” for plant. It refers to every naturally

occurring chemical presents in plants. In plants, phytochemicals act as a natural defense system

22

for host plants and provide colour, aroma and flavour. Phytochemicals are present in a variety of

plants utilized as important components of both human and animal diets. These include fruits,

seeds, herbs and vegetables. Herbs and spices are harmless sources for obtaining natural

antioxidants (Okwu, 2004).

Phytochemicals found in plant foods have both adverse effects and health benefits. For example,

phytic acid, lectins, phenolic compounds (tannins), saponins and enzyme (amylase and protease)

inhibitors have been shown to reduce the availability of nutrients and cause growth inhibition,

while phytoestrogens and lignans have been linked with infertility problems. However, phytic

acid, lectins, phenolic compounds, amylase inhibitors and saponins have also been shown to

reduce the blood glucose and insulin responses to starchy foods and/or the plasma cholesterol

and triglycerides. In addition, phytic acid, phenolics, saponins, protease inhibitors,

phytoestrogens and lignans have been related to reduce cancer risks. Because anti-nutrients can

also be mitigating agents, they need re-evaluation and perhaps a change in name in the future

(Sidhu & Oakenful, 1986).

2.5.1. Phytic Acid

Phytic acid (myoinositol 1, 2, 3,4,5,6, hexakis-dihydrogen phosphate; PA) is present in foods in

concentrations ranging from 0.1 to 6.0%. It is found as crystalline globoid inside protein bodies

in the cotyledon of legumes or oilseeds or in the bran region of the cereal grains (Reddy et al.,

1982). Salts of phytic acid, designated as phytates, are found in plants, animals and soil. Phytate

is ubiquitous among plant seeds and grains, comprising 0.5 to 5 percent (w/w). It is primarily

present as a salt of the mono- and divalent cations K+, Mg 2+, and Ca 2+ and accumulates in the

seeds during the ripening period. Phytate is regarded as the primary storage form of both

phosphate and inositol in plant seeds and grains (Loewus, 2002). Because phytate is a naturally

occurring compound formed during maturation of plant seeds and grains, it is a common

constituent of plant-derived foods. Depending on the amount of plant-derived foods in the diet

and the grade of food processing, the daily intake of phytate can be as high as 4500 mg (Reddy,

2002).

23

Figure 2.2: Proposed structures of phytic acid. Source: Reddy (2002)

Health benefits of phytate

A significant negative relationship was observed between the blood glucose responses to

different starchy foods, expressed as glycemic index, and the intake of phytic acid (Yoon et al.,

1983). The addition of PA (0.2-9%) to the diet of rats significantly reduced the plasma

cholesterol and triglyceride levels (Sharma, 1984). This was suggested to be related to the ability

of PA to bind to Zn and thus lower the plasma Zn to copper (Cu) ratio; lower ratios tend to

predispose humans to cardiovascular disease. Certain minerals such as iron and copper catalyze

oxidative enzymes that generate free radicals, resulting in undesirable oxidative damage such as

cell membrane damage (produces leaky cells). Because phytates have the ability to chelate

minerals that participate in undesirable oxidative reactions, phytates have been suggested to have

protective effects. The ability of phytates to chelate the divalent minerals makes them a natural

antioxidant.

Phytate as an antinutrient

The major concern about the presence of phytate in the diet is its negative effect on mineral

uptake. Minerals of concern in this regard would include Zn 2+, Fe 2+ / 3+, Ca 2+, Mg 2+, Mn 2+, and

Cu 2+ (Vucenik, 2003). Its highly negatively charged structure at a wide range of pH values

makes it very reactive with other positively charged ions such as minerals, forming insoluble

complexes which are less available for digestion and absorption in the small intestine. This is the

main reason why PA has traditionally been considered as an antinutrient. The adverse effect of

PA in mineral availability depends on a number of factors including the concentration of PA and

the strength of its binding with different minerals. For example, zinc (Zn) forms one of the

strongest mineral complexes with PA (Evans & Martin, 1988).

24

PA can also react directly with the positively charged group or indirectly with the negatively

charged group of the proteins mediated by a positively charged mineral ion such as calcium. It

can bind with starch either directly by hydrogen bonding with the phosphate group or indirectly

through the proteins to which it is associated with. The formation of these complexes is likewise

thought to reduce the solubility and digestibility of the proteins or starch and several in-vitro

studies have indeed shown reductions in protein digestibility by PA and in-vivo (Atwal et al.,

1980).

2.5.2. Phenolic compounds

Phenolic compounds encompass a wide variety of compounds characterized by the presence of

an aromatic ring with one or more hydroxyl groups and a variety of substituents. Flavonoids

have a basic C6C3-C6 structure and include the anthocyanin pigments, flavonols, flavanoids and

isoflavones. They occur mostly as glycosides except the flavanols which tend to polymerise to

condensed tannins. The tannins could be classified either as condensed or hydrolysable. Most

condensed tannins are polymers of flavan-3-ols (catechins) or flavan-3, 4-diols

(leucoanthocyanins) while most hydrolysable tannins are glucose or polyhydric alcohol esterified

with gallic acid (gallotannins) or hexahydrodiphenic acid (ellagitannins). The stable dilactone of

the latter is ellagic acid (Deshpande et al., 1984). Tannins may be classified as polyphenolic

substances (Shimelis and Rakshit, 2007).

Health benefits of Phenolic compounds

Several phenolics (e.g. chlorogenic acid, gallic acid, caffeic acid, tannin acid, catechin) have

been shown to inhibit the mutagenic effects of both direct-acting carcinogens (e.g.

benzo(a)pyrene diol epoxide) and carcinogens that require metabolic activation (e.g. aflatoxin B)

and to trap nitrite, thus reducing the nitrosating species and preventing the endogenous formation

of carcinogenic nitroseamines (Stich & Rosin, 1984).

Anti-nutritional effect of Phenolic compounds

The anti-nutritional and toxic effects of phenolic compounds, particularly the tannins, have been

categorized as depression in food/feed intake, formation of the less digestible tannin-dietary

protein complexes, inhibition of digestive enzymes, increased excretion of endogenous protein,

malfunctions in digestive tract, and toxicity of absorbed tannin or its metabolites. Increased risks

25

of cancer of the mouth and esophagus have been linked to dietary tannins in some

epidemiological studies. Although the depressed food intake has been related in part to the

astringent taste of the phenolic compounds, the adverse effects of tannins have traditionally been

attributed to their ability to bind with proteins and hence inhibit their digestion and absorption

(Butler, 1989).

Tannins decreased feed consumption in animals, bind dietary protein and digestive enzymes to

form complexes that are not readily digestible. Tannins form another group of phenolic

compounds, usually divided into hydrolysable tannins and condensed tannins

(proanthocyanidins), and are caustic and bitter-tasting. They bind and precipitate proteins,

decreasing enzyme- resistant substrates formed by interaction between tannins and

protein/starch. Digestibility of the substrates is compromised by interaction between tannin and

the enzymes (Deshpande and Salunke, 1982), the digestibility of protein and carbohydrate.

2.6. Avocado oil processing methods and oil characteristics

2.6.1. Avocado oil processing methods

Avocado is one of the few cultivated fruits in which oil is a main component on dry basis. The

oil content is in the range of 15-30% depending on the variety, and is mainly mono-unsaturated

with the predominant fatty acid being oleic acid. According to Werman and Neeman (1987), of

all fruits only olive and palm can rival the avocado in oil content.

Avocado oil is valued as edible oil due to its health-enhancing qualities and is especially used in

the treatment of connective tissue diseases. This oil is of good quality because the processed fruit

from which the oil is obtained is still intrinsically sound and is only termed second grade because

of its appearance (black or brown spots, rough skin, shape and size), which is not appealing to

the consumer (Eyres et al., 2006).

The avocado oil can be extracted in different ways. It is contained in a finely-dispersed emulsion

in the cells of the fruit pulp. Hence, the extraction process requires rupturing not only the cell

walls, but also the structure of the emulsion (Lewis et al., 1978). Traditionally, this oil used to be

obtained by mashing the pulp in water, then heating and skimming off the supernatant oil. Later,

for cost reasons, most producers started to extract oil from dried fruits by means of solvents

26

(Sadir, 1972; Human, 1987; Martinez Nieto et al., 1988). Two main methods are in use to extract

avocado oil for industrial production. According to the second method, fruits are dried and

pressed at high temperature, subsequently oil is extracted by means of organic solvents. In the

first method, oil is separated from fruits by centrifugal or pressing forces, then oil cells are

submitted to mechanical and enzymatic destruction (Human, 1987; Werman and Neeman, 1987;

Martinez Nieto et al., 1988; Bizmana et al., 1993). The first method was developed in order to

cut energy costs and minimise the air pollution caused by organic solvents. Nevertheless, in both

cases, the crude avocado oil still needs to be refined before final consumption and use in the

cosmetic industry, where it is particularly appreciated for its high vitamin E content and

emollient properties, although it is considered marginal as a food product (Eyres et al., 2001).

The first attempt to develop a method to produce cold-pressed oil intended to obtain high-quality

edible oil was made back in the late 1990’s by a New Zealand company in collaboration with

Alfa Laval (Eyres et al., 2001). In the follow paragraph let compare shortly the main extraction

methods: chemical extraction by solvent, traditional mechanical extraction, microwave assisted

extraction, cold-pressed mechanical method and ultrasound assisted extraction.

Chemical extraction by solvents

Organic solvent extraction is the most widespread. Warm air drying of the pulp followed by

hexane solvent extraction yields 95% oil (oil extracted/oil content). The resulting oil is brownish

with a high pigment content and needs to be refined for most applications. Refining consists of

three steps: deacidification to remove free fatty acids which are less than 1% in good-quality

fruits; bleaching to remove chlorophylls and their degradation products, pheophytins, as well as

carotenoids; de-odourisation. When oil is sold crude, it is generally winterised at 5°C and

drummed in lacquer-lined drums (Human, 1987; Martinez Nieto et al., 1988).

Although hexane extraction is a mild, well-known extraction method, large amounts of solvent is

needed which is expensive and environmentally hazardous. Carbon dioxide is a non-toxic and

environmentally compatible fluid for the extraction of edible oils (Garcia et al., 1996). Carbon

dioxide is an inert solvent which is non-reactive and does not form other type of chemical

compounds. When the extraction process completes, the carbon dioxide will be returned back to

gas phase and released to air. Therefore it does not leave any residual in the oil and pure oil can

27

be obtain. But this new technology is very expensive due to its sophisticated operation and

control. Consequently, it is not yet widely used. Supercritical carbon dioxide (SC-CO2)

extraction has been proven to be a viable alternative for hexane as avocado oil extracted with

these two methods have been shown to have similar fatty acid profiles (Botha, 2004). The

micro-component content and composition as well as oxidative stability of avocado oil extracted

with SC-CO2 have, however, not been determined. Furthermore, the effects of progressive

extraction on the micro-component distribution and oxidative stability are not known.

Traditional mechanical extraction

The mechanical method has been used traditionally in locations where drying facilities and/or

solvent extraction units cannot be installed. However these processes have poor yields and

frequently require the use of chemical aids.

Avocado oil extraction was generally obtained by peeling and destoning the fruit, mashing the

pulp and eventually drying it, then heating the paste with hot water with chalk and/or NaCl, and

spinning, pressing or skimming off (by natural decantation) the oil (Werman and Neeman, 1987;

Bizmana et al., 1993). The centrifugation/pressing yield is 60-80% (oil extracted/oil content)

depending on the fruit variety.

An extensive literature describes the mechanical method and compares different process

conditions in relation to yield and oil quality. After peeling and de-stoning, the pulp is mashed

with hot water. Werman and Neeman (1987) recommend a dilution ratio of 1/3 and a 30-min

treatment at 75°C. Bizmana et al. (1993) found the best combination with a dilution ratio of 1/5

and a 5-min treatment at 98°C. Traditionally, the mechanical method gives low yields, which can

however be increased by maintaining the pH between 4.0 and 5.5 by adding chalk (CaCO3,

CaSO4) or salt (NaCl) to the paste before centrifugation. The presence of monovalent and

divalent cations activates enzymes with pectinase activity, therefore at certain concentrations the

cellulolytic and proteolytic activities are unaffected. The addition of salts favours the extraction

from difficult pastes (Dominguez et al., 1994). Bizimana et al. (1993) reported good results with

an addition of 5% (w/w) CaCO3 or CaSO4. NaCl improves oil extraction only at a low

concentration (<15%), but it causes a significant corrosion of the equipment (Werman and

Neeman, 1987). Also when the traditional mechanical method is used, the resulting oil normally

28

needs to be refined depending on the desired use. The refining system is the same described in

the previous paragraph.

In a complete review about avocado oil (Jacobsberg, 1988), the author maintains that the

mechanical extraction method compared with the chemical method and without chemical aids

offers the best-quality oil, but it has poor cost/benefit ratio.

Cold-pressed extraction

This is an alternative way to extracting oil from plant. This ancient method uses mechanical

pressure to force the oils out from the leaves. In the late 1990’s, a processing company in New

Zealand began production of cold-pressed avocado oil (CPAO) to be sold as culinary oil for

salads and cooking (Eyres et al., 2001). CPAO is not refined and maintains the chemical,

organoleptic and flavour profile of the fruit flesh. In the 2008/2009 season, the New Zealand

processors produced more than 150,000 liters of CPAO with approximately 3% of the avocado

crop grown for oil production (Wong et al., 2010). Today CPAO is produced also in Chile, South

Africa, Kenya, Israel, Samoa and other countries.

Microwave assisted extraction

More recently has been demonstrated that oil extracted from pressed and microwave-dried

avocado pulp presented the lowest acid and peroxide values and the highest oxidative stability in

contrast with the oil from ethanol extraction. Microwaves have been used in assisting the

extraction of essential components and oil due to their environmentally friendly and economical

traits. In microwave assisted extraction (MAE), rapid generation of heat and pressure within the

biological system forces out compounds from the biological matrix, producing good quality

extracts with better target compound recovery. The efficiency of the MAE process depends on

time, temperature, solid-liquid ratio, type and composition of solvent used (Hemwimon et al.,

2007). Combining microwave drying and pressing of avocado pulp seems to be able to led to a

superior quality avocado oil (Santana et al., 2015).

29

Ultrasound assisted extraction (UAE)

Introduction to sonochemistry: Sonochemistry studies the effects of ultrasonic sound waves on

chemical and physical reactions. Ultrasound is a sound which is at a frequency beyond the range

of human hearing (>20 kHz). Ultrasonic devices use transducers which convert electrical energy

to sound energy to produce an ultrasonic field. The use of ultrasound can be divided into two

separate areas; diagnostic and power ultrasound (Mason, 1990).

Diagnostic ultrasound is the most commonly known form of ultrasound. This employs high

frequency sound waves from 3-10 MHz as a diagnostic tool in the field of medical imaging.

Sound waves at this level of low power and high frequency do not leave permanent effects to the

medium that it is exposed to, making it ideal for non-invasive. The most common known use is

in foetal imaging; however it also has applications for detecting cancerous growth within the

body (Phull and Mason, 1999). Ultrasound uses a pulse/echo technique to produce an image

from within the body (Mason, 1990).

Power ultrasound employed for the disruption of microalgae, focuses on low frequency sound

waves that range from 20-100 kHz. The range of practical uses for power ultrasound is truly

enormous. It is employed in many industries such as engineering and biological sciences for uses

ranging from emulsification to impregnating dyes into leather (Sivakumar et al., 2007). Power

ultrasound induces permanent changes in the medium they are exposed to due to a mechanism

called acoustic cavitation (Mason, 1990).

Cavitation: When a liquid is exposed to low frequency sound waves (20-100 kHz) acoustic

cavitation bubbles are produced. These are microscopic bubbles which in a matter of

milliseconds form, grow and collapse, creating microscopic “hot spots” of extremely high

pressures and heat upon their collapse. These “hot spots” are of great interest because of the

range of their potential applications. The extreme conditions created by these “hot spots” lead to

excited states, bond breakage and free radical production within the medium. The advantages of

this from an industrial point of view is that these effects can lead to higher yields of output,

shorter reaction times and less hazardous conditions needed for chemical reactions to take place

(Bonrath, 2003).

30

In addition to cavitation, mechanical forces associated with ultrasound have also been shown to

disrupt cell walls and cause the release of compounds such as lipids (Cravotto et al., 2008).

Shock waves are an example of a physical mechanical force associated with ultrasound due to

waves of extreme pressure created outside the cavitation bubble after collapse (Mason and

Lorimer, 2002). When a cavitation bubble is formed near a solid surface, for an example the cell

wall of a microalgal cell, the cavitation bubble will form and collapse in a non-spherical shape.

When collapsing in this shape the bubble will form a high pressured localized liquid jet against

the solid surface. The combination of shock waves and liquid jet formation cause intense shear

forces within the liquid media to break open cell walls.

The use of ultrasound in combination with solvents, also known as Ultrasound Assisted

Extraction (UAE) to extract products from plant cells has been employed recently to extract

numerous products from plant cells. UAE is often found to increase the efficiency of the

extraction process. An example of this is the extraction of anti-oxidants from Rosmarinus

offcinalis in which UAE is more effective than traditional extraction and carried out at a lower

temperature with less solvent (Paniwnyk, 2009). UAE has also been employed to extract oil from

oleaginous crops such as soy bean, vegetables and seaweed. This technique resulted in an

increase in extraction efficiency of up to 50% from each source by comparison to the soxhlet

method (Cravotto et al., 2008).

Cravotto et al. (2008) developed an UAE method to extract oils from milled seaweed (SW),

macroalgae and milled pure soybean germ (SG). Firstly, lipids were extracted from the two

species using hexane in a separating funnel and by soxhlet extraction using hexane. The amount

of lipid extracted from SW was 2% - dry biomass for the separating funnel method and 4.8% for

the soxhlet extraction which took 4 hours. For SG, the separating funnel extracted a lipid content

of 3.5% of the dry weight and the soxhlet extraction removed 8.6%, but this method took 8

hours. Cravotto et al. (2008) employed a number of ultrasonic devices which were developed

and produced in-house. Generally, they recovered a lipid yield up to 24.7% for seaweed and

17.7% for soybean germ through changing some processing parameters.

Lou et al. (2010) developed an UAE method to extract oils from the chickpea plant. This

involved mixing 1g of crushed chickpea powder with 8.5 ml of hexane: isopropanol (3:1) in a 50

31

ml beaker. The sample was immersed in a 40 kHz ultrasonic cleaning bath (DL-360B, Shanghai

Zhixin Instruments Co. Ltd.) and sonicated for 90 minutes at 50°C. The ultrasonic power

entering this system was 230 W. After extraction the solvent was evaporated using a rotary

evaporator and the remaining oil quantified. Results indicated that the amount of lipids extracted

from the dry weight peaked at 80% after 30 minutes. This result was 35% higher than the amount

of lipids extracted using the same method but without ultrasound.

2.6.2. Factors that influence avocado oil yields

Avocado fruits with high oil content must be used in the production of oil. Various factors

however are known to affect the oil content of fruits and percentage oil yield during extraction

which may include:

Variety/Cultivar: - Different cultivars vary in oil content upon maturity and only those with high

oil content should be considered. Because the oil is contained in the pulp or flesh, cultivars with

high proportion of flesh and minimum seed and peel should also be selected (Human, 1987).

Many studies have confirmed the Hass cultivar to be superior in quality with all the favourable

attributes (Human, 1987).

Maturity stage: - The time at which the fruits of any given cultivar is harvested was noted by

Arpaia et al. (2006) to have the greatest impact on the oil content of the fruits. Maturity is when

the fruit is most suitable for human consumption and not for processing. Some cultivars mature

early while others mature much later and understanding this becomes very important for

choosing when to harvest. However it is understood that when avocado fruits mature their

moisture content lower while their oil increases and leaving the fruits on the trees much

longer after maturity tend to increase oil content (Human, 1987).

Location and growth conditions: - The study of avocado postharvest quality by Arpaia et al.

(2006) also noted differences in oil content for the same cultivar due to different locations and

growth conditions such as soil fertility. Sun exposed fruits were also found by Woolf et al.

(1999), to yield higher levels of oil than those fruits in the shade.

Particle Size: Oil extraction yield was higher as particle size decreased and contact time

increased. The effect of particle size is associated to an increase in cellular damage as particle

32

size decreases. This favors removal of the oil on the particle surface and diffusion of n-hexane

within the particle (Şaşmaz, 1996). Pre-extraction heat treatment causes expansion and rupture of

cell structures, which enhances material plasticity and permeability, it also facilitates oil release

and thus increase yield (Li, Bellmer, & Brusewitz, 1999).

Solvent to Sample ratio: The meal: solvent ratio marks the difference in oil yield up to a certain

extent. A high initial extraction rate is attributed to rapid solution of the oil on the solid’s surface

and a higher conduction mass transference force anticipated by the high solvent concentration. A

slower rate can be attributed to a lower motive force resulting from a lower solvent

concentration. Extraction with solvents is a mass transfer process in which materials (oils) are

moved from one phase to another to separate one or more compounds from a mixture (Giraldo et

al., 2010). The meal: solvent ratio is one of the most important variables in the extraction

process, such that at higher solvent proportions the mass transfer coefficient increases, producing

greater oil extraction. Material transfer from a particle by solvent effect is called leaching or

percolation. Leaching involves a complex mechanism that implies transfer of a solvent to the

surface of solid particles, penetration of the solvent into the solid matrix, incorporation of the

solute into the solvent by diffusion, and transfer of the solute into the bulk solvent (Adu-

Amankwa, 2006).

2.6.3. Avocado oil characteristics

Most of the beneficial attributes associated with eating avocados are mostly preserved in the oil

and for this reason is very valuable. The composition and in particular properties of the oil

varies according to how it is produced whether it be crude, virgin or refined according to the

method and number of successive operations involved in its production (Simental and Escalona,

2004).

According to Eyres et al. (2006); Botha (2004); Human (1987) study, avocado oil shows very

similarity with olive oil which has Acidity Value (as oleic) 2.0 – 0.08 %, Peroxide value 3.3-0.1

meq/kg fat), Iodine value 87-75 (from GLC), Specific Gravity 0.912- 0.916 (25oC). The acidity

value and peroxide values indicate stability in terms of minimal hydrolysis and lipase activities.

The oil is also free of cholesterol and carbohydrate.

33

The high peroxide value in crude oil might be as a result of the effect of moisture, atmospheric

oxygen and light on the oils leading to a progressive increase in the peroxide value. The

hydroperoxide formation may subsequently decompose into secondary oxidation products on

storage, majority of which have unpleasant odours or flavour (Jambunathan and Reddy, 1991).

The Iodine value is indicating a high degree of unsaturation. A typical avocado oil is comprised

mostly of monounsaturated fatty acids (74%), 11% polyunsaturated fatty acids and about 13%

saturated (Arpaia et al., 2006). These percentages vary slightly with cultivars and other

influential factors but the oil is very similar to olive oil. It is this high level of monounsaturated

fat which gives the desirable effect of being “anti-cholesterol” as it prevents the formation of

clots the major cause of coronary heart disease.

The high iodine value indicates dehydrogenation. It is a measure of unsaturation in lipid, which

again determines the degree of flow. Decrease in iodine value indicates lipid oxidation and this

might be due to metallic ions present among other factors, which enhances or promotes oxidation

after the formation of hydro-peroxide (Ruize et al., 1995).

2.7. Concluding remarks

Avocado is one of the climacteric fruits of an excellent nutritional quality with low sugar content

which makes avocado very recommendable source of high energy food for those who are

diabetic. It is classified into three horticultural races which are: Mexican, Guatemalan and West

Indian. The quality of avocado are mainly composed of physical properties (color, texture and

physical disorders), chemical (dry matter, TSS, TA, oil content and moisture content) and

sensory properties (flavor).

In Ethiopia it is one of the main fruits produced and exported including banana, citrus fruits,

mango, papaya and grape fruits (Zeberga, 2010). Owing to its shortest introduction to Ethiopia,

these days the crop is produced in several countries where Ethiopia stands the 10th leading

producer and 6th most important consumer in the world (FAOSTAT, 2010).

The lipid content in avocados varies greatly with the cultivar, and the same is observed for fatty

acid composition, which depends on growth rate and variety (Tango et al, 2004). Since Ethiopia

has a large agricultural sector and there is increasing interest in the growth of small and

34

developing farmers, if a new market for avocado fruit can be created by production of high

quality avocado oil at a premium price, more small and developing farmers would be interested

in cultivating avocado fruit. This will in turn benefit the agricultural and economic sectors in the

long term. Oil production can also create an alternative market for the commercial farmer,

which has the benefit of less risk, compared to the fresh fruit market where visual appearance of

the fruit is very important. Various factors however are known to affect the oil content of fruits

and percentage oil yield during extraction which may include: cultivar, maturity, Location and

growth conditions, Solvent to Sample ratio, particle size, type of solvent, extraction time etc.

Avocado pulp is enriched with oil which can be extracted by different extraction methods. A

number of new methods for extracting oils have been investigated in recent years, including

mechanical compression (Karaj and Müller, 2011), ultrasonic extraction (Ozkan et al., 2007),

microwave extraction (Kumaran and Karunakaran, 2007) and supercritical fluid extraction (Louli

et al., 2004). Compared with traditional Soxhlet extraction, ultrasonic extraction provides higher

selectivity, is less time-consuming, has lower energy consumption and reduced emissions (Ward,

et al., 1985). It is also environmentally friendly because most of the extraction solvent can be

recovered, and the equipment is inexpensive. So, application of ultrasonic extraction for

extraction of oil from avocado and optimization of its extraction condition should be done.

The composition and in particular properties of the oil varies according to how it is produced

whether it be crude, virgin or refined according to the method and number of successive

operations involved in its production (Simental & Escalona, 2004). Avocado oil shows very

similarity with Olive oil which has Acidity Value (as oleic) 2.0 – 0.08 %, Peroxide value 3.3-0.1

meq/kg fat), Iodine value 87-75 (from GLC), Specific Gravity 0.912- 0.916 (25oC).

35

CHAPTER THREE

3. Materials and Methods

3.1 Materials

3.1.1 Raw Material collection, transportation, storage and sample preparation

Avocado Fruit: Avocado fruit (Hass and Fuerte variety) were picked and collected at the mature

green stage of development from each cultivar of different stands on a farm of WARC

(Wondogenet Agricultural Research Center), SNNP regional State of Ethiopia. These fruits were

packed by carton box and transported to Food Engineering Laboratory, Addis Ababa Institute of

Technology, and some of them were transferred to Food and Human Nutrition laboratory, Addis

Ababa University and other food laboratory. Some of the fruits were stored at room temperature

for about a week and ripened. To keep ripening uniformity overlapping of fruits on storage were

avoided. The unripe and ripe avocado fruits were wiped off any dust, washed with distilled

water, dried and weighed. The pulp of the fruit was cut into halves from the stem to the tip end.

The seed and skin were removed and weighed, care was being taken to free the skin from

adhering to the pulp. The pulp and seed was oven dried, crushed and ground for chemical

property, proximate, mineral and phytochemical analysis. Also, its pulp was needed for oil

extraction.

Equipments:-The equipment used for the experiment were: knife, Computerized UV/Vis

Spectrophotometer (model UV-752), condenser, sieves, volumetric flasks, vacuum rotary

evaporator (Fisatom, model 801), drying oven (Tecnal, model DHG-9203A), furnace

(Gallenkamp, modelSX-2.5-10), coffee grinder (Mouliner,AR1044), tray, centrifuge (Labtech,

model AVI-558) refrigerator(WestPoint, model WRES-358.X), soxhlet, Sonicator, Kjeldhal,

Erlenmeyer flasks, sensitive balance (Electronic balance,FA2004B), pH meter, measuring

beaker, quantitative filter paper which is equivalent to what man No. 42., Laboratory

thermometer, spoon, pipette, micropipette, vortex mixer(DLAB ,model MX-SVB6F035754), test

tubes, cuvettes (1cm, 2ml plastic or glass), racer, Kjeldhal flask (model KDN-102F).

36

Chemicals:- The chemicals used in the experiment were: Hydrogen Peroxide, copper sulfate,

Vanillin reagent, Petroleum ether, hexane, Ethanol, Methanol, Folin Ciocalteu Reagent, Gallic

acid, D-catechin, sodium carbonate, ascorbic acid, aluminum chloride (AlCl3), Sodium nitrite

(NaNO2), Sodium hydroxide (NaOH), sulfuric acid, HCl, KI, sodium thiosulfate,

phenolphthalein indicator, boric acid, acetylene, acetic acid and starch were purchased and used

during the investigation.

3.2. Framework of the thesis

In this research the pulp and seed of two avocado cultivars (Hass and Fuerte) were selected and

their physicochemical properties, proximate, mineral and phytochemical composition were

determined. Furthermore, effects of different oil extraction methods on the yield of oil were

investigated and some of their extraction parameters were optimized. The extracted oil were

characterized and analyzed including its fatty acid profile.

Figure 3.1 Frameworks of the thesis

Pulp Seed

Mineral

Constituents

Mg

Ca

Fe

Zn

Na & K

Chemical

Properties

DM

TSS

pH

TTA

Physical

Properties

Weight

Flesh to

seed ratio

Oil chemical properties

PV

FFA

IV

AV, MVM & Alk.

Avocado variety (unri-

pe and ripe fuerte, hass)

)

Proximate

Analysis

Moisture

content

Protein

Ash

Crude fiber

Crude fat/ Oil

Carbohydrate

Phytochemical

Analysis

Total

Flavonoids

Total phenols

Phytates

Tannins

Oil Extraction using UAE

and soxhlet Extraction Oil Yield

Seed FA profile

Seed

37

3.3. Processing Methods

Two extraction techniques (Soxhlet and Ultrasonic) were used to obtain avocado oils that were

subjected to fatty acid and oil characteristic analyses. The effects of different extraction

conditions on the yield of oil were determined. Results were compared to evaluate the efficiency

of the extraction methods. Gas chromatography was used to determine the fatty acid composition

of the oil.

3.3.1. Extraction of oil using soxhlet Extraction

The extraction was carried out according to AOAC (2005) with some modifications. To

implement each of the experimental runs designed with the aid of Design Expert using the Box-

Behnken design of the response surface methodology, the dried and ground avocado pulp (ten

gram per sample) was packed inside a thimble bag and placed inside the thimble chamber of the

250 ml Soxhlet extractor. The extractor itself was placed inside a thermostatic water bath. A

round bottom flask containing n-hexane as well as a condenser was fixed to the extractor. The

flask was heated to a temperature above the boiling point of the solvent. The solvent then

vaporized and passed through the prepared sample to remove its oil. The mixture obtained

(solvent and oil) moved directly into the round bottom flask. The process was allowed to

continue for the specified time, as obtained from the experimental design. Thereafter, the oil

extracted was recovered by distilling the solvent using rotary evaporator. At the end of each

experiment, the yield of the oil was obtained using the following relationship.

𝑂𝑖𝑙 𝑌𝑖𝑒𝑙𝑑 =𝑊3 − 𝑊1

𝑊2∗ 100% (3.1)

Where, W1 is the weight of flask, W2 is the weight of sample, W3 is the weight of flask and

extracted oil

3.3.2. Extraction of oil using Ultrasound Assisted Extraction

The extraction was carried out according to Cravotto et al. (2008) and Lou et al. (2010) with

some modifications. Fresh avocado pulp were dried in an oven at 60°C until their weights were

constant, and they were then ground into powders and separated by particle size of 1.4mm, 2mm

and 2.6mm mesh. The general method for extraction involved weighing avocado powder (10 g)

into a 250 ml flask. Then the extraction solvent (hexane) was added depending on the solvent to

38

sample ratio and the flask was placed in the ultrasonic water bath. The following extraction

conditions were investigated for optimization: particle size of avocado pulp powder, extraction

time and solvent–solid ratio. After the extraction, the solution was reduced on a rotary evaporator

and the solvent was recovered. The avocado oil was weighed and the extraction yield was

calculated as above stated.

3.4. Analytical Methods

3.4.1. Physicochemical analyses

The weight of the whole fruit, separated pulp, seed, seed coat and peel were measured using a

weighing balance. The flesh to seed ratio was also done.

Percentage weight loss: Weights of avocado fruit were r`ecorded before and after storage at room

temperature, using an electronic balance (Sartorius BP 6100, R & M marketing, UK). The

difference in the weight was expressed as the percentage weight loss (Anthony et al., 2003).

Dry Matter Content Determination: The dry matter (DM) was determined according to the Lee

method (Lee, 1981). The petridishes used for the moisture determination were dried at 130 οC for

1hr using drying oven. The dishes were removed and kept in a desiccator for about 30 minutes.

The mass of empty dishes were measured as M1. This was regulated until constant weight was

obtained. About 10g of the sample was weighed using analytical balance in to the dish and

recorded as M2. The sample was mixed thoroughly and dried at 60 οC until it reached a constant

weight. And it was taken and kept in a desiccator to cool. After cooling the weight was taken as

M3 and then kept in oven for another 15 minutes. Then it was removed and allowed to cool in a

desiccator and again the weight was taken. This process was repeated until constant weight was

obtained. Then, the dry matter content was calculated using the following formulae:-

Dry Matter (%) = 𝑀3 − 𝑀1

𝑀2 − 𝑀1∗ 100 (3.2)

Where, M1 = weight of the dish, M2 = weight of the dish and the sample before drying, M3 =

weight of the dish and the sample after drying

39

Total soluble solids (TSS): Avocados were peeled and diced. About ten (10) g sample of

avocado pulp and seed was blended with distilled water (40 ml) in a homogenizer (Black &

Decker, BX 250, Hunt Valley, USA) for 2 min. The homogenate was filtered through a muslin

cloth and few drops of the filtrate was used to measure TSS using a hand-held Refractometer

(ATC-1E, ATAGO Co. Ltd., Japan). The readings were taken and results expressed in ºBx.

(AOAC, 1994).

pH: pH of the filtrates were measured using a digital pH meter (PC 510, EUTECH Instruments,

Singapore) (Anthony et al., 2003).

Titratable acidity (TA) (% acid): Ten (10) ml samples of filtrates prepared for the TSS test was

pipetted to a 50-ml beaker and titrated against 0.1 N NaOH with phenolphthalein as the pH

indicator until pink color was obtained. TA was calculated as number of milliliters of 0.1 N

NaOH multiplied by an appropriate conversion factor. The conversion factor of 0.075 was

chosen based on tartaric acid, a predominant acid in avocado. TA, expressed as % tartaric acid

(AOAC, 1994) can be calculated as follows:

𝑇𝐴 (%) =ml NaOH x N(NaOH) x acid meq.factor

𝑚𝑙 𝑗𝑢𝑖𝑐𝑒 𝑡𝑖𝑡𝑟𝑎𝑡𝑒𝑑∗ 100 (3.3)

3.4.2. Proximate Composition Analyses

Moisture Content Determination (AOAC 925.09, 2000)

The aluminum dishes used for the moisture determination were dried at 130οC dried for 1hr

using drying oven. The dishes were removed and kept in a desiccator for about 30 minutes. The

weight of empty dishes were measured as M1. This was regulated until constant weight was

obtained. About 5g of the sample was weighed using analytical balance in to the dish and

recorded as M2. The sample was mixed thoroughly and dried at 60οC until constant weight

obtained. And it was taken and kept in a desiccator to cool. After cooling the weight was taken as

M3 and then kept in oven for another 15 minutes. Then it was removed and allowed to cool in a

desiccator and again the weight was taken. This process was repeated until constant weight was

obtained. Then, the moisture content was calculated using the following formulae:-

40

MC (%) = 𝑀2 − 𝑀3

𝑀2 − 𝑀1∗ 100 (3.4)

Where, M1 = Mass of the dish, M2 = Mass of the dish and the sample before drying, M3 = Mass

of the dish and the sample after drying.

Crude Protein Analysis (AOAC 979.09, 2000)

About 0.5g of powdered sample was weighed on analytical balance and transferred to the

digestion flask. Then 6ml of H2SO4 and 3.5ml of 30% H2O2 was added in to the digestion flask

step by step. The tubes were shaken observing a violent reaction. After this violent reaction

disappeared 3g of the catalyst mixture (Cu2SO4: K2SO4) was added in to the digestion flask. The

solution was then digested at 370 οC for 4hr. After digestion was completed, the content in the

flask was diluted by distilled water and concentrated sodium hydroxide (40%) was added to

neutralize the acid and to make the solution slightly alkaline.

(NH4)2SO4 + 2NaOH 2NH3 +2H2O +Na2SO4

The ammonia was then distilled into a receiving flask that consisted solution of excess boric acid

(4%). The borate ion was formed as a result of the reaction of the boric acid and the ammonia

and this was titrated with standard acid (0.1N HCl) until the green color changes to pink.

The total nitrogen content was calculated using the following formulae:

NH3 + H3BO3 NH4+ + H2BO3

-

Nitrogen(%) = 𝑉𝐻𝐶𝑙∗𝑁𝐻𝐶𝑙 ∗14.01

𝑀∗ 100 (3.5)

% Protein = % N ∗ 6.25 (3.6)

V HCl = Volume of HCl consumed until the end point of titration, N HCl = Normality of HCl,

14.01= Molecular weight of nitrogen, M = Weight of sample on dry basis

Crude Fat Determination (AOAC 4.5.01, 2000)

The flasks used for the extraction were washed and then dried in drying oven at 92 οC for 1hr

and cooled in a desiccator. The weight of the cooled flasks were measured by analytical balance

and recorded as M1. About 2g of the powdered sample was weighed in to each thimble lined with

cotton at their bottom. The thimbles with its sample content were placed in to the Soxhlet

extraction apparatus. Then 50 ml of petroleum ether was added in to each flask and the

41

extraction process was done for about 4hr followed by removing this flask with its content

from the Soxhlet, it was placed in drying oven at 92οC for 1hr. The flasks with their contents

were then placed in a desiccator for 30 minutes. The weight of each flask together with its fat

contents was measured as M2. Then, the total lipid amount was calculated using the following

formulae:-

Fat(%) = 𝑀2 − 𝑀1

𝑀∗ 100 (3.7)

Where, M2= weight of flask and extracted lipid, M1= weight of dried flask, M = weight of

sample on dry basis.

Crude Fiber Determination (AOAC 962.09, 2000)

About 1.6g of the sample was weighed in 600 ml beaker using analytical balance. About 200 ml

of 1.25% H2SO4 solution was added to each beaker and allowed to boil for 30 minutes by

stirring and rotating it periodically on a hot plate. During boiling the level was kept constant by

addition of hot distilled water. After 30 minutes 20ml of 28% potassium hydroxide was added

and again allowed to boil for another 30 minutes. The level was still kept constant by addition of

hot distilled water. Then after 30 minutes the solution was filtered through crucibles containing

calcite sand by placing it on (Buchner funnel fitted with No.9 rubber stopper). During filtration

the sample was washed with hot distilled water followed by 1% H2SO4, hot distilled water, 1%

NaOH and finally with acetone. The crucible with its content was dried for 2h at 130 ±2οC in

drying oven and cooled in a desiccator and weighed as M1. Then it was ashed for 30 minutes at

550οC in muffle furnace and was cooled in a desiccator. Finally, it was weighed as M2. The

difference in the weights M1-M2 represents the weight of fiber (Raghuramulu et al., 2003).

Crude Fiber(%) = (100 − (Moisture + Fat)) ∗ (𝑀1 − 𝑀2)

M∗ 100 (3.8)

Where, M1= weight of the crucible, the sand and fiber M2= weight of the crucible and the sand

M = Weight of sample on dry basis

Crude Ash Determination (AOAC 923.03, 2000)

The porcelain dishes used for the analysis were washed by dilute hydrochloric acid on boiling

and washed with distilled and de-mineralized water respectively. Then dried at 120 οC in an

42

oven and ignited at 550 οC in furnace for 30 minutes. Then the dishes were removed from

furnace and cooled in a desiccator. The weight of the dish was measured as M1. About 2.5g of

sample powder was being weighed in to the porcelain dish and recorded as M2. The sample was

charred at 120 οC for 4h in a hot plate, until the whole content becomes carbonized. Then the

sample was placed in a furnace at 550 οC until free from carbon and the residue appears grayish

white after 5h. The sample was removed from the furnace and placed in a desiccator.

Finally the mass was weighed as M3. And the total ash content was calculated with the following

formulae:-

Crude Ash (%) = 𝑀3 − 𝑀1

𝑀2 − 𝑀1∗ 100 (3.9)

Where, M1=weight of the dried dish/ crucible, M2= weight of the dish and the sample, M3=

weight of the dish and the ash

Total and Utilizable Carbohydrate Determination

The total carbohydrate was calculated by difference and Utilizable carbohydrate was calculated

by difference with the exclusion of crude fiber. This is expressed mathematically as follows:

% Total carbohydrate = (100 − (% MC + % protein + % ash + % fat + %fiber (3.10)

% Utilizable carbohydrate = (100 − (% MC + % protein + % ash + % fat (3.11)

Total energy calculation in kilo Calories

The total energy content in each sample was calculated as follows:

Total energy (kcal) = ((Carbohydrate × 4) + (Protein × 4) + (Lipid × 9)) (3.12)

3.4.3. Mineral Composition Analyses

The ash was dissolved by 5 ml of 6 N HCl at low temperature on hotplate for about 2 hrs. Then,

7 ml of 3 N HCl was added and heated on a hot plate until the solution boils. The digest was

cooled and filtered through a filter paper (42 mm, whatmann) in to a 50 ml volumetric flask.

Then 5 ml 3 N HCl was added to the dishes and heated to dissolve the residue in the dishes and

then transferred to the volumetric flask. Then the filter paper was washed thoroughly and the

washing was collected in the flask made to the mark. After wards the mineral concentration was

43

determined by AAS. For calcium determination 2.5 ml of 10 % Lanthanum chloride solution was

added to the flask. Then diluted to 50 ml mark with de-ionized water. The blank was prepared

by taking the same amount of reagents through the steps all of the above without sample.

The instrument was set based on the instruction given in the manual. The calibration solutions

and the reagent blank solutions were measured first. Then the samples were run following the

calibration values. The calibration curve was prepared for the required metal by plotting

the absorption values against the metal concentration in ppm. The mineral contents of each

sample were calculated using the following formulae:-

Metal content (mg/100g) = (𝑎−𝑏)∗𝑉

10∗𝑤∗ 100 (3.13)

Where; W = weight of the sample in g, V = volume in ml of the extract, a= concentration in ppm

of sample solution, b= concentration in ppm of blank solution.

3.4.4. Phytochemical Analyses

Phytate

The phytate content in the sample was determined according to the method described by Latta

and Eskin (1980) method, and later modified by Vaintraub and Lapteva (1988) with some

modifications. About 0.05g of seed and 0.4g of pulp of dried sample was extracted with 10ml of

2.4% HCl in methanol for 1hr at ambient temperature and centrifuged (3000 rpm) for 30

minutes. The clear supernatant was used for the phytate estimation. About 2ml of Wade reagent

(0.03% solution of FeC13.6H2O containing 0.3% sulfosalicylic acid in water) was added to

3ml of the sample solution and the mixture was centrifuged. The absorbance at 500 nm was

measured using spectrophotometer. The phytate concentration was calculated from the difference

between the absorbance of the control (3ml of water + 2ml Wade reagent) and that of the assayed

sample. The concentration of phytate was calculated using phytic acid standard curve and the

results were expressed as of phytic acids in mg per 100g dry weight.

To prepare the phytic acid standard curve, a series of standard solution was prepared containing

5–40 mg/ml phytic acid in water. About 3ml of the standards was pipetted into 15ml centrifuge

tubes with 3ml of water used as a zero level. To each tube was added about 1ml of the wade

44

reagent, and the solution was mixed on a vortex mixer for 5s. The mixture was centrifuged for 10

minutes and the supernatant read at 500ηm was read by using water as a blank.

phytic acid in µg/g = [(𝐴𝑠 − 𝐴𝑏) − 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡] ∗ 10

𝑠𝑙𝑜𝑝𝑒 ∗ 𝑊 ∗ 3 (3.14)

Where, As= sample absorbance, Ab= blank absorbance, W= weight of sample

Tannin analysis

Tannin content was determined by the method of Burns (1971) with some modifications. About

two gram of sample was weighed in a screw cap test tube and extracted with 10ml of 1% HCl in

methanol for 24 hours at room temperature with mechanical shaking. After 24 hours shaking, the

solution was centrifuged at 1000rpm for 5 minutes. About 1ml of supernatant was taken and

mixed with 5 ml of vanillin-HCl reagent (prepared by combining equal volume of 8%

concentrated HCl in methanol and 4% vanillin in methanol).

D-catechin was used as standard for condensed tannin determination. A 40mg of D-catechin was

weighed and dissolved in 1000 ml of 1% HCl in methanol, which was used as stock solution.

About 0, 0.2, 0.4, 0.6, 0.8 and 1 ml of stock solution was taken in test tube and the volume of

each test tube was adjusted to 1ml with 1% HCl in methanol. About 5ml of vanillin HCl reagent

was added into each test tube. After 20 minutes, the absorbance of sample solutions and the

standard solution were measured at 500nm by using water to zero the spectrophotometer, and the

calibration curve was constructed from the series of standard solution using SPSS-20. A standard

curve was made from absorbance versus concentration and the slope and intercept were used for

calculation. Concentration of tannin was read in mg of D-catechin per 100gm of sample.

Tannin inmg

100g=

𝑠𝑎𝑚𝑝𝑙𝑒 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 − 𝑖𝑛𝑡𝑒𝑟𝑐𝑒𝑝𝑡

𝑠𝑙𝑜𝑝𝑒 ∗ 𝑑𝑒𝑛𝑠𝑖𝑡𝑦 ∗ 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 ∗ 10∗ 100 (3.15)

Total Phenol and Total Flavonoid Determination

Extraction: The phenolic compounds were extracted according to the method reported by

Singleton and Rossi (1965) with slight modifications. The avocado pulp and seed was ground

45

into powder in a mortar and about 5g of ground sample was accurately weighed in a screw-

capped tube.

The phytochemicals were extracted with 50ml of methanol and vortexed for 1 min. The samples

were digested overnight, filtered and the filtrate was evaporated using rotary evaporator and then

stored mixing with methanol based on the obtained yield. The supernatant was used for

determination of total phenolics and total flavonoids.

Determination of total phenolic content: Total phenolic contents of the fruit pulp and seed

extracts were measured using a modified colorimetric Folin-Ciocalteu method (Singleton and

Rossi, 1965) with further slight modifications. The Folin–Ciocalteu (F–C) reagent is sensitive to

reducing compounds, polyphenols and thus produces a blue colour complex. Fruit extracts (0.5

ml) were placed in a test tube. Folin-Ciocalteu reagent (2.5 ml) was added to the solution and

allowed to react for 3 min. The reaction was neutralized with 2 ml of sodium carbonate (7.5 %).

Absorbance at 765 nm was read after 30 min. Gallic acid was used as standard and data were

expressed as mg Gallic acid equivalents (GA)/ g FW.

Determination of total flavonoid content: The total flavonoid content of the samples was

measured using a colorimetric method (Zhishen et al., 1999; Dewanto et al., 2002). The

methanolic extract (250 µl) was mixed respectively with 1.25 ml DI water and 75 µl of 5%

NaNO2 solution, then allowed to mix for 6 min. After addition of 150 µl of 10% AlCl3 solution

and mixing for 5 min, the reaction was initiated by adding 0.5 ml of 1 N NaOH and the total

volume was made up to 2.5 ml with DI water. Sample absorbance was read at 510 nm using a

UV/vis spectrophotometer. (+)- Catechin standard was used as standard and total flavonoid

content was expressed as µg (+)-catechin equivalents (CE)/g FW.

3.4.5. Quality characterization of extracted avocado oil

Determination of peroxide value (PV): AOAC 965.33

About Five grams of the oil was dissolved in 30 ml of glacial acetic acid: chloroform (3:2, v/v).

0.5 ml of saturated KI was added and iodine was liberated by the reaction with the peroxide. The

solution was then titrated with standardized sodium thiosulphate using starch indicator. The

peroxide value (PV) was determined as follows:

𝑃𝑉 (𝑚𝐸𝑞/𝐾𝑔) =(S − B) x N x 1000

Sample weight (g) (3.16)

46

Where S = Sample titre value, B = Blank titre value, M = Molarity of Na2S2O3.

Determination of Iodine value (IV)

Iodine value of the oil was assayed according to the titration method of Pearson (1970). About

two gram of oil sample was weighed into a dry 250ml glass stopper bottle and 10ml of carbon

tetrachloride was added to the oil. About 20ml of Wij’s solution was then added and allowed to

stand in the dark for 30 min. 15 ml of 10% potassium iodide and 100 ml of water were added and

the resulting mixture was then titrated with 0.1 N sodium thiosulphate solution using starch as

indicator just before the end point. A blank determination was carried out alongside the oil

samples. The iodine value was calculated as follows:

𝐼𝑜𝑑𝑖𝑛𝑒 𝑣𝑎𝑙𝑢𝑒 (𝐼𝑉) =(B − S) x Nx 12.69

Sample weight (g) (3.17)

Where B = blank titre value, S = sample titre value, N = Normality of Na2S2O3, 12.69 =

Conversion factor from Meq. Na2S2O3 to gram iodine, molecular weight of iodine is 126.9 g

Determination of percentage free fatty acids (%FFA): AOAC 940.28

About two grams of well-mixed sample was accurately weighed into a conical flask in to which

10 ml of neutralized 95% ethanol and phenolphthalein were added. This was then titrated with

0.1 N NaOH, shaking constantly until a pink colour persisted for 30 s. The percentage free fatty

acid was calculated from Equation 3.18:

%𝐹𝐹𝐴 =V × N × 2.82 mg

Sample weight (g) (3.18)

Where V=Volume of NaOH, N= Normality of NaOH, 2.82=Conversion factor for oleic acid

Acid value determination: AOAC 940.28

Acid value is defined as the milligrams of KOH required for neutralization of free fatty acids

present in one gram of oil. About ten ml of neutral alcohol was added to two gram of avocado oil

sample and titrated against KOH solution. The acid value was calculated using the formula:

AV =(B−S)∗N∗56.11

M (3.19)

Where, B, S = is the volume in ml of standard KOH required to titrate the blank and the sample

respectively, N = is the normality of KOH solution/NaOH solution, M = is the weight in g of the

oil (sample), 56.1 = molecular weight of KOH.

47

Moisture and volatile matters – oven method: AOAC 926.12

About 5g prepared test sample were weighed in to a vessel which has been previously dried for

30 minutes at 105oC and weighed. The sample within the vessel was dried to constant weight at

105 oC and cooled in the desiccators for 30 minutes and weighed. % loss in weight was reported

as moisture and volatile matter.

% Loss in weight =𝑊1 − 𝑊2

𝑊1∗ 100 (3.20)

Where W1 = Weight before drying, W2= Weight after drying

Determination of Soap Content: ISO 10539

About 40g of the test portion was weighed and added in to the test tube, which has been

previously well rinsed with the test solution (prepared by adding 0.5ml of the bromophenol

blue indicator to each 100ml of the aqueous acetone just before use and titrating with 0.01N

acid until it is just yellow in color). About 1 ml of water, warmed on the steam bath was

added to it and shaken vigorously. About 50 ml of the neutralized aqueous acetone was then

added and after warming on the steam bath, the test tube was well shaken and the contents

allowed standing until they separate into two layers. If soap is present in the oil, the upper

layer will be colored green or blue. Then, 0.01 N HCl shall be added preferably from micro

burette, until the yellow color is restored. The process of warming and shaking was continued

until the yellow color in the upper layer remains permanent. In order to readily perceive

difference in color between the layers, a blank determination was carried out. Dissolved soap,

as sodium oleate, was calculated as follows:

% by mass =0.304 ∗ V

M (3.21)

Where, V = volume of 0.01N HCl, ml; M = weight of the test portion used, g

Fatty Acid Analysis of extracted avocado oil

Conversion of Triglycerides to Fatty Acid Methyl Ester (FAME): Fatty acids were transformed

to their methyl esters (FAME) following AOAC method. About 0.5g of lipids were weighed and

immediately resuspended in 5 ml of chloroform and stored at -20°C. To determine its fatty acid

48

composition, a 50 µl subsample of the lipid-in- chloroform was treated with 100µl of 0.5 N

sodium methoxide in methanol (prepared with a solution of dimethoxypropane and methanol

(95:5, v/v). Esterification of fatty acids to fatty acid methyl esters (FAME) was completed after

standing at room temperature for 15 minutes. Sulfuric acid (400 µl of 0.125 N) was added and

fatty acid methyl esters were recovered in 7.5 ml of petroleum ether (boiling point 60-80°C).

Gas Chromatography Analysis: About one µl of fatty acid methyl esters in petroleum ether was

injected into the gas chromatograph (Hewlett Packard model 5890A), equipped with a Supelco

fused silica capillary column No. 11484-02A, catalogue No. 2-4019 (30 m x 0.25mm ID x 0.2

µm film Mfg.) and a flame ionisation detector (FID). The temperature was 100°C initially, then

increased by 15°C per minute to 190°C and held at 190°C for 25 minutes. Injector and detector

temperatures were at 200 and 220°C respectively. The fatty acid peaks in lipid samples were

identified by comparison with the retention times of fatty acids in the standard mixture, and the

amount calculated as a percentage of the total lipids and as grams of fatty acid per 100 grams of

fruit (fresh weight).

3.5. Experimental Design and Statistical Data Analysis

Response Surface Methodology: Response surface methodology is a powerful technique that

allows optimization and determination of the best conditions to maximize the desired responses.

There are different multilevel designs that have been used for the optimization of variables in

many studies such as central composite design (CCD) and Box–Behnken design (BBD) (Grosso

et al., 2014).

The Box-Behnken design was selected in this study as it has a higher efficiency compared with

CCD and is more efficient than a factorial design (Ferreira et al., 2007). The optimum

conditions were found by analyzing the response surface plots aiming for the highest reachable

response variable for each independent parameter.

There were two investigations: characterization of avocado and Optimization of different oil

extraction conditions. For characterization of avocado there were, avocado variety (Hass and

Fuerte) of pulp parts and seed parts and condition of avocado (unripe and ripe). For Optimization

there were two extraction methods:

49

1) Ultrasound assisted extraction with three factors and three levels: Particle size (1.4mm, 2mm,

2.6mm), Solvent to sample ratio (5:1, 10:1, 15:1) and extraction time (30, 60, 90 min).

2) Conventional soxhlet extraction with three factors and three levels: Particle size (1.4mm,

2mm, 2.6mm), and sample to solvent ratio (15:1, 20:1, 25:1) and Extraction time (4, 6, 8 hour).

Data were statistically analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s

test using SPSS (version 20) and design expert. After obtaining the responses (oil yield) from

each of the experiments, the values (the responses) were entered into the appropriate column in

Design Expert, and the response together with the factors considered were analysed, and a

quadratic models relating the percentage oil yield to the factors considered were developed for

the extraction. In order to get a model that would best fit the experimental results and take care

of effects of all the factors, the developed models were modified. The modifications carried out

resulted in improvements in the performances of the models. Using the modified models, a

numerical optimization was carried out and the optimum conditions of the factors investigated

for the extraction were obtained.

50

CHAPTER FOUR

4. Results and Discussion

4.1.Physical properties of avocado

The Physical properties of avocado were shown in Table 4.1 below. From Table 4.1, the average

weight of Hass avocado variety was determined to be 183.92 ± 47.58g using replicates of five

fruits; this is less than the value of 0.374kg of Hass variety of avocado as reported by FAO,

(1997). From this cultivar its pulp, seed, seed coat and peel comprises 78.82±4.35%,

8.14±4.29%, 0.33±0.04% and 12.71±3.45 % respectively, which yields higher pulp based on the

total fruit weight.

The average weight of Fuerte avocado variety was determined to be 263.98±58.85g using

replicates of five fruits; this is greater than the weight value of Hass variety of avocado studied

here. From this variety its pulp, seed, seed coat and peel comprises 70.78±2.63% 19.83±2.69%,

0.73±0.36% and 8.67±2.64% respectively, which yields lower pulp than Hass variety. This fruit

comprises pulp to seed ratio of 3.63±0.6 which is less than that of the Hass variety (13.57±10.4).

In a study conducted by Tango et al. (2004), the avocado pulp content ranged between 52.9 and

81.3% of the fruit weight, in the several varieties analyzed which is similar to this findings. Up

on ripening Fuerte avocado cultivar shows greater weight loss than Hass variety.

Table 4.1 Physical properties of avocado

Fruit

variety

Wt. of

fruit (g)

% of

pulp

% of

seed

% seed

coat

% of

peel

Pulp to

Seed ratio

Weight

loss after 7

days

Hass 183.92±47.58 78.82±4.35 8.14±4.29 0.33±0.04 12.71±3.45 13.57±10.43 6.38±1.31

Fuerte 263.98±58.85 70.78±2.63 19.83±2.69 0.73±0.36 8.67±2.63 3.63±0.61 10.33±2.09

Data are means of replicates of five fruits determination ± standard deviation

4.2. Chemical properties of avocado pulp and seed

The results of the dry matter contents of avocado pulp and seed have been shown in Table 4.2.

From Table 4.2, dry matter contents of the avocado pulp were 35.08±00, 22.19±0.19, 31.2±0.09

and 22.24±0.01% for unripe hass, unripe fuerte, ripe hass and ripe fuerte respectively. These indicated

that dry matter content of Hass pulp is greater than that of fuerte pulp for both unripe and ripe

and it was decreased as the fruit ripened for Hass, but nearly similar for Fuerte. Dry matter

51

contents of the seed were 48.4±1.02, 45.48±1.17, 45.91±0.03 and 38.17±0.23 for unripe hass, unripe

fuerte, ripe hass and ripe fuerte respectively. These indicated that dry matter content of Hass seed is

greater than that of fuerte seed and it was decreased for the ripened fruit. Villa-Rodriguez et al.

(2011) found that the dry matter had increased from 31.65 to 36.52% over eight days at 15°C and

thereafter decreased to 32.91 on the day 12. High dry matter content implies that the fruit

contains high oil percentage and low moisture content.

The result of the pH value of avocado pulp and seed is shown in Table 4.2. The obtained pH

value of the pulp were 6.67±0.02, 6.58±0.11, 6.75±0.04 and 6.69±0.01for unripe hass, unripe

fuerte, ripe hass and ripe fuerte respectively. The study revealed that hass pulp has greater pH

than fuerte pulp and it is slightly increased as the fruit ripened. The results in the Table 4.2

shown that the pH value of avocado seed were between 4.68±0.11 and 5.24±0.03. The minimum

pH corresponds to UFS and the maximum belongs to RHS. UHP and RFP had a pH value of

4.96±0.06 and 5.19±0.12 respectively. The seed had lower pH than the pulp and it was greater for

Hass seed. The obtained pH value of avocado pulp is comparable to the result (6 to 6.5) and (6.4

to 7.0) reported by Soliva-Fortuny et al. (2004) and Lange and Kader (1997) respectively. Since

pH of fruit is nearest to 7 it implies that avocado pulp is neutral (neither acidic nor basic).

The analysed total soluble solid of avocado pulp were 6.55±0.04, 5.70±0.04, 5.77±0.06 and

4.95±0.22°Bx for unripe Hass pulp, unripe Fuerte pulp, ripe Hass pulp and ripe Fuerte pulp

respectively. These indicate that pulp of Hass avocado variety contains higher TSS than Fuerte,

which may be due to the difference in variety and maturity level. From the obtained result there

was a slight decrease as the fruit ripens for both varieties which may due to the consumption of

stored carbohydrate during respiration process. Liu et al. (1999b) demonstrated that ripening of

avocados at 20°C resulted in a considerable decline in the TSS in the peel and flesh. Similar

pattern was also observed by Rathore et al. (2007). It was observed that total soluble solids

contents were increased from 10 to 16.23 % up to 6 day of their storage and thereafter, a gradual

decrease up to (6.13 %) was observed after 15th days of storage period. The increase in TSS

might be due to the alteration in cell wall structure and breakdown of complex carbohydrates

into simple sugars during storage. This increase and decrease in TSS are directly correlated with

hydrolytic changes in starch and conversion of starch to sugar being an important index of

ripening process in mango and other climacteric fruit and further hydrolysis decreased the TSS

52

during storage (Kays, 1991). The obtained avocado pulp TSS was superior to those reported for

‘Hass’ avocados of American origin (5.1±0.1°Brix) (Arias et al., 2012), but lower than those

reported for ‘Hass’ avocados from New Zealand (~9° Brix) (Burdon et al., 2007). So, this low

sugar content makes it an ideal food source for diabetics.

The total soluble solid of avocado seed was 3.39±0.01, 3.26±0.08, 3.35±0.04 and 3.25±0.01 for

unripe Hass seed, unripe Fuerte seed, ripe Hass seed and ripe Fuerte seed respectively. These

indicate that seed of Hass avocado cultivar contains higher TSS than Fuerte, which may be due

to the difference in variety and maturity level. From the obtained result there is nearly similar

TSS between unripe and ripe fruit seed varieties. The obtained avocado seed TSS was lower than

its pulp and those reported for Algarvian Hass avocados (3.54±1.97°Brix) reported by Ana et al.

(2013).

The result of the total titratable acidity of avocado pulp and seed has been reported in Table 4.2.

The pulp of Hass avocado cultivar contains lower TA than Fuerte and there was a decrease in TA

of ripened fruit for both varieties which may be due to the consumption of organic acid during

respiration as the fruit ripens and increasing its pH. The obtained acidity of the pulp was found to

be superior to that exhibited by ‘Hass’ avocados of American origin (0.04±0.01% citric acid)

(Arias et al., 2012), but lower than the value of Algarvian Hass avocados (1.07±0.02) for the ripe

pulp as reported by Ana et al. (2013). The difference might be due to the difference in

environmental location, maturity stage and harvesting season.

According to Hernández-Muñoz et al. (2006) the total acidity is a measure of the organic acid

content. The predominant acid found in avocados is tartaric acid although, theoretically, every

species capable of donating a proton, including fatty acids, also contribute to the total acidity of

the fruit (Omar et al., 2012). It has been suggested that during storage, fruits utilize organic acids

for metabolic activities and this results in a decrease in the TA content during the storage periods

which is similar with the present findings.

The total acidity tends to decrease during the ripening period as a result of the breathing process

or conversion in to sugars. These results coincided with those Doreyappa-Gowda and Huddar

(2001), who reported the similar pattern in different varieties of mango fruit stored at 18-34οC

under gone a series of physico-chemical changes during ripening and the major changes were

53

considerably increased in pH from 2.85 to 4.38 and decreased in acidity from 2.71 to 0.04%

during ripening. These results further correspond with Srinivasa et al. (2002), who found that

titratable acidity values of Alphonso mango either packed in carton or control sample also

showed a decreasing trend from 2.17% to 0.08% on 12 day when stored at ambient temperature

27±1οC and 65% RH.

The total acidity of avocado seed were 2.60±0.04, 2.88±0.04, 2.45±0.03 and 2.44±0.04 for unripe

Hass seed, unripe Fuerte seed, ripe Hass seed and ripe Fuerte seed respectively, which indicates

that the seed of Hass avocado cultivar contains lower TA than Fuerte and there is a decrease in

TA as the fruit ripens for both varieties which may be due to the consumption of organic acid

during respiration as the fruit ripens and increasing its pH. In case of comparing total acidity of

the pulp and seed it is superior in its seed due to highly presence of organic acid and low pH in

its seed and the obtained total acidity of the seed was lower than the result of Algarvian Hass

seed (2.67±0.17) reported by Ana et al. (2013).

Table 4.2 Chemical properties of avocado pulp and seed

consti

tuents

Avocado variety

Hass Fuerte

Pulp Seed Pulp seed

Unripe ripe unripe Ripe unripe Ripe unripe ripe

DM 35.08±

0.00 d

31.2±

0.09 e

48.4±1

.02 a

45.91±

0.03 b

22.19±

0.19 f

22.24±

0.01 f

45.48±

1.17 b

38.17±

0.23 c

pH 6.67±0.

02a

6.75±

0.04a

4.96±0

.06 bc

5.24±0

.03 b

6.58±0.

11a

6.69±0.

01a

4.68±0

.11 c

5.19±0

.12b

TSS 6.55±0.

04a

5.77±

0.06b

3.39±0

.01d

3.35±0

.04 d

5.70±0.

04b

4.95±0.

22 c

3.26±0

.08 d

3.25±0

.01 d

TA 1.30±0.

03 e

0.65±

0.02 g

2.60±0.

04 b

2.47±0.

06 bc

1.78±0.

06 d

0.90±0.

04 f

2.88±0.

02 a

2.44±0.

02 c

* Data are means of duplicate determination ± standard deviation

The mean values followed by different superscript letters in the same row are significantly different (p ≤

0.05) according to the Tukey’s multiple range test.

4.3. Proximate composition of avocado pulp and seed

Tables 4.3 showed the proximate analysis of unripe pulp, unripe seed, ripe pulp and ripe seed of

both Hass and Fuerte avocado varieties. From the result, Fuerte avocado pulp had higher

moisture content than Hass and the moisture content was slightly decreased as the fruit ripened

for fuerte, but increased for Hass variety. The moisture content of avocado seed were

54

51.60±1.02, 54.52±1.17, 54.09±0.02, and 61.83±0.23% for unripe Hass seed, unripe Fuerte seed,

ripe Hass seed and ripe Fuerte seed respectively. Its ripe contains high moisture content relative

to unripe. The obtained moisture content for fuerte was nearly similar with the result (77.72%)

reported by Orhevba and Jinadu (2011).

The obtained protein content of avocado pulp were 2.92±0.04, 2.25±0.05, 2.84±0.08 and

1.77±0.03% for unripe Hass pulp, unripe Fuerte pulp, ripe Hass pulp and ripe Fuerte pulp

respectively. These values were closer to the values of 2g and 1.72g obtained by USDA, (2009)

and FAO, (1989) respectively. The protein content of Hass pulp was greater than that of Fuerte

pulp for both unripe and ripe fruits and its content was decreased as the fruit ripened. The result

was nearly similar with California and Florida avocado, where the protein content ranged from

1.21 to 2.26% as reported by Hall et al. (1955) and Slater et al. (1975) respectively. The study

revealed that avocado seed had high protein content than its pulp for both Hass and Fuerte

cultivars, which indicates the possibility of its seed in the development of some products. The

protein content of unripe fuerte seed is greater than that of unripe Hass seed and its content

decreases as the fruit ripens for fuerte seed, but shows a slight increase for Hass seed.

The evaluated ash content of avocado pulp were 0.42±0.00, 0.27±0.00, 0.37±00 and 0.27±00%

for the corresponding unripe hass pulp, unripe fuerte pulp, ripe hass pulp and ripe fuerte pulp

respectively. This shows that ash content of Hass pulp was greater than that of fuerte pulp and

decreased as the fruit ripens for Hass pulp, but constant for Fuerte pulp. The obtained result was

nearly similar to the range (0.4-1.68%) reported by FAO (1989) for fuerte variety. Similar to the

pulp, ash content of Hass seed was greater than that of fuerte, but its content decreased as the

fruit ripened for seed of both varieties. Similar pattern was also observed by Olaeta et al. (2007)

with slight variation, who reported decrease of ash content for ripe Fuerte seed but increase for

ripe Hass seed. Generally, the ash content of studied avocado seed and pulp were ranged

between 0.46 to 0.77 and 0.27 to 0.42% of the fresh-weight material respectively. This ash

content of avocado pulp and seed was significant in that it contains nutritionally important

minerals.

The fat content of avocado pulp were 22.80±0.00, 14.48±0.08, 22.07±0.11 and 15.03±0.00% for

the corresponding unripe hass pulp, unripe fuerte pulp, ripe hass pulp and ripe fuerte pulp

55

respectively; this is higher than the value of 9.1% reported by Ikhuoria and Maliki (2007). The

obtained lipid contents of the fuerte pulp was comparable to the result (15.39%) reported by

Pushkar et al. (2001), but lower than the result (22.1 %) and (20%) reported by Tango et al.

(1972) and Freitas et al. (1993) respectively. The obtained result indicate that pulp of Hass

avocado cultivar contains higher oil content than Fuerte, which may be due to the difference in

variety and maturity level. From the obtained result there is a slight decrease for the ripe fruit of

Hass cultivar, but a slight increase for fuerte cultivar. This may be due to a slight increment of

moisture content in Hass and a slight decline of it in Fuerte cultivar, since as the moisture content

increases the fat/oil content decreases. Lipid concentrations ranging from 15 to 30% in Fuerte,

Bacon and Hass cultivars were also reported by Biale and Young (1971). Also the fat content of

seed is higher in Hass cultivar, but in both the fat content of seed is lower than the result reported

by Olaeta et al. (2007), who observed the higher oil content for seeds coming from ripe

avocados. The obtained result was superior for fuerte seed, but lower for Hass seed than the

result reported by Olaeta et al. (2007), which might be due to the maturity level and location.

Also, the obtained seed lipid content were lower than the result (1.87%) reported by Pushkar et

al. (2001).

The obtained crude fiber content of avocado pulp were ranged between 1.46±0.01 and 4.50±0.09

g/100g The total dietary fiber content of the avocado pulp in both cultivars was higher than the

value of 0.4 g/100g reported by Moreno et al. (2003), but lower than the value of 6.3g/100g

reported by Gondim et al. (2005). The fiber content of avocado seed was 4.46±0.78, 4.73±0.03,

3.72±0.23 and 4.84±0.01g/100g for unripe Hass, unripe Fuerte, ripe Hass and ripe Fuerte seed

respectively. The seed of Hass cultivar contains lower fiber content than fuerte, and its content

increases as the fruit ripens for both varieties. Similar pattern was observed by Olaeta et al.

(2007) eventhough reported as Hass seed contains higher fiber content. The obtained seed fiber

content was superior to the one reported by Olaeta et al. (2007).

The total carbohydrate content of avocado pulp were ranged between 2.92±0.36 and 4.44±0.14%

where avocado hass pulp contains greater total carbohydrate content than fuerte pulp and its

content shows a slight decrease as the fruit ripens for both cultivars. The total carbohydrate

content of avocado seed were 39.11±1.91, 35.42±1.21, 37.51±0.26 and 30.44±0.33% for unripe

Hass seed, unripe Fuerte seed, ripe Hass seed and ripe Fuerte seed respectively. This indicates

56

that avocado hass seed contains greater total carbohydrate content than fuerte seed and shows a

slight decrease as the fruit ripens for both cultivars, which implies that starch stored during fruit

growth was decomposed and consumed during fruit ripening as a result of metabolism.

Table 4.3 Proximate composition of avocado pulp and seed of Hass and Fuerte variety in wet

basis.

Constituents

Avocado variety

Hass Fuerte

Pulp Seed Pulp seed

unripe ripe Unripe ripe unripe Ripe unripe ripe

MC (%) 64.92±

0.00c

68.81±

0.09b

51.60±

1.02a

54.09±0.

02e

77.82±0.

19a

77.74±0.

04a

54.52±

1.17e

61.83±

0.23d

Protein (%) 2.92±0.

04 c

2.84±0.

08c

2.96±0.

12bc

3.21±0.0

0b

2.25±0.0

6d

1.77±0.0

3e

3.58±0.

00a

1.94±0.

09e

Ash (%) 0.42±0.

01e

0.37±0.

00f

0.77±0.

01a

0.55±0.0

0c

0.27±0.0

g

0.27±0.0

1g

0.73±0.

01b

0.46±0.

01d

Fat (%) 22.80±

0.00a

22.07±

0.11b

1.21±0.

00 e

0.92±0.0

0 f

14.48±0.

08d

15.03±0.

00c

1.14±0.

00 e

0.57±0.

00 g

Fiber (%) 4.50±0.

09 a

2.99±0.

1 bc

4.46±0.

78 a

3.72±0.2

3ab

1.46±0.0

1d

2.17±0.0

0cd

4.73±0.

03 a

4.84±0.

01a

Total carboh-

ydrate (%)

4.44±0.

05 e

2.93±0.

18 f

39.11±

0.65a

37.51±0.

23b

3.72±0.1

3ef

3.02±0.0

3 f

35.42±

0.03c

30.44±

0.08d

Total Energy

(kcal)

234.62

±0.38a

221.65

±0.06b

179.19

±3.07c

171.16±

0.91d

154.17±

0.42f

154.42±

0.00f

166.25

±0.11e

134.65

±0.04g

* Data are means of duplicate determination ± standard deviation. The mean values followed by different

superscript letters in the same row are significantly different (p ≤ 0.05) according to the Tukey’s multiple

range test.

4.4. Mineral composition of avocado pulp and seed

Diet is responsible for several existing problems relating to human health. Deficiency diseases

could be prevented by sufficient intake of specific micronutrients that are involved in many

biochemical processes. Vegetables and fruits are particularly important sources of minerals

(Milton, 2003). Diets high in fruits and vegetables are also linked to decrease risk of diseases

such as diabetes and cancer and daily consumption of these foods is being encouraged (Leterme,

2002).

Tables 4.4 showed the mineral analysis of unripe pulp, unripe seed, ripe pulp and ripe seed of

both Hass and Fuerte variety. Hass pulp contains more magnesium content than Fuerte pulp and

its content was decreased for ripened fruit. The obtained magnesium content of the seed of

57

unripe Hass, unripe fuerte, ripe Hass and ripe Fuerte cultivars were 2.02 ±0.02, 1.89 ±0.01,

1.91±0.00 and 1.59±0.00mg/100g respectively. Also, like the result of the pulp, the magnesium

content of the seed of Hass cultivar was greater than that of Fuerte and its content was decreased

for ripe fruit. While the magnesium content was compared for pulp and seed, it was higher in

seed part for both cultivars. The magnesium content of both varieties was lower than that of

avocados varieties grown in US which is 29mg/100g (USDA 2010a).

The obtained calcium content of the avocado pulp of unripe Hass, unripe fuerte, ripe Hass and

ripe Fuerte cultivars were 15.35±0.07, 11.02±0.01, 13.55±0.10, and 13.69±0.10mg/100g

respectively. These indicate that Hass pulp contains more calcium content than Fuerte pulp and

its content was decreased as the fruit ripened for Hass pulp but increased for fuerte. The obtained

calcium content of the seed of unripe Hass, unripe fuerte, ripe Hass and ripe Fuerte cultivars

were 24.77±0.02, 16.42±0.06, 23.43±0.09 and 21.00±0.05 respectively. Also, like the result of

the pulp, the calcium content of the seed of Hass cultivar was greater than that of Fuerte and its

content was decreased for ripe Hass but increased for Fuerte seed. While the calcium content was

compared for pulp and seed, it was higher in seed part for both cultivars. The calcium content of

both varieties was higher than that of avocado varieties grown in US which is 12mg/100g (USDA

2010a).

The studied avocado pulp had iron content of 1.12±0.00, 0.53±0.01, 0.96±0.01 and

0.92±0.01mg/100g for unripe Hass pulp, unripe Fuerte pulp, ripe Hass pulp and ripe Fuerte pulp

respectively. These indicate that Hass pulp contains more iron content than Fuerte pulp and its

content was decreased as the fruit ripened for Hass pulp but increased for fuerte. The iron

content of avocado seed were 1.00±0.03, 0.57±0.01, 1.05±0.01and 0.83±0.00mg/100g for unripe

Hass seed, unripe Fuerte seed, ripe Hass seed and ripe Fuerte seed respectively. Also, like the

result of the pulp, the iron content of the seed of Hass cultivar was higher than that of Fuerte and

its content was slightly increased for ripe fruits of both cultivar seed. While the iron content was

compared for pulp and seed, it was nearly similar for both cultivars. The iron content of both

varieties was higher than that of avocados varieties grown in US which is 0.55mg/100g (USDA

2010a).

58

The zinc content of avocado pulp were 0.72±0.02, 0.44±0.00, 0.55±0.00, and 0.43±0.00

mg/100gm for unripe Hass pulp, unripe Fuerte pulp, ripe Hass pulp and ripe Fuerte pulp

respectively. These indicate that Hass pulp contains more zinc content than Fuerte pulp and its

content was decreased for the ripened fruit of Hass pulp but slightly increased for fuerte. The

zinc content of avocado seed were 0.36±0.01, 0.27±0.00, 0.46±0.00 and 0.40±0.00 mg/100g for

unripe Hass seed, unripe Fuerte seed, ripe Hass seed and ripe Fuerte seed respectively. Like the

result of the pulp, the zinc content of the seed of Hass cultivar was higher than that of Fuerte and

its content was increased for ripe seed. While the zinc content was compared for pulp and seed, it

was higher in pulp part for both cultivars. The zinc content of both varieties was nearly similar to

that of avocados varieties grown in US which is 0.64mg/100g (USDA, 2010a).

The results in the Table 4.4 show that the K contents of avocado pulp were between 516.03±4.44

and 783.29±2.31mg/100g. The results show that ripening increased the K content of avocado

pulp. Also, the K contents of avocado seed were between 502.11±10.64 and 594.51±15.34

mg/100g. The minimum K content corresponds to unripe hass seed and the maximum belongs to

unripe fuerte seed. Ripe hass pulp and ripe fuerte pulp had a K content of 506.64±0.23 mg /100g

and 558.99 ±3.32 mg/100g respectively. The K contents of avocado pulp were higher than

avocados varieties grown in US and California which is 485 and 507mg/100g respectively (USDA,

2010a).

Table 4.4 Mineral composition of pulp and seed of avocado variety (mg/100g, WB).

Mineral

Constitu

ents

Avocado variety

Hass Fuerte

Pulp Seed pulp seed

Unripe Ripe Unripe ripe unripe ripe unripe ripe

Mg 1.46±0.0

1 d

1.26±0.0

0 e

2.02

±0.01a

1.91±0.0

0 b

0.91±0.0

0 f

0.92±0.0

1 f

1.89

±0.01b

1.59±0.

00 c

Ca 15.35±0.

08e

13.56±0.

11f

24.77±0.

02a

23.43±0.

08b

11.02±0.

01g

13.69±0.

11f

16.42±0.

06d

21.00±

0.06c

Fe 1.12±0.0

0a

0.96±0.0

1cd

1.00±0.0

3bc

1.05±0.0

1b

0.53±0.0

1f

0.92±0.0

1d

0.57±0.0

1f

0.83±0.

01e

Zn 0.72±0.0

2a

0.55±0.0

0b

0.36±0.0

1e

0.46±0.0

1c

0.44±0.0

0c

0.43±0.0

0cd

0.27±0.0

2f

0.40±0.

00d

Na 9.81±0.0

0 f

9.95±0.0

3 e

11.59±0.

25 b

12.67±0.

01 a

8.75±0.0

8 g

5.33±0.0

1 h

10.81±0.

28 c

10.58±

0.07d

K 770.64±

0.16 b

783.29±2

.31 a

502.11±1

0.64 h

506.64±

0.23 g

516.03±

4.44 f

639.32±

1.01 c

594.51±

15.34 d

558.99

±3.32 e

59

* Data are means of duplicate determination ± standard deviation. The mean values followed by

different superscript letters in the same row are significantly different (p ≤ 0.05) according to the Tukey’s

multiple range test.

The sodium content of the avocado pulp were 9.81±0.00mg/100g, 8.75±0.08mg/100g,

9.95±0.03mg/100g and 5.33±0.01mg/100g for the corresponding UHP, UFP, RHP and RFP

respectively; these were higher than avocados varieties grown in US and California which is 7

and 8mg/100g (USDA, 2010a). Avocado seed had sodium content values varied from 10.58±0.07

to 12.67±0.01mg/100g. The minimum corresponds to RFS and the maximum to RHS. The UHS

and UFS had a Na content of 11.59±0.25mg/100g and 10.81±0.28 mg/100g, respectively.

Significant (P > 0.05) difference was observed between all the samples in zinc content. The

results of zinc content of the seed samples were higher than that of the pulp.

In general, a variation in mineral distribution was noted between varieties. The composition and

concentration levels of these nutrients varied significantly among cultivars. Potassium was the

most abundant macro-mineral and Calcium was the second most common mineral. Appreciable

amounts of sodium and magnesium were also noted. With regard to micronutrients (Table 4.4),

the concentration of iron and zinc was the lowest of quantified minerals observed in the varieties

studied.

4.5. Phytochemical composition of avocado pulp and seed

The investigated phytate content of an avocado pulp and seed are given in Table 4.5. The

investigated phytate content of avocado pulp were 4.22±0.06, 3.05±0.13, 2.73±0.03, and

2.53±0.13% (i.e. its concentration ranged between 2.53 and 4.22%) for unripe Hass pulp, unripe

Fuerte pulp, ripe Hass pulp and ripe Fuerte pulp respectively. The phytates content of Hass pulp

is greater than that of Fuerte pulp for both unripe and ripe fruits and its content decreased for the

ripe fruit. Also the phytates content of avocado seed were 49.12±0.25, 34.78±0.12, 45.82±0.36

and 37.68±0.10 (ranged between 34.78 and 49.12%) for unripe Hass seed, unripe fuerte seed,

ripe Hass seed and ripe fuerte seed respectively. The study revealed that avocado seed contains

high phytates content than its pulp for both Hass and Fuerte cultivars.

Tannins affect nutritive value of food by forming a complex with protein (both substrate and

enzyme) thereby inhibiting digestion and absorption (Oboh and Elusian, 2007). The tannin

60

determinations of the two avocado pulp and seed are shown in table 4.5. The tannin content of

avocado pulp were 0.99±0.02, 0.67±0.08, 0.68±0.06 and 0.81±0.02% for corresponding UHP,

UFP, RHP and RFP respectively. This shows that tannin content of Hass pulp is greater than that

of fuerte pulp and decrease when the fruit ripen for Hass pulp, but a slight increase for Fuerte

pulp. The tannin content of avocado seed were 13.72±3.21, 14.66±7.62, 7.31±0.41 and

11.24±0.89% for unripe Hass, unripe Fuerte, ripe Hass and ripe Fuerte respectively.

The investigated Phenol content of an avocado pulp and seed are given in Table 4.5. The Phenol

content of avocado pulp were 0.03±0.03, 0.04±0.02, 0.02±0.04 and 0.01±0.01mg/100g for unripe

Hass pulp, unripe Fuerte pulp, ripe Hass pulp and ripe Fuerte pulp respectively. This shows that

Phenol content of Fuerte pulp is greater than that of Hass pulp when unripe stage but lower when

they are in ripe form. The Phenol content of avocado seed were 0.42±1.11, 0.23±0.39, 0.07±0.02

and 0.17±0.92mg/100g for unripe Hass, unripe Fuerte, ripe Hass and ripe Fuerte respectively.

This Phenol content of seed is greater than that of its pulp. The results reveal that the pulp has

lower levels of phenolics than those found in Mexican ‘Hass’ avocados (4.9±0.7 mg GAE/g FW)

and Algarvian avocado of ‘Hass’ variety (4.102 ±0.69 mg/g) (Ana et al., 2013), except

comparable for UFP. The obtained unripe pulp phenolic levels in other cases are superior to

those reported for the Hass fruit of Turkish provenance (1.20±0.02 g/kg FW) (Golukcu and

Ozdemir, 2010). Also the seeds has lower levels of phenolics than those found in Mexican

‘Hass’ avocados (51.6±1.6 mg GAE/g FW) (Wang et al., 2010) but higher levels than Algarvian

avocado of ‘Hass’ variety (7.040±0.13mg/g) (Ana et al., 2013).

The obtained Flavonoid content of an avocado pulp and seed are given in Table 4.5. The

Flavonoid content of avocado pulp were 0.01±0.00, 0.01±0.06, 0.02±0.04, and

0.01±0.04mg/100g for unripe Hass pulp, unripe Fuerte pulp, ripe Hass pulp and ripe Fuerte pulp

respectively. This shows that Flavonoid content of Fuerte pulp is greater than that of Hass pulp

for unripe fruit but lower for ripe one. The Flavonoid content of avocado seed were 0.27±1.12,

0.07±0.25, 0.06±0.42 and 0.05±0.01mg/100g for unripe Hass, unripe Fuerte, ripe Hass and ripe

Fuerte respectively. This Flavonoid content of Hass seed is greater than that of Fuerte seed and

its content decreased for ripe fruit. The results revealed that in the avocado seed the highest

levels of total phenolics and flavonoids are found rather than its pulp. This agrees with the results

61

reported for avocados cultivated in Mexico (Wang et al., 2010) and Algarvian avocado of ‘Hass’

variety (Ana et al., 2013).

Table 4.5 Phytochemical composition of pulp and seed in wet basis mg/100g

Phytoch-

emical

Constit-

uents

Avocado Variety

Hass Fuerte

Pulp Seed Pulp Seed

Unripe Ripe Unripe Ripe Unripe Ripe Unripe Ripe

Phytates 4.22±0.

06e

2.73±0.0

3f

49.12±0.

25a

45.82±0.

36b

3.05±0.

13f

2.53±0.1

3f

34.78±0.

12d

37.68±0.

10c

Tannins 0.99±0.

02b

0.68±0.0

6b

13.72±3.

21a

7.31±0.4

1ab

0.67±0.

08b

0.81±0.0

2b

14.66±7.

62a 11.24±0.

89ab

Phenol 0.03±0.

03ef

0.02±0.0

4fg

0.42±1.1

1a

0.07±0.0

2d

0.04±0.

02e

0.01±0.0

1g

0.23±0.3

9b

0.17±0.9

2c

Flavonoid 0.01±0.

00d

0.02±0.0

4d

0.27±1.1

2a

0.06±0.4

2bc

0.01±0.

06d

0.01±0.0

4d

0.07±0.2

5b

0.05±0.0

1c

Table 4.5 Phytochemical composition of pulp and seed in wet basis mg/100g

* Data are means of duplicate determination ± standard deviation. The mean values followed by

different superscript letters in the same row are significantly different (p ≤ 0.05) according to the Tukey’s

multiple range test.

Remarkably, the total phenolic, flavonoid, and tannin contents of the seed were greater than in

the pulp (Table 4.5). Previous studies also found that total phenolic and flavonoid contents of the

seed far exceeded those of the pulp and these compounds possess strong in vitro antioxidant

activity and antimicrobial potential (Rodríguez-Carpena et al., 2011; Vinha et al., 2013).

Therefore, the avocado seed, as a byproduct, could be an interesting and inexpensive raw

material for a functional food ingredient or an antioxidant additive (Rodríguez-Carpena et al.,

2011).

4.6. Yield of avocado oil extracted using soxhlet and ultrasound assisted extraction

Results obtained from the experiments (observed and predicted) are summarized in Table 4.6 and

4.7. The experimental parameters and levels of the independent variables (particle size, solvent

to sample ratio and extraction time) investigated in this study and the results on the basis of the

Box-Behnken experimental design were shown in Table 4.6 and 4.7. Each of the 17 designed

experiments were done and the results were analysed. Five duplicates for each are included at the

centre of the design. The predicted values were obtained from the model fitting technique and

were seen to be sufficiently correlated to the observed values. The following quadratic model

62

equation (in coded factors) that correlates the yield of oil of ultrasound and soxhlet to various

process parameters is given by Eq. (4.1 & 4.2).

Final Equation in Terms of Coded Factors: for ultrasound

Yield =+59.61-3.92 * A+4.54 * B+10.65 * C-2.76 * B2-6.00 * C2 (4.1)

Final Equation in Terms of Coded Factors: for soxhlet

Yield =+64.69-3.49 * A+4.88 * B+10.29 * C-4.94 * B2-8.11 * C2+2.92 * B * C (4.2)

From the experimental results in Table 4.6 & 4.7 and equation (4.1 & 4.2), the second order

response functions representing Y is the response for oil yield, A the coded value of particle

size, B the coded value of solvent to sample ratio and C the coded value of extraction time. The

closer the value of R2 to unity, the better the empirical models fit the actual data. On the other

hand, the smaller the value of R2, the lesser will be the relevance of the dependent variables in

the model in explaining the behaviour of variations (Cao et al., 2008). Thus, the predicted values

match the observed values reasonably well, with R2 of 0.97 and 0.96 for ultrasound and soxhlet

respectively.

Statistical analysis obtained from the analysis of variance (ANOVA) for response surface

reduced quadratic model is shown in Table A2. The value of “P>F” for models is less than 0.05,

indicated that the model is significant which is desirable as it indicates that the terms in the

model have a significant effect on the response. The value of P<0.0001 indicates that there is

only a 0.01% chance that a “model F-value” this large could occur due to noise. Generally P-

values lower than 0.01 indicate that the model is considered to be statistically significant at the

99% confidence level (Ravikumar et al., 2005). Values greater than 0.1000 indicate the model

terms are not significant.

63

Table 4.6 Actual and predicted oil Yield value by soxhlet extraction

Standard

Order

Run

Order

Factor 1

A: Particle

Size

Factor 2

B: Solvent:

Sample ratio

Factor 3

C:Extraction

time

Actual

Value

Predicted

Value

Residual

1 15 1.4 15 6 59.5 58.37 1.13

2 17 2.6 15 6 47.2 51.39 -4.19

3 13 1.4 25 6 67.9 68.12 -0.22

4 8 2.6 25 6 60.5 61.14 -0.64

5 5 1.4 20 4 45.6 49.78 -4.18

6 14 2.6 20 4 42.1 42.81 -0.71

7 6 1.4 20 8 69.7 70.36 -0.66

8 7 2.6 20 8 65 63.38 1.62

9 16 2 15 4 42.4 39.41 2.99

10 2 2 25 4 45.2 43.31 1.89

11 12 2 15 8 54.2 54.13 0.069

12 3 2 25 8 68.7 69.73 -1.03

13 11 2 20 6 65.8 64.69 1.11

14 10 2 20 6 65.3 64.69 0.61

15 4 2 20 6 65.4 64.69 0.71

16 1 2 20 6 64.7 64.69 0.01

17 9 2 20 6 66.2 64.69 1.51

It can be observed that the variable with the largest effect on oil yield is the linear term of

extraction time (C) followed by the linear term of solvent to sample ratio (B), quadratic term of

extraction time (C2), linear term of particle size (A) and quadratic term of solvent to sample ratio

(B2) for ultrasound extraction and for soxhlet linear term of extraction time (C) followed by

quadratic term of extraction time (C2), the linear term of solvent to sample ratio (B), linear term

of particle size (A) and quadratic term of solvent to sample ratio (B2) (see Table A1 and A2 of

“F” ANOVA under appendixes). However, the quadratic term of particle size (A2) and all the

interaction terms (AB, AC and BC) are found to be insignificant (p>0.05), except BC on soxhlet

extraction. Regression analysis of the experimental data also shows that solvent to sample ratio

and extraction time had significant positive while particle size has negative linear effects on oil

yield (Eq. 4.1). The insignificant model terms can be removed and may result in an improved

model.

64

Table 4.7 Actual and predicted oil Yield value by ultrasound extraction

Standard

Order

Run

Order

Factor 1

A:particle

size

Factor 2

B:solvent:

sample ratio

Factor 3

C:extraction

time

Actual

Value

Predicted

Value

Residual

1 4 1.4 5 60 56.76 56.22 0.54

2 8 2.6 5 60 45.17 48.39 -3.22

3 6 1.4 15 60 65.79 65.31 0.48

4 13 2.6 15 60 57.12 57.47 -0.35

5 1 1.4 10 30 44 46.88 -2.88

6 11 2.6 10 30 39.98 39.05 0.93

7 10 1.4 10 90 66.09 68.18 -0.69

8 9 2.6 10 90 60.42 60.34 0.081

9 17 2 5 30 38.8 35.66 3.14

10 14 2 15 30 43.56 44.75 -1.19

11 12 2 5 90 56.5 56.95 -0.46

12 2 2 15 90 67.1 66.04 1.06

13 5 2 10 60 60.55 59.61 0.94

14 16 2 10 60 59.24 59.61 -0.38

15 7 2 10 60 61.84 59.61 2.23

16 3 2 10 60 60.09 59.61 0.48

17 15 2 10 60 58.9 59.61 -0.71

From Table 4.6, the soxhlet extraction parameters that provided the highest yield was optimized

as follows: particle size of 1.4 mm, Solvent to solid ratio of 20: 1 and extraction time of 8hr

which yields 69.7 g oil/100g DW. Also, from Table 4.7, the ultrasonic extraction conditions was

optimized as follows: particle size of 2 mm, Solvent to solid ratio of 15: 1 and extraction time of

90 min which yields 67.1 g oil/100 g DW.

A comparably higher yield of avocado oil was obtained using the soxhlet extraction (Table 4.6),

than ultrasonic extraction (Table 4.7), which is in agreement with a study performed by Ricardo

et al. (2014), Döker et al. (2010) and Mageshni Reddy et al. (2012). But, this extraction method

65

used high organic solvent and was more time consumer (8hr compared with 90 min). In other

words, ultrasonic extraction yields interesting amount by using less solvent and consuming less

time compared to soxhlet extraction. Yields by ultrasound and soxhlet techniques lay between

38.8 to 67.1% (Table 4.7) and 42.1 to 69.7% (Table 4.6) respectively. The comparison between

oil extraction yield (69.7%) from avocado pulp by soxhlet and the oil content percentage in the

initial matrix (70.75±0.35%), shows that almost all (about 98.5%) of the oil in the initial matrix

was extracted. Soxhlet extraction yield was higher because the solvent was almost pure in each

reflux solvent in the equipment in addition to use of high organic solvent and long time. Thus,

the concentration gradient between solid matrix and solvent was always high and the equilibrium

of extraction was constantly modified to enable oil solubility.

4.6.1. Effect of raw material particle size on oil yield

The effect of particle size is associated to an increase in cellular damage as particle size

decreases. This favors removal of the oil on the particle surface and diffusion of n-hexane within

the particle (Şaşmaz, 1996). The length of diffusion pathways will also decrease when the

particle size decreases, resulting in the increase of mass transfer rate for the process (Pronyk, and

Mazza, 2009). This can be noted in this results, which show that extraction yield reduced as

particle size increased, and improved with smaller particle sizes (Figures 4.1a and b).

In this study, two types of extraction techniques i.e. ultrasound extraction and soxhlet extraction

were considered. The range of particle size is 1.4mm to 2.6mm separated by sieving. Figure 4.1a

and b illustrates the effect of raw material particle sizes on the yields of oil extracted by

ultrasound and soxhlet techniques, respectively. The extraction yield of oil was better for smaller

particles (1.4mm) compared to larger ones (2.6mm). Operating with solvent to sample ratio of

10:1 in a 60 minutes extraction, an increase of about from 55.7 % to 63.5% of the oil yield was

obtained when raw material particle size is reduced from 2.6mm to 1.4mm. Similar trend is

observed for soxhlet extraction as illustrated by Figure 4.1(b), which shows reduction of oil yield

as particle size increased. The results show that among the three raw material particle size, 2mm

for ultrasound extraction and 1.4mm for soxhlet extraction was most effective (optimum)

condition.

66

Figure 4.1 One Factor Plot of effect of Particle Size on oil yield for (a) ultrasonic extraction (b)

soxhlet extraction.

The results concluded that smaller particle sizes are better for extraction processes. This is

attributed to the larger total surface area presented by smaller particles for extraction (Cheah et

al., 2010). Furthermore, solvent penetration path length decreases when the particle size

decreases as the specific surface area of the raw material increases thus influences solubility

(Ghoreishi, and Shahrestani, 2009).

4.6.2. Effect of solvent to solid ratio on oil yield

The dissolving of oil into the solvent is a physical process. When the amount of extraction

solvent is increased, the chance of the desired oil coming into contact with the solvent leading to

higher leaching rates (Zhang et al., 2007). When the ratio of solvent to solid ratio is higher, it

means that the difference of the concentration between the bulk solution and the solutes becomes

higher. Thus, more oil can leach out if a higher volume of solvent is used (Cacace and Mazza,

2003). This can be noted in this results, which show that extraction yield increased as solvent to

sample ratio increased (Figures 4.2a and b).

The effect of solvent to solid ratio on the oil yield is shown in Figure 4.2a and b. Three different

ratios of solvent to solid, i.e., 5:1, 10:1 and 15:1 for USAE and 15:1, 20:1 and 25:1 for soxhlet

extraction were used. The results show that among the three, 15:1 for ultrasound extraction and

20:1 for soxhlet extraction was most effective. The increase of extraction yields with the increase

DESIGN-EXPERT Plot

Yield

X = A: particle size

Design Points

Actual Factors

B: solvent:sample ratio = 10.00

C: extraction time = 60.00

1.40 1.70 2.00 2.30 2.60

38.796

45.872

52.948

60.024

67.1

A: particle size

Yiel

d

One Factor Plot DESIGN-EXPERT Plot

Yield

X = A: particle size

Design Points

Actual Factors

B: solvent:sample ratio = 20.00

C: extraction time = 6.00

1.40 1.70 2.00 2.30 2.60

42.1

49

55.9

62.8

69.7

A: particle size

Yie

ld

One Factor Plot

a b

67

of solvent to solid ratio is consistent with mass transfer principles. Smaller volumes of solvent

can lead to incomplete target extraction while larger volumes can make the extraction procedure

becomes complex and wasteful (Touati and Meniai, 2011).

Figure 4.2 One Factor Plot of effect of solvent to sample ratio on oil yield for (a) ultrasonic

extraction and (b) soxhlet extraction.

Therefore, suitable solvent to solid ratio is preferred in order to achieve higher extraction yields.

Based on the figure 4.2a and b, the optimum solvent to solid ratio is 15:1 and 20:1 for ultrasound

extraction and soxhlet extraction, respectively.

4.6.3. Effect of extraction time on oil yield

It is important to determine the duration of the extraction process required to extract the desired

compounds. Typically, this would be the time at which equilibrium of solvent concentration

between inner and outer cells is established (Zhang et al., 2007). Extraction time is crucial in

solvent extraction of oil as appropriate extraction time can result in time and cost saving. This

could have been due to the longer amount of time the solute and solvent were in contact with

each other. Longer contact time favored the system to have more mass transfer. However,

excessive extraction time would be unnecessary as the solvent and sample would be in final

equilibrium after certain duration. This is based on Fick’s second law of diffusion by then; the

rate of extraction of compounds would decelerate (Teixeira et al., 2014).

DESIGN-EXPERT Plot

Yield

X = B: solvent:sample ratio

Design Points

Actual Factors

A: particle size = 2.00

C: extraction time = 60.00

5.00 7.50 10.00 12.50 15.00

38.796

45.872

52.948

60.024

67.1

B: solvent:sample ratio

Yie

ld

One Factor Plot DESIGN-EXPERT Plot

Yield

X = B: solvent:sample ratio

Design Points

Actual Factors

A: particle size = 2.00

C: extraction time = 6.00

15.00 17.50 20.00 22.50 25.00

42.1

49

55.9

62.8

69.7

B: solvent:sample ratio

Yie

ld

One Factor PlotWarning! Factor involved in an interaction.

a b

68

The effects of extraction time on the yield of crude oil are shown in figure (4.3a and b) for

ultrasonic and soxhlet extraction, respectively. As shown in the figure 4.3a and b below, the

highest extraction yield content is obtained at time of 90min with a value of 64.26% for

ultrasonic extraction and at extraction time of 8hr with a value of 66.87% for soxhlet. In both

cases as extraction time increases (from 30 min to 90min and 4hr to 8hr) the yield also increases

(from 42.97 to 64.26% and 46.29% to 66.87% for ultrasonic and soxhlet extraction, respectively)

at fixed levels of both particle size and solvent to sample ratio as illustrated in the figure 4.3a and

b. Generally, as expected, a longer extraction time led to a higher percentage yield of oil. This

could be because a longer time gave the ultrasound wave more time to disrupt the cell walls and

release the cell contents as well as for soxhlet favored the system to have more mass transfer.

Figure 4.3 One Factor Plot of effect of extraction time on oil yield for (a) ultrasonic extraction

and (b) soxhlet extraction.

4.6.4. Interaction effect of solvent to sample ratio and extraction time on oil yield

Interaction graph, three dimensional and contour plots for interaction effect of solvent to sample

ratio and extraction time towards oil yield are shown in Figure 4.3. The oil yield increased as the

extraction time increased to its high level (8h) and with solvent to sample ratio to its central level

(20:1). Therefore, increasing solvent to sample ratio towards its high level (25:1) above central

DESIGN-EXPERT Plot

Yield

X = C: extraction time

Design Points

Actual Factors

A: particle size = 2.00

B: solvent:sample ratio = 10.00

30.00 45.00 60.00 75.00 90.00

38.796

45.872

52.948

60.024

67.1

C: extraction time

Yie

ld

One Factor Plot DESIGN-EXPERT Plot

Yield

X = C: extraction time

Design Points

Actual Factors

A: particle size = 2.00

B: solvent:sample ratio = 20.00

4.00 5.00 6.00 7.00 8.00

42.1

49

55.9

62.8

69.7

C: extraction time

Yie

ld

One Factor PlotWarning! Factor involved in an interaction.

a

b

69

point couldn’t have significant effect on oil yield rather consumption of solvent, and the stronger

influence of extraction time occurred when extraction time was at its high level.

Figure 4.4 graph of effect of solvent to sample ratio and extraction time on oil yield for soxhlet

extraction (a) Interaction graph (b) contour plot (c) 3D surface

DESIGN-EXPERT Plot

Yield

X = B: solvent:sample ratio

Y = C: extraction time

Design Points

C- 4.000

C+ 8.000

Actual Factor

A: particle size = 2.00

C: extraction time

Interaction Graph

B: solvent:sample ratio

Yie

ld

15.00 17.50 20.00 22.50 25.00

37.3352

46.4426

55.55

64.6574

73.7648

DESIGN-EXPERT Plot

Yield

Design Points

X = B: solvent:sample ratio

Y = C: extraction time

Actual Factor

A: particle size = 2.00

Yield

B: solvent:sample ratio

C: e

xtra

ctio

n ti

me

15.00 17.50 20.00 22.50 25.00

4.00

5.00

6.00

7.00

8.00

44.565549.725

54.8845

60.0441

65.2036

55555

DESIGN-EXPERT Plot

Yield

X = B: solvent:sample ratio

Y = C: extraction time

Actual Factor

A: particle size = 2.00

39.4059

47.1452

54.8845

62.6238

70.3631

Yield

15.00

17.50

20.00

22.50

25.00

4.00

5.00

6.00

7.00

8.00

B: solvent:sample ratio

C: extraction time

a

c

b

70

4.7. Quality characterization of the extracted avocado oil

The highest percentage oil yield was achieved by soxhlet (69.7%) and lower yield was by

ultrasonic extraction (67.1%). The oil yield differences of the extraction method could be

attributed to the differences in their chemical properties and their fatty acid composition. The

avocado oil obtained from soxhlet and ultrasonic extraction was analysed for various parameters

as shown in Table 4.8. The oil extracted by soxhlet extraction comprises lower moisture and

volatile matter, free fatty acid, acid value, alkalinity and Peroxide value than the oil extracted by

Ultrasound assisted extraction. The obtained result was superior to the result reported by Woolf

et al. (2009) where Moisture <1%, FFA <0.5%, AV <1% except in the range for PV (<4).

Acid Value: AV indicated the free fatty acids present in fats and oils. High degree acid value can

be related with degree of oxidation during preparation or storage. The good quality of oil

generally has low acid value. Since SEO had lower acid value (2.31±0.01 mgKOH/g) than UEO

(2.65±0.00 mgKOH/g), it indicated that the quality of SEO was better than UEO. These results are

found within the range of minimum (1) and maximum (7) values for the acid value properties of

avocado oil reported by Eckey (1954). The analyzed oil also comprises higher acid value than

the result (1.23 ± 0.02) reported by Pushkar et al. (2001), the result (1.23) reported for the Fuerte

cultivar (Bora et al., 2001) and (1.46) reported for avocado grown in Mexico (Moreno et al.,

2003). The low acid value obtained for avocado oil in this study therefore suggests that the oil is

edible and less susceptible to rancidity. The variation may be due to the differences in variety of

plant, cultivation climate, ripening stage, solvent type, extraction time and temperature.

Free fatty acid: The percentage free fatty acid (FFA) value of oil is a crucial parameter in

determining the quality of oil because the lower the FFA, the higher the quality of the oil

especially in terms of its edibility. The percentage free fatty acid of UEO (1.33±0.00 g/100g), is

slightly higher than SEO (1.16±0.86 g/100g). Low FFA content of the oil is also indicative of low

susceptibility to enzymatic hydrolysis and could be an advantage over oils with high free fatty

acids value which can become off-flavor during storage (Bailey, 1954).

Peroxide Value: Peroxide value is a measure of the content of hydro-peroxides in oil which are

the primary reaction product formed in the initial stages of oxidation of oil and therefore gives an

indication of the likely occurrence of the process of lipid peroxidation (Onwuka, 2005). The

71

peroxide value of the UEO (2.74±0.36) was higher than that of the SEO (0.23±0.00). Nearly

similar peroxide values of the pulp oil were found by other researchers, such as 2.5 by Soares et

al. (1991), but a lower value than UEO and higher value than SEO (1.4), was reported by Bora et

al. (2001), (1.40 ± 0.215) was reported by Pushkar et al. (2001). Much higher values of peroxide

values, varying from 3.7 to 12.74, were reported by Moreno et al., (2003). These differences may

be due to fruit production in different places, climate, and soil composition. The extent of

oxidative activity in oil may be estimated by Peroxide Value (PV). The number of peroxides

present in vegetable oils reflect its oxidative level and thus tend to become rancid. The peroxide

value of UEO was higher than SEO, indicating that UEO is unstable against oxidation than SEO.

A product with peroxide value above 10 meq kg–1 is classified at high oxidation state

(Moigradean et al., 2012). This result may be due to the presence of chlorophylls as photo-

sensitizers which have not been removed in crude avocado oil samples. Besides, avocado oil

should exhibit a high rate of oxidation due to its high content of unsaturated fatty acids.

Unsaturated fatty acids easily react with oxygen to form peroxides. Peroxide values of avocado

oil have been reported in the range of 5.1-12.3 meq kg–1 (Quinones-Islas et al., 2013).

Iodine Value: The iodine value (IV) indicates the unsaturation degree of fats and oils (Lusas et

al., 2012). Higher iodine value is attributed to high unsaturation. The slightly higher iodine

value (78.6) of SEO gives the indication that SEO contains more unsaturated fatty acid than

UEO (75.3). These results are lower than the range of 82-95 reported by Moreno et al. (2003) but

consistent with some reports stating that the number of IV for avocado oil was in the range of 65-

95 (AOCS, 1998). The results obtained for the iodine value also agree with the data (77.6)

reported by Bora et al. (2001) for avocado oil.

Fatty acid Profile of avocado oil

Oil quality is a significant concern of consumers, particularly for the contents of oleic and

linoleic acids which are proven as healthy sources of oil for human body. Avocados store mainly

oleic, palmitic, palmitoleic, and linoleic acids (Tango et al., 2004). Thus, oil analyses was

performed to determine the content of aforesaid and other fatty acids in the present study. The

FA composition of oils obtained for the different extraction methods are presented in Table 4.8.

The analyzed oil consisted of four different FAs; two unsaturated FAs and two saturated FAs.

MUFAs, oleic (C18:1) and SFA, palmitic (C16:0) acids, are predominant constituents of

72

avocado oils. Linoleic (C18:2) acid was only detected in oil extracted by soxhlet and for

saturated fatty acids, butyric acid was only detected in oils extracted by ultrasound extraction.

The lower percentages of FAs produced by USAE could be due to short extraction time and low

solvent to sample ratio. USAE is relatively rapid; increasing the extraction time could increase

the yield as well as fatty acid composition.

Mazliak (1971), reports eight fatty acids were identified in the pulp oil of cultivar Fuerte, while

Southwell et al. (1990) reported the presence of only six fatty acids. But in this study, only four

fatty acids in the pulp oil of Hass cultivar were identified. In all the cultivars C18:1 (oleic acid)

was always the principal fatty acid followed by C16:0 (Palmitic) and C18:2 (linoleic). Similar to

these reports, in this study the major fatty acid observed was C18:1 followed by C16:0 and

C18:2. This also agrees with a previous report by Sinyinda (1998), Haiyan et al. (2007) and

Moreno et al. (2003). However, the concentrations of these fatty acids in the pulp oil were

different from the cited reports. In this study the observed principal fatty acid concentration was

46.46-59.59%, 23.66-24.66% and 7.62% for oleic, palmitic and linoleic acid respectively. These

fatty acid concentration value were nearly similar (in the range) with that of reported by Woolf et

al. (2009), which was: Palmitic acid (16:0) 10%-25%, Oleic acid (18:1) 60%-80%, Linoleic acid

(18:2) 7%-20%. But, Martinez et al. (1988), reported a range of 60-65, 15-19 and 11-12%,

respectively for the same fatty acids in the oil from Bacon, Fuerte, and Hass cultivars. This

quantitative difference is may be due to the difference in fruits geographical location and other

factors such as maturity, ripening stage and solvent type.

Table 4.8. Quality characteristics of oil extracted by UAE and Soxhlet Extraction

Parameters USAE SOXHLET Unit

Moisture and volatile matter 8.47±0.09 2.27±0.01 %(g/100g)

Free Fatty Acid 1.34±0.01 1.17±0.01 %(g/100g)

Acid Value 2.65±0.00 2.32±0.01 mgKOH/g

Alkalinity (Soap Content) 0.11±0.00 0.004±0.00 %(g/100g)

Peroxide Value 2.75±0.01 0.23±0.01 meq/kg

Iodine value 75.3±0.14 78.6±0.14 (g/100 g)

palmitic acid (16:0) 24.01 23.66 %

oleic acid (18:1) 46.46 59.59 %

Linoleic acid (18:2) ND 7.62 %

Butyric acid (4:0) 1.38 ND %

Results are expressed as the mean of duplicate ± Standard deviation. ND: Not Detected

73

Generally, Soxhlet extracted avocado oil had the lowest acid, peroxide values, FFA, MVM than

ultrasonic extracted oil. Brazilian legislation for oils and fats (BRASIL, 2005) does not propose

specific standards for avocado oil. However, acid and peroxide values are standardized for

pressed and crude oils, where the maximum allowed acid value is 4.0mgKOH/g and the peroxide

value cannot exceed 15mEq O2/kg. In contrast, Mexico does have specifications for avocado oil

(NMX-F-052-SCFI-2008); the maximum allowed acid and peroxide values are 1.5% oleic acid

and 10mEqO2/kg, respectively. In the present work, oils from both processes presented acid and

peroxide values below the limits set by Brazilian legislation.

Figure 4.5. GC-MS result for ultrasound extracted oil.

74

Figure 4.6 GC-MS result for soxhlet extracted oil.

As the lipid content in avocados varies greatly with the variety and the same is also observed for

fatty acid composition, which depends on growth rate and variety (Tango et al., 2004). Avocados

store mainly oleic, palmitic, palmitoleic, and linoleic acids. Concerning the region of cultivation,

differences may reside not only between countries, as suggested by Landahl et al. (2009), but

also between different geographical locations within the same country, due to variation in

climate, soil composition, and other environmental factors. Therefore, the choice of an avocado

cultivar for oil extraction might be based on lipid content as well as on fatty acid composition

(which is related to the intended use of the oil) (Isabelle Santana1 et al., 2015).

75

CHAPTER FIVE

5. Conclusions and Recommendations

5.1. Conclusions

This study was aimed to characterize the pulp and seed of the Hass and Fuerte varieties of the

avocado fruit. From the result it was concluded that the higher pulp yield, lipid (22.80±0.00%),

protein (2.92±0.04%), ash (0.42±0.01%) and fiber(4.50±0.09%) concentrations found in the Hass

variety of unripe fruit which make this fruit to be more palatable and suitable for pulp oil

extraction, while higher moisture content (77.82±0.19%) was found in unripe fuerte. The seed of

Fuerte variety was accounted with higher moisture (61.83±0.23%), ash, protein (3.58±0.00%)

and fiber (4.76±0.01%) contents than those of the Hass variety. Also, higher TSS and lower TTA

was observed in the pulp of Hass cultivar while this was nearly similar in its seeds.

The results clearly showed that all the studied phytochemical concentration was higher in

avocado seed than its pulp. All Phytate, phenol and flavonoid was also higher in unripe Hass

seed cultivar except tannin which was higher in unripe Fuerte seed. In case of its pulp Phytate

and tannin concentration was higher in unripe Hass avocado cultivar while total phenol and

flavonoid was higher in unripe Fuerte cultivar which reveals that phytochemical concentration

was higher mostly in unripe fruit.

The results also show that a variety of fatty acids was detected in the pulp oils extracted using

soxhlet and ultrasonic from Hass avocado cultivar in the present study. Relatively high contents

of oleic (59.59) and linoleic (24.01) acids present in the avocado pulp oil reveal that this variety

was rich sources of ω-6 fatty acids and it is needed to reduce LDL and maintain HDL, which is

responsible in reducing cancers and cardiovascular diseases.

Also, in the present study, ultrasonic and soxhlet extraction of avocado oil was optimized. The

yield of oil increased with decreasing particle size, increasing extraction (Ultrasonication) time

and solvent to solid ratio. The comparison of the two techniques shows that the soxhlet

extraction provided the greater yield (69.7%). This fact may be explained because extractions

were conducted above the boiling point of the solvents, takes longer extraction time and

consumes more solvents. It may also occur because this technique uses practically pure solvent

76

in each refluxing which makes the mass transfer easier. Eventhough the method presented, UAE,

gives lower quality and quantity, but it has the advantage of easy operation, shorter extraction

time and reduced solvent consumption and can be used for sequential extraction of fatty acids.

Gas chromatography analysis showed that the fatty acid composition of the avocado oil obtained

by ultrasonic extraction was nearly similar to that of oil obtained by Soxhlet extraction. So,

Ultrasonic extraction is a useful and environmentally friendly extraction method that could be

applied to the production of other plant oils and active substances.

5.2. Recommendations

From the study result, both avocado cultivars appeared to be promising in nutrition and

phytochemical point of view and also the optimization of extraction condition was gave an

attractive yield. Based on the result found the following recommendations were made.

The composition of avocado fruit may vary with location, season, variety and maturity especially

the principal (fat) content. Therefore, characterization of avocado fruit from other location as

well as other variety from the present study’s location should be conducted.

Since avocado seed was rich in phytochemicals and somewhat proteins, further research should

be conducted on it in order to utilize it in medicinal purposes and formulate some products

including animal feed rather than wasting it and also if bioactive compounds of avocado is

needed it should be extracted from unripe fruit since ripening decreases the content of

phytochemicals.

These findings clearly prove that USAE has enormous potential as a novel method of oil

extraction. More research on the effects of other parameters affecting the efficiency of USAEs

could help make this technique more economically feasible for industrial applications.

The power of ultrasonic device I used could not be adjusted and its temperature was controlled

by replacing the water in the bath. But if the temperature is self-controlled and the power was

adjusted to examine the effect of ultrasonic power and temperature on the extraction yield and

quality of oil it will better.

77

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Appendixes

Appendix A1. ANOVA for Response Surface Quadratic Model of oil yield from ultrasound

extraction

Response: Yield from ultrasound

ANOVA for Response Surface Quadratic Model

Analysis of variance table [Partial sum of squares]

Sum of Mean F

Source Squares DF Square Value Prob > F Model 1406.36 9 156.26 52.67 < 0.0001 significant

A 122.77 1 122.77 41.38 0.0004

B 165.13 1 165.13 55.65 0.0001

C 906.66 1 906.66 305.57 < 0.0001

A2 6.21 1 6.21 2.09 0.1911

B2 30.70 1 30.70 10.35 0.0147

C2 148.33 1 148.33 49.99 0.0002

AB 2.14 1 2.14 0.72 0.4238

AC 2.33 1 2.33 0.78 0.4054

BC 8.53 1 8.53 2.87 0.1339

Residual 20.77 7 2.97

Lack of Fit 15.36 3 5.12 3.79 0.1155 not significant

Pure Error 5.41 4 1.35

Cor Total 1427.13 16

The Model F-value of 52.67 implies the model is significant. There is only a 0.01%

chance that a "Model F-Value" this large could occur due to noise.

Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case

A, B, C, B2, C2 are significant model terms. Values greater than 0.1000 indicate the model terms

are not significant.

91

Appendix A2. ANOVA for Response Surface Quadratic Model of oil yield from soxhlet

extraction

Response: Yield from soxhlet extraction

ANOVA for Response Surface Quadratic Model

Analysis of variance table [Partial sum of squares]

Sum of Mean F

Source Squares DF Square Value Prob > F

Model 1590.23 9 176.69 33.13 < 0.0001 significant

A 97.30 1 97.30 18.25 0.0037

B 190.13 1 190.13 35.65 0.0006

C 846.66 1 846.66 158.76 < 0.0001

A2 14.65 1 14.65 2.75 0.1415

B2 98.63 1 98.63 18.50 0.0036

C2 270.49 1 270.49 50.72 0.0002

AB 6.00 1 6.00 1.13 0.3240

AC 0.36 1 0.36 0.068 0.8025

BC 34.22 1 34.22 6.42 0.0390

Residual 37.33 7 5.33

Lack of Fit 36.06 3 12.02 37.92 0.0021 significant

Pure Error 1.27 4 0.32

Cor Total 1627.56 16

The Model F-value of 33.13 implies the model is significant. There is only a 0.01% chance that

a "Model F-Value" this large could occur due to noise.

Values of "Prob > F" less than 0.0500 indicate model terms are significant. In this case A, B, C,

B2, C2, BC are significant model terms. Values greater than 0.1000 indicate the model terms are

not significant.

92

Appendix B1. Standard calibration curve of D-catechin for the determination of tannin content.

Appendix B2. Standard calibration curve of phytic acid for the determination of phytate content

of pulp.

y = 0.2483x + 0.008R² = 0.9928

0

0.05

0.1

0.15

0.2

0 0.2 0.4 0.6 0.8

Absorban

ce

Concentration

abs

abs

Linear (abs)

y = -0.0076x + 0.3543R² = 0.9873

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 10 20 30 40

Absorban

ce

Concentration

Series1

93

Appendix B3. Standard calibration curve of phytic acid for the determination of phytate content

of seed.

Appendix B4. Standard calibration curve of gallic acid for the determination of total phenolic

content.

y = -0.0094x + 0.4433R² = 0.9947

0

0.1

0.2

0.3

0.4

0.5

0 10 20 30 40

Absorban

ce

Concentration

abs

abs

Linear (abs)

0.08

0.214

0.426

0.624

0.855

0.994

y = 0.1026x + 0.0023R² = 0.9952

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15

Absorban

ce

Concentration

abs

abs

Linear (abs)

94

Appendix B5. Standard calibration curve of quercetin for the determination of total flavonoid

content.

Appendix C. Some of the pictures and photos taken during research was conducted.

Collecting raw material Hass avocado variety

Cut and sliced avocado preparing sample

y = 0.1027x + 0.128R² = 0.9973

0

0.5

1

1.5

0 5 10 15

Absorban

ce

Concentration

abs

abs

Linear (abs)

95

Filtration samples for phenol and flavonoid determination

Measuring for phytate determination ashing fat determination

For Protein determinatin Titration Oil product