UNIVERSITY OF GONDAR

COLLEGE OF NATURAL AND COMPUTATIONAL SCIENCES DEPARTMENT OF CHEMISTRY

ELEMENTAL AND POLYPHENOL CONTENTS OF GREEN COFFEE BEANS FROM CENTRAL GONDAR ZONE, ETHIOPIA

BY: TESFAHUN SISAY

ADVISOR: BEWKETU MEHARI (PhD)

A THESIS SUBMITTED TO DEPARTMENT OF CHEMISTRY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN CHEMISTRY (ANALYTICAL)

OCTOBER 2020

GONDAR, ETHIOPIA

UNIVERSITY OF GONDAR COLLEGE OF NATURAL AND COMPUTATIONAL SCIENCES DEPARTMENT OF CHEMISTRY

ELEMENTAL AND TOTAL POLYPHENOL CONTENTS OF GREEN COFFEE BEANS FROM CENTRAL GONDAR ZONE, ETHIOPIA

BY: TESFAHUN SISAY APPROVED BY THE EXAMINING BOARD

Name Signature Date

------Chairman

------Advisor

------Internal Examiner

------External Examine

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DECLARATION First of all, I declare that this thesis is a result of my genuine work and I submit this thesis to University of Gondar in partial fulfillment for the requirement of a degree of master of science. The thesis is deposited at the library of the university to be made available to borrowers for reference. I solemnly declare that I have not so far submitted this thesis to any other institution anywhere for the award of any academic degree, diploma, or certificate. Brief quotations from this thesis are allowed without requiring special permission provided that an accurate acknowledgement of the source is made.

Name of the author: Tesfahun Sisay Signature ______

Place: University of Gondar, Gondar

Date of submission: ______

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ACKNOWLEDGEMENTS

First and above all I thank God and saint Mary for giving me strength and patience in doing this research work. His power has done many things for me. I am grateful to my Advisors Dr.Bewketu Mehari for his valuable professional comments, guidance and overall support starting from research proposal up to thesis write up. His moral support and continuous guidance enabled me to complete my work successfully. For me, really it was a good opportunity to do this thesis work with Dr.Bewketu, a man with patience, full of knowledge. I would like to extend my gratitude to Mr. Mulugeta Legese for the technical support of laboratory work and for valuable professional comments; Mr. Legese Terefe for the technical support of laboratory work and also the Department of Chemistry for overall support. I would also like to thank the Genda Wuha town administration for giving the scholar and the financial support of this work. I would like to take the opportunity and privilege to acknowledge the overall support and unreserved assistance of my wife Eskedar Abaya also like to mention special appreciation for her love of my lovely baby Atinasiya Tesfahun, specially for her smile face and sweaty laugh that she invite me all the time when I go back from school to home. Lastly, I would like to thank my heart-felt gratitude to Tirngo Gebre, Eneye Abaya and Mandefro Alebachew for their material, financial and moral support to continue my study in the right track.

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TABLE OF CONTENTS

Contents page

ACKNOWLEDGEMENTS ...... i

LIST OF TABLES ...... v

LIST OF FIGURES ...... vi

LIST OF ABBREVIATIONS ...... vii

ABSTRACT ...... viii

CHAPTER ONE ...... 1

1. INTRODUCTION ...... 1

1.1. Background of the study ...... 1 1.2. Statement of the problem ...... 3 1.3. Significance of the study ...... 4 1.4. Objectives of the study ...... 4 1.4.1. General objective ...... 4

1.4.2. Specific objectives ...... 4

1.5. Scope of the study ...... 5 CHAPTER TWO ...... 6

2. LITERATURE REVIEW ...... 6

2.1. Coffee production and consumption ...... 6 2.2. Coffee production in Ethiopia ...... 8 2.3. The chemical composition of coffee ...... 10 2.4. Source (origins) of polyphenol ...... 11 2.5. Different types of phenolic compounds ...... 11 2.5.1. Phenolic acid in coffee ...... 11

2.5.2. Caffeine in coffee ...... 13

2.5.3. Chlorogenic Acids (CGA) in Coffee ...... 13

2.5.4. Trigonelline in Coffee ...... 14

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2.6. Physiological and psychological effects of caffeine on the body ...... 15 2.7. The uses of polyphenols ...... 16 2.8. Impact of total polyphenol on health...... 17 2.9. Elemental composition in green coffee ...... 18 2.10. The health benefits of metals in coffee ...... 19 2.11. Analytical techniques for the determination of total polyphenol ...... 22 2.12. Instrumental method for elemental analysis...... 22 CHAPTER THREE ...... 24

3. MATERIALS AND METHODS ...... 24

3.1. Description of the study area and Coffee Samples ...... 24 3.2. Coffee Sample Preparation...... 25 3.3. Apparatus and Equipment ...... 25 3.4. Chemicals and Reagents...... 25 3.5. Extraction of Polyphenols ...... 25 3.6. Determination of Total Phenolic Content (TPC) ...... 26 3.7. Standard Solution Preparation ...... 26 3.8. Sample Digestion ...... 26 3.9. Instrument Calibration for the Analysis of Metals ...... 27 3.10. Method Validation...... 29 3.11. Statistical Analysis ...... 29 CHAPTER FOUR ...... 30

4. RESULTS AND DISCUSSION ...... 30

4.1. Total Polyphenol ...... 30 4.1.1. Instrument Calibration ...... 30

4.1.2. Total Polyphenol Contents of Green Coffee beans ...... 30

4.1.3. Comparison of the Total Phenolic Contents with Coffee from other origins ...... 32

4.1.4. Statistical comparison (ANOVA) of the total polyphenol contents of coffees from the different sampling districts ...... 33

4.2. Elemental composition of the coffee beans...... 33 4.2.1. Optimal conditions for sample digestion ...... 33

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4.2.2. Instrument calibration ...... 34 4.2.3. Method performance...... 34

4.2.4. Method detection and quantification limits ...... 35

4.3. The elemental concentration of green coffee beans ...... 37

4.3.1. Comparison of the elemental contents of green coffees with other literature values ..... 39 5. CONCLUSION ...... 41

6. RECOMMENDATION ...... 42

7. REFERENCES ...... 43

APPENDIXES ...... 48

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LIST OF TABLES Table 1: annual worldwide coffee production (million bags of 60 kg) ...... 10 Table 2: Chemical composition of coffee ...... 11 Table 3: Optimization of green coffee bean digestion procedure ...... 27 Table 4: Instrumental operating conditions for determination of metals using FAAS ...... 28 Table 5: The concentration of total polyphenols (TP) determined in green coffee beans grown in different districts of Central Gondar zone ...... 32 Table 6: ANOVA table obtained from the analysis of the variation of mean total polyphenol contents among green coffee beans from the four districts studied. Difference is significant when p < 0.05...... 33 Table 7: linearity range, regression equation and correlation coefficients of the calibration curve ...... 34 Table 8: The method detection limits and method of quantification the metals of interest in (mg/kg) for green coffee samples (n = 23) ...... 36 Table 9: Recoveries of metals in samples ...... 37 Table 10:The concentration (Mean ± SD) of metals from sampling districts ...... 38 Table 11: Comparison of the elemental contents of green coffee with other literature values .... 39

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LIST OF FIGURES Figure 1: Caffeic acid, gallic, p-hydroxybenzoic, protocatechuic, vanillic, and syringic acids ... 12 Figure 2: The chemical structure of caffeine (1, 3, 5-trimethylxanthine) ...... 13 Figure 3:The chemical structure of CGA...... 14 Figure 4: The chemical structure of trigonelline ...... 15 Figure 5: Map of green coffee sample collection districts ...... 24 Figure 6: The calibration curve of Gallic acid ...... 30 Figure 7: The total polyphenol content in each districts of sampling site ...... 31

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LIST OF ABBREVIATIONS

AAS Atomic absorption Spectroscopy

UV ultra-violet

CGA Chlorogenic acid

TG Trigonelline

CF Caffeine

TPC Total polyphenol

C. Arabica Coffee arabica

GAE Gallic acid equivalent

ANOVA Analysis of variance

LOD Limit of detection

LOQ Limit of quantification

RSD Relative standard deviation

SB Standard deviation of blank

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ABSTRACT The total polyphenol and some essential metal contents of twenty three green coffee bean samples, collected from four coffee growing districts (Gondar Zuria, Takusa, Tach Armachiho and Chilga) in Central Gondar Zone, Ethiopia, were quantified using UV/Vis-spectrometer and flame atomic absorption spectrometry, respectively. A previously reported procedure was employed for the extraction of total polyphenols using 80% aqueous methanol and maceration for 24 h. For the analysis of metals a wet-digestion procedure was optimized. Accordingly, a 0.5 g of green coffee bean powder was digested on a hot plate with HNO3: HClO4 (4:4; v/v) at 240 °C for 3.5 h. The accuracy of the optimized procedure was evaluated. The recovery of the stated parameters ranged from 85 to 116.3%. The levels of metals determined in the green coffee beans were relatively higher than those reported in the literature.the average metal concentration of green coffee from Central Gondar Zone was show the following trend mg/kg: Ca (3,163.5 ±65.8), Mg (853.5 ±2.65), Cu (46 ± 11.1), Zn (11.8 ± 1.4), Fe (91.75 ± 33.6) and Mn (22.2 ±1.5) .The polyphenol contents of the coffee beans ranged from 43.1 to 46.73 mgGAE/g. Analysis of variance (p < 0.05) noted that there was no significant difference in the concentrations of all the metals, except for copper, as well as the total polyphenols among the four sampling districts. In general, this study revealed that the coffee bean samples from the Central Gondar Zone contained substantial amounts of total polyphenols and essential elements like calcium.

Keywords: Central Gondar Zone, Green coffee, Metals, Total polyphenol, Wet-digestion

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CHAPTER ONE 1. INTRODUCTION 1.1. Background of the study

Coffee is the most commercialized food product, and widely consumed beverage in the world. Coffee trees are grown around Asia, Africa, South America and Caribbean region. Among commonly available coffee species, only two coffee species: Arabica (coffea Arabica), and Robusta (coffea Canephora), account for 75% and 25%, respectively (Asfaw et al., 2019).

The name coffee is derived from the name of the province Keffa where shepherds from Abyssinia (Ethiopia) discovered the coffee beans in the 6th century (Chilakala et al., 2018). It is an important plantation crop which belongs to the Rubiaceae and genera Coffea family; they are shrubs or small trees. The coffee plant is usually a woody perennial tree that is growing at a region of higher altitudes. Coffee is one of the most consumed drinks in the world that is ranked after petroleum as the second most traded global commodity, and it is exported to more than 167 countries; and the annual local consumption in Ethiopia is estimated to be more than 9.0 million ton in recent years (Sabah et al., 2019).

It is a popular beverage all over the world, and has characteristic taste and aroma. The most important bioactive compounds of coffee include the following: phenolic compounds (such as chlorogenic acids and derivatives), methylxanthines (caffeine, theophylline and theobromine), diterpenes, (including cafestol and kahweol), nicotinic acid (vitamin B3) and its precursor trigonelline, magnesium and potassium. This variability of the coffee chemical constituents should be determined using sensitive, precise and accurate analytical methods to examine the quality, aroma and properties of green coffee beans, instant coffees and coffee brew (Grzes´kowiak et al., 2014). Typical compounds in coffee, e.g. caffeine, trigonelline and chlorogenic acid, contribute to the acidity and confer astringency and bitterness, thereby influencing coffee flavor. They are also the component of total polyphenols, and are known to be biologically active. For example, caffeine, an alkaloid, stimulates the central nervous system, heart rate and respiration; and chlorogenic acid, a family of esters formed between caffeic acid and quinic acid (Arai et al., 2015), exhibits various biological properties including anti-bacterial, antioxidant and anti-carcinogenic activities, particularly hypoglycemic and hypolipidemic effects (Arai et al., 2015).

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Arabica coffee and Robusta coffee are the most important types among 70 different species of genera Coffee that have been reported so far. These two species differ in origin, caffeine contents, appearance, and their quality and flavor. Mild Arabica comes from upland and mountain areas of East Africa (Ethiopia) and Robusta from the lowland of Central and West Africa (Grzes´kowiak et al., 2014). Arabica coffee is favored by a hedonic trend of consumers as compared to Robusta coffee (Sabah et al., 2019). Both Arabica coffee and Robusta coffee generally have significant positions in production and export.

Besides the genetic factors, the chemical composition of green coffee beans depends largely on growing environmental conditions. Climate and altitude, among others, are well-known to play an important role in affecting the ripening process of coffee cherries. Moreover, the flavor and aroma of coffee are believed to be affected by the presence of various volatile and nonvolatile chemical constituents such as proteins, amino acids, fatty acids, and phenolic compounds, as well as by the action of enzymes on some of these components (Dechassa et al., 2018).

Coffee is believed to have multifarious beneficial health effects, usually attributable to its high antioxidant activity˗ ability to inhibit the process of oxidation. The antioxidant activity of coffee depends on the chemical composition, mainly due to chlorogenic, ferulic, caffeic, and n-coumaric acids contained in it. In addition, it was observed that the antioxidant activity of coffee varies according to the degree of roasting. Maximum antioxidant activity was measured for the medium- roasted coffee (Jae-Hoon et al., 2014). In roasted coffee, melanoidins, brown pigments with strong antioxidants property, are synthesized. Nitrogen- and/or sulfur-containing heterocyclic compounds, flavor imparting compounds, are formed by the Maillard reaction; and such compounds exhibit antioxidant activity of caffeine and trigonelline (Delores et al., 2000). The coffee constituents that have received due attention from flavor chemists, are the heterocyclic compounds because of their characteristic roasted flavors. There are almost 350 heterocyclic compounds, including thiophenes, thiazoles, oxazoles, pyrroles, pyrazines, imidazoles, and furans that have been identified in coffee. Phenylalanines which are formed during the roasting process show high antioxidant activity, as do heterocyclic compounds (Yashin et al., 2013 ).

In the past few decades, there has been growing evidence that oxidative stress and specific human diseases can be prevented by including in the diet of plant foods that contain large amounts of

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antioxidants, e.g. polyphenols. This is because they can act as free radical scavengers, radical chain reaction inhibitors, metal chelators, oxidative enzyme inhibitors and antioxidant enzyme cofactors (Karadag et al., 2009). In this respect, coffee is one of the sources of polyphenolic species like chlorogenic, ferulic, caffeic and n-coumaric acids that can prevent the formation of free radicals, leading to bond breakage in DNA and deformation of our genetic makeup. Therefore, it is well- known in circumventing prolonged oxidative stress that inevitably leads to chronic disease like cancer, cardiovascular diseases, diabetes and premature aging (Yashin et al., 2013 ).

Coffee also contains various mineral elements, such as Na, B, Mg, Fe, Ca, and K, with good nutritional values. These elements are absorbed in various proportions from the soil and accumulated by the plant. In this respect, climate, elevation, underlying geology, type of soil and its chemical composition, and the application of fertilizers and pesticides are the factors, affecting the types and proportions of such elements (Habte et al., 2016). Hence, the determination of total concentrations of elements in coffee helps to assess its nutritive quality and judge its possible side effects to human health upon their presence at higher doses (Sabah et al., 2019)

Therefore, the focus of this study is to: determine the total polyphenol and elemental (Ca, Cu, Fe, K, Mg, Mn, and Zn) contents of green coffee beans from different districts in the Central Gondar Zone by using UV/Visible spectrophotometry and FAAS; and investigate the effects of their geographic source on the levels of total polyphenols and the six elements under study.

1.2.Statement of the problem

Ethiopia is widely regarded as the birthplace of coffee and it is a leading Arabica coffee producer in Africa, ranking the fifth largest Arabica coffee producer and tenth in coffee export worldwide. Arabica, the original bean, is still the only one grown in Ethiopia, but an impressive selection of different Arabica varieties with distinct flavor profiles are produced. These varieties have diverse tastes, depending on their geographical origin. Factors including climate, altitude and soil type contribute to the unique characteristics of each variety. Coffees cultivated in different regions of the country are also genetically divers (Tolessa et al., 2019).

Ethiopia is known by not only as a coffee exporter country, but also its people highly consumed coffee regularly. Coffee closely associated with the social life of the people. Drinking coffee has

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been indicated to minimize diabetes risk, cancers and liver disease because coffee contains sufficient amounts of chlorogenic, caffeic, quinic acids and other chemicals that enable it to protect diseases. But, also several diseases have been alleged to be caused by coffee consumption. In Ethiopia, researches have mainly been focused on Oromia and South Nations, Nationalities and Peoples regional states. Although the Amhara Region is not a traditional coffee-growing area in the country, coffee production in the region has been increasing from time to time. Studies conducted in recent years showed that there is a huge potential in selected areas of the region where different coffee varieties are growing. However, the chemical compositions of coffees grown in the region, particularly in the Central Gondar Zone, have yet to be analyzed. Thus, this research is initiated with the aim of determining the contents of total polyphenols and elements in green coffee beans collected from different districts in the Central Gondar Zone.

1.3. Significance of the study

The relevance of the study is the provision of analytical information on the variability of polyphenol and element composition of green coffee beans from Central Gondar districts. The analytical data can be compared with the findings of other researchers on green coffee beans from different countries and different locations of Ethiopia.

1.4. Objectives of the study

1.4.1. General objective

This study intended to investigate the total polyphenol and elemental content of green coffee from various districts in the Central Gondar Zone.

1.4.2. Specific objectives

 To determine the total polyphenol content of green coffee beans from Gondar Zuria, Tach Armachiho, Chilga and Takusa districts using UV-visible spectrophotometry;  To determine the variations in the level of polyphenol in green coffee beans from the studied districts;  To compare the determined polyphenol contents with values reported for green coffee beans that grows in different Ethiopian regions and other countries;  To determine the elemental content of green coffee from the studied districts by using FAAS;

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 To compare the elemental composition of coffee that grows in the different districts; and  To compare the elemental content of coffee from the studied districts with values reported for green coffee beans from different Ethiopian regions and other countries.

1.5. Scope of the study

In Amara region there is a lot of coffee that grows in different zones. However, this research was limited to the determination of the total polyphenol and elemental content of green coffee from Gondar Zuria, Tach Armachiho, and Chilga and Takusa districts of Central Gondar zone. The rest of the coffee cultivation areas of the Zone were not covered under this study.

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CHAPTER TWO 2. LITERATURE REVIEW 2.1. Coffee production and consumption

There are different types of coffee in the world. Among different types of coffee, the major economic species are coffea Arabica and coffee Robusta. Arabica accounts 80% of the world coffee trade, and Robusta most of the remaining 20% (Aychehet al., 2008). Coffea species are well‐ known tropical plants, which are mostly used for preparing the famous beverage called coffee (Brigitta et al., 2017). For the last 50 years the production as well as consumption of coffee throughout the world has increased dynamically and nowadays it is the most popular drink next to water (Illy et al., 2003).

The primary center of origin of C. Arabica coffee is the highlands of southwestern Ethiopia and the Boma plateau of South Sudan ( Krishnan et al., 2017 ). Coffee is grown in more than 80 countries in tropical and subtropical regions of the world and is exported in green or roasted beans to more than 165 countries. The crop accounts for 75% of export revenue and provides livelihoods for smallholder coffee producers around the world (Tolessa et al., 2018 ). Currently there are six countries determined as the world's largest coffee producers i.e. Brazil, Colombia, Vietnam, Indonesia, Mexico, and India. Ethiopia is a leading Arabica coffee producer in Africa, ranking the fifth largest Arabica coffee producer and tenth in coffee export worldwide (Nasir et al., 2017). Nevertheless, the ten largest coffee producing countries are responsible for approximately 80% of the world production. Of this percentage, South America participates with around 43%, Asia with 24%, Central America 18%, and Africa with 16%. Brazil, Vietnam, Colombia, and Indonesia are respectively the first, second, and third largest world producers, responsible for more than half of the world supply of coffee (Teixeira et al., 2011). Coffee is a main dietary source of polyphenol and phenolic acid due to its high polyphenol and phenolic acid content. Coffee is consumed by around 40 % of the world's population. For many people, especially in Western countries, coffee drinking is a part of their lifestyle and an everyday habit. The coffee beverage can be consumed for many reasons, with high antioxidant properties, weight loss, mood enhancing and increase alertness, effectiveness against hyper-tension, and anticancer properties, including its stimulatory effects resulted from the presence of caffeine, rich phytochemistry, health benefits, and primarily excellent taste and aroma due to a habitual consumption of coffee, its chemical composition,

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namely the presence of essential, non-essential and toxic elements, has to be known and kept under control in terms of its safety, and to assists its quality, nutritional value, and certain sensorial properties (Szymczycha-Madeja et al., 2012 ).

Polyphenols are the most common phytochemicals in human dirt and comprise a variety of compounds with a great diversity of structures, ranging from simple molecules to polymers with higher molecular weight. Polyphenols are plant secondary metabolites present in all plant tissues, and their primary role is to protect plants from insects, ultraviolet radiation, and microbial infections and to attract pollinators (Mrduljaš et al., 2017).

The coffee beverage is a rich source of bioactive compounds especially polyphenols, such as phenolic acids, mostly chlorogenic (in green beans) and cafeic (occurring after roasting). Other phenolic acids in coffee beans are: ferulic and p-coumaric. These compounds contribute to the total polyphenol intake in diet and are beneficial to consumer health (Hallmann et al., 2019). The profile and content of bioactive compounds depend mainly on roasting parameters, which range from 160 to 240 °C and from 8 to 24 min. The color of beans is a main parameter to describe a degree of roasting and is classified as light, medium and dark roasted coffee. During the roasting process, a decrease in polyphenolic compounds is observed and it is connected with the degradation of chlorogenic, malic and citric acid, which influences the total antioxidant activity. However, the formation of melanoidins and quinic acid during thermal processes can maintain or even enhance the antioxidant capacity. The lighter (brown) roasted coffee had the highest antioxidant activity compared to dark roasted coffee and also unroasted coffee the origin, harvesting, processing, and preparation of the beverage influence the total antioxidant activity (Hallmann et al., 2019).

Polyphenols are secondary compounds widely distributed in the plant kingdom. They are divided into several classes, i.e. phenolic acids (hydroxybenzoic acids and hydroxycinnamic acids), flavonoids (flavonols, flavones, flavanols, flavanones, isoflavones, proanthocyanidins) stilbenes, and lignans, which are distributed in plants and food of plant origin. Phenolic are an important constituent of fruit quality because of their contribution to the taste, color and nutritional properties of fruit. There is evidence that phenolic substances act as antioxidants by preventing the oxidation of LDL (low density lipoprotein) lipoprotein, platelet aggregation, and damage of red blood cells. Additionally, phenolics act as: metal chelators, antimutagens and anticarcinogens, antimicrobial

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agents and clarifying agents. They are responsible for red wine color, astringency and bitterness and contribute to its sensory profile. They are derived from the fruit and vine stems, or by the yeast metabolism. In addition, phenolic serves as important oxygen reservoirs and substrates for browning reactions (Gharras et al., 2009).

2.2. Coffee production in Ethiopia

Coffee trees are indigenous to Africa, and can still be found growing wild in the hills of Ethiopia. Today, coffee is produced in several countries and it is the base of economy for many these countries such as Central America (Mexico, Guatemala, El Savador, Nicaragua, Costa Rica, and Panama), South America (Venezuela, Colombia, Ecuador, Peru, Bolivia, Paraguay, and Argentina), India, East Africa (Ethiopia, Kenya, Tanzania, Zambia, and Mozambique), Western and Central Africa (Ivory Coast, Cameroon, Uganda, and Angola), Brazil, Malaysia (Vietnam, Sri Lanka, Sumatra, Java, etc.). The two biggest producers by far are Brazil and Colombia, followed by Indonesia, Vietnam and Mexico. Ethiopia is the 3rd largest coffee producer in Africa after Uganda and Ivory coast. Coffee grows in most parts of Ethiopia, Oromia and SNNP region comprise the largest coffee cultivated area. Wolega, EliAbaboura, Jimma, Bench Maji, Sidamo, Gedeo, East and west Harrarge, South and North Omo are the largest coffee cultivated areas of Ethiopia. Coffee Arabica is native to the southwestern highlands of Ethiopia. It is the most cultivated coffee species throughout the world. About 90% of the world’s coffee production is coffee Arabica and 9% is Robusta. Coffee Arabica grown at higher altitude, requires less rain and its beans have a lower caffeine content than that of Robusta. It is mainly grown in Central and Southern America, East Africa, India. It is the most important foreign currency earner for more than 80 developing countries. It is the only species occurring in Ethiopia. Coffee Canephora is widely grown in Western and central Africa, Malaysia, Brazil, and India. It originated in the humid lowland forest of tropical Africa, which stretches from Guinea to Uganda and Angola. It has a stronger flavor than Arabica with a fully body and a woody after test, which is useful in creating blends and especially usefull in instant coffee. It is grown at lower altitude (Gudu et al., 2009).

Ethiopia is the birthplace of coffee and it was discovered earlier. In the tenth century, Ethiopian nomadic mountain peoples may have been the first to recognize the coffee it has stimulating effect, although they ate the red cherries directly and did not drink it as a beverage initially. Then the mystic Sufi pilgrims of Islam spread coffee throughout the Middle East. From the Middle East

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these Ethiopian coffee beans spread to Europe and then throughout their colonial empire including Indonesia and the Americas. More than 1,000 years ago, coffee was a goatherd in Ethiopia southwestern highlands plucked a few red berries from some young green trees growing there in the forest and tasted them to check it has the flavor and make a feel-good effect to the consumers at that time. In addition, as David Beatty discovers in words and pictures, the Ethiopian province where they first blossom Kaffa gave its name to coffee. The story of coffee was beginning in Ethiopia, and the country is the original home of the coffee plant, coffee Arabica, which still grows wild in the forest of the highlands of Ethiopia. While nobody is sure exactly how coffee was originally discovered as a beverage plant, it is believed that its cultivation and use began as early as the 9th century in Ethiopia. Some authors claim that it was cultivated in Yemen earlier, around AD 575. It originated in Ethiopia, from where it traveled to Yemen about 600 years ago, and from Arabia began its journey around the world. Coffee is vital to the cultural and socio-economic life of Ethiopians. It sustains the livelihoods for over 15 million and provides important income from casual labor and for many additional poor rural peoples. It contributes 25%-30% of the country's foreign exchange earnings and Ethiopia has huge potential to increase coffee production as it endowed with suitable elevation, temperature, and soil fertility, indigenous quality plantation materials, and sufficient rainfall in coffee growing belts of the country (Amamo et al., 2014 ).

In Ethiopia coffee is produced under four broad production systems: forest coffee (8-10%), semi forest coffee (30-35%), cottage or garden coffee (50-57%) and modern coffee plantations (5%). Ethiopian coffee is predominantly produced by small-scale farmers using traditional farming systems and thus considered as organic by real and known for its superior quality ( Kufa et al., 2011 ).

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Table 1: annual worldwide coffee production (million bags of 60 kg)

Countries Production 2004 2005 2006 2007 2008 2009 Brazil 39.272 32.944 42.512 36.07 45.992 39.470 Vietnam 14.370 13.842 19.340 16.467 18.500 18.00 Colombia 11.573 12.564 12.541 12.504 8.664 9.500 Indonesia 7.536 9.159 7.483 7.777 9.350 9.500 Ethiopia 4.568 4.003 4.636 4.906 4.350 4.850 India 4.592 4.396 5.159 4.460 4.372 4.827 Mexico 3.867 4.225 4.200 4.150 4.651 4.500 Guatemala 3.703 3.676 3.950 4.100 3.785 4.100 Peru 3.425 2.489 4.319 3.063 3.872 4.00 Honduras 2.575 3.204 3.461 3.842 3.450 3.750 Coted`Ivoire 2.301 1.962 2.847 2.598 2.353 1.850 Nicaragua 1.130 1.718 1.300 1.700 1.615 1.700 El Salvador 1.437 1.502 1.371 1.621 1.547 1.500 Other 15.713 15.779 16.019 16.138 15.680 15.455 countries Total 116.062 111.463 129.138 119.396 128.181 123.002

2.3. The chemical composition of coffee

Coffee is the complex mixture of potential “neutriceuticals”. Its chemical composition is determined by a complex interaction of agricultural factors, roasting, blending and brewing. Studies have demonstrated that the chemical composition of coffee beans can discriminate Arabica from Robusta, country of origin and organic from conventional system of cultivation.The main components of coffee beans have been known over half of the century. As it was stated by an ICS, coffee beans constituents, in order of abundance (are: 8 % phenolic polymer (pulp), 6 % polysaccharides, 4 % chlorogenic acids, 3 % minerals, 2 % water, 1 % caffeine, 0.5 % organic acids, 0.3 % sugars, 0.2 % lipids, and 0.1 % aromas. The aroma of green coffee contains some 250 different volatile molecular species, whereas roasted coffee gives rise to more than 800. On the other hand, some recent studies on the chemical compositions of coffee, has been identified over 600 different substances in green coffee, while roasted coffee contains much larger than the green once. The most important once are; minerals, lipids, caffeine, proteins, fats, carbohydrates and water. The chemical composition of coffee varies according to species (arabica or robusta),

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country origin (Ethiopia, Brazil, Kenya, etc.), system of cultivation (organic or conventional) and the way it exist (raw or roasted). Roasting of coffee gives; taste and aroma, A change in the chemical composition, change the size of the bean in a certain way, Loss of weight due to evaporation of water in about 14 – 23 % and loss of volatile substances, increase in volume of coffee beans, Change of bean color, of course the color change depends on the intensity and duration of roasting. In general, roasting of green coffee does not change noticeably, the mineral content, but their relative content increases when water and volatile organic compounds disappear (Gure et al., 2006). Table 2: Chemical composition of coffee g %/100 g on dry mas C. Arabica C. Robusta Green (raw) Green (raw Caffeine 0.9 – 1.2 1.5 – 2.4 Minerals 3.0 – 4.2 4.0 – 4.5 Proteins 11.0 – 13.0 4.0 – 4.5 Fat 12.0 – 18.0 9.0 – 13.0 Oligosaccharides 6.0 – 8.0 5.0 –7.0 Water 10.0 – 13.0 10.0– 13.0 2.4. Source (origins) of polyphenol

Phenolic compounds are commonly found in both edible and non-edible plants, and they have been reported to have multiple biological effects, including antioxidant activity. Crude extracts of fruits, herbs, vegetables, cereals, and other plant materials rich in phenolic are increasingly of interest in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food. These phenolic compounds may be classified into different groups as a function of the number of phenol rings that they contain and of the structural elements that bind these rings to one another. Distinctions are thus made between the phenolic acids, flavonoids, stilbenes, and lignans (Gharras et al., 2009).

2.5. Different types of phenolic compounds

2.5.1. Phenolic acid in coffee

The name phenolic acid describes phenols that possess single carboxylic acid functionality. Phenolic acids contain two distinguishing constitutive carbon frame works:hydroxycinnamic and

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hydroxybenzoic structures. The numbers and position of the hydroxyl groups on the aromatic ring creates a variety of structures and compounds. Hydroxybenzoic acids have common C6–C1 structure and include Gallic, p-hydroxybenzoic, protocatechuic, vanillic, and syringic acids. Hydroxycinnamic acids, on the other hand are aromatic compounds with a three carbon side chain (C6–C3) with caffeic, ferulic, p-coumaric, and sinapic acids being the most common. Phenolic acids in plants have been connected with diverse functions, such as nutrient uptake, protein synthesis, enzyme activity, photosynthesis, and structural components. Caffeic acid is known to selectively block the biosynthesis of leukotrienes, components involved in immuno-regulation diseases, asthma, and allergic reactions. Caffeic acid and some of its esters might possess antitumor activity against colon carcinogenesis. And act as selective inhibitors of human immunodeficiency virus type I integrase. Chlorogenic acid has been found to inhibit lipid peroxidation in rat liver induced by carbon tetrachloride, a potent liver carcinogen. Caffeic and ferulic acid was found to detoxify carcinogen metabolites of polycyclic aromatic hydrocarbons ( Ajila et al., 2010). Caffeic acid, Gallic, p-hydroxybenzoic, protocatechuic, vanillic, and syringic acids as presented in Figure 1

OH

HO OH OH

O O Cinnamic acid

Caffeic acid

O OH O

O OH O HO OH H3C OH HO OH HO Gallic acid Ferulic acid

P-Coumaric acid

Figure 1: Caffeic acid, Gallic, p-hydroxybenzoic, protocatechuic, vanillic, and syringic acids

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2.5.2. Caffeine in coffee

Quality of coffee depends on caffeine contents. Caffeine content of a coffee mixture is usually used to indicate its coffee level. The world's primary source of caffeine is the coffee "bean" (which is actually the seed of the coffee plant), from which coffee is brewed. Caffeine content in coffee varies widely depending on the type of coffee bean and the method of preparation used; even beans within a given bush can show variations in concentration. Arabica coffee normally contains less caffeine than the Robusta variety. The caffeine content varies widely from about 100 μg/mL (100 ppm) to over 1000 μg/mL in certain types of coffee. This variation in caffeine content depends on the type of coffee, environment and soil type. In its pure state, it is an intensely bitter white powder.

Its chemical formula is C8H10N4O2, its systematic name is 1, 3, 5-trimethylxanthine. Pure caffeine occurs as odorless, white, fleecy masses, glistening needles of powder. Its molecular weight is 194.19g, melting point is 236 0C, point at which caffeine sublimes is 178 0C at atmospheric pressure, pH is 6.9 (1% solution), specific gravity is 1.2, volatility is 0.5%, vapor pressure is 760 mm Hg at 178 0C, solubility in water is 2.17%, vapor density 6.7. Caffeine is a pharmacologically active substance and depending on the dose, can be a mild central nervous system stimulant, improve cardiac performance, increase brain circulation, and exhibit vasodilator and diuretic effect. It is also increase heartbeat rate, dilate blood vessels and elevate levels of free fatty acids and glucose in plasma.4, 5 Caffeine is added to soft drinks as a flavoring agent, it is part of the overall profile of soft drinks, which consumers enjoy for refreshment, taste and hydration ( Gebeyehu et al., 2017 ).

O CH3 H3C N N

O N N

CH3

Figure 2: The chemical structure of caffeine (1, 3, 5-trimethylxanthine)

2.5.3. Chlorogenic Acids (CGA) in Coffee

Chlorogenic acids (CGA) are the main phenolic compounds in coffee and coffee has one of the highest concentrations of CGA of all plant constituents. CGA is an ester of trans cinnamic acids,

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such as caffeic acid, ferulic and p-coumaric acids with quinic acid. Figure 3 shows the chemical structure of CGA. They are believed to have antioxidant properties which are suggested to play an important role in protecting food, cells and any organ from oxidative degenerative. Reports indicate that diets rich in CGA compounds play a great role in preventing various diseases associated with oxidative stress such as cancer, cardiovascular, aging and neurodegenerative disease (Sabally et al., 2013).

On the other hand CGA contributes a great role in the formation of pigments, taste and flavor of coffee beans, which determines the quality and acceptance of the beverages. Previous research reports have indicated the relation between the composition of the CGA and quality of coffee beans. CGA are known to be important determinants of coffee flavor. They contribute to the final acidity and confer astringency and bitterness to the beverage. As a result of maillard and strecker’s reactions, bitterness increases during roasting due to release of caffeic acid and formations of lactones and other phenol derivatives responsible for flavor and aroma. The level of CGA has an inverse association with coffee quality with higher contents observed in lower quality coffee samples (Sabally et al., 2013).

HO CO2H

O

OH HO O

OH

OH

Figure 3: The chemical structure of CGA

2.5.4. Trigonelline in Coffee

Trigonelline is a nitrogenous compound, a pyridine alkaloid that is derived from the methylation of the nitrogen atom of nicotinic acid (niacin). Trigonelline is the second main alkaloid found in green coffee beans. During the roasting process, trigonelline suffers severe thermal degradation generating a series of volatile compounds responsible for flavor formation and aroma production and it is used as roasting-level discriminator in both Arabica and Robusta coffees (´kowiak et al., 2014 ).

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Trigonelline (N-methyl betaine of pyridine-3-carboxylic acid) present in coffee seeds undergoes thermal degradation during the roasting process. Part of the trigonelline is thereby transformed into nicotinic acid (anti pellagra factor), the degree of conversion being strongly dependent on time and temperature of roasting .Although the trigonelline content seems to be dependent on the coffee species, and there are variations in the data of the trigonelline content due to different analytical methods (Gonzalez et al., 1996).

COOH

N+

CH3

Figure 4: The chemical structure of trigonelline

2.6. Physiological and psychological effects of caffeine on the body

Caffeine stimulates the central nervous system, an effect that may begin as early as 15 minutes after ingesting the caffeine and can last for as long as six hours. There are several types of side effects produced such as physiological effect, energy metabolism, psychoactive and neurological effect. Caffeine is rapidly and completely absorbed from gastrointestinal tract within a short period of time from consumption and then distributed throughout the body .However, it is not removed from the circulation until metabolized initially into paraxanthine, theophylline and theobromine then into derivative of uric acid and diaminourcil. Thus, the plasma half-life of caffeine in man, that is, the time required for its level to be diminished by 50% as a result of biotransformation and excretion is 5-6 hours. Most experts agree that drinking 600 mg (around 6 cups of brewed coffee) or more of caffeine per day may cause side effects. Some examples of the side effects of excessive caffeine intake include difficulty concentrating, insomnia, muscle tremors, fast heartbeat, jitteriness, heartburn, nervousness, stomach upset and irritability. Excessive caffeine intake during pregnancy has been linked to low birth weight, premature delivery and miscarriage. The Food and Drug Administration recommends that women who are pregnant or trying to become pregnant consume no more than 200 mg of caffeine a day. Excessive caffeine consumption should be avoided by people who are being treated for certain conditions including depression, anxiety or

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insomnia, heart problems, gastro esophageal reflux disease, high blood pressure and kidney disease. In these cases, decaffeinated drinks may be chosen over caffeinated ones. On the other hand, besides the physiological and psychological effects of caffeine, the chemical analysis of caffeine in coffee beans has been used as an additional tool for evaluating coffee quality. It has been reported that higher caffeine contents is associated with less quality samples compared to other Arabic samples. Caffeine is a central nervous system stimulant which has been enjoyed by humans for many years through consumption of foods and beverages containing caffeine. However, coffee is one of the most consumed beverages throughout the world. Due to the importance of caffeine level in coffee simple analytical methods are required in order to characterize and identify the amount of caffeine in coffee beans. Due to the above mentioned facts many physical and chemical methods have been developed for the determination of caffeine in coffee beans including spectroscopic techniques. The literature survey indicated that caffeine is determined in coffee samples mostly with the use of HPLC and UV-Vis spectrophotometer. However, relatively the above methods use solvents that are expensive and toxic which are chlorinated compounds that lead us to the chance of causing cancer (Weldegebreal et al., 2016).

2.7. The uses of polyphenols

Polyphenols are mainly attributed to their antioxidant properties, since they can act as chain breakers or radical scavengers depending on their chemical structures. Polyphenols might also trigger changes in the signaling pathways and subsequent gene expression. It is possible that the distinct chemical and receptor-mediated activities of polyphenols might result in similar outcomes via different pathways. Under some circumstances, polyphenols can exhibit pro-oxidative effects. Depending on their particular structures, polyphenols exhibit a wide range of properties. They include yellow, orange, red, and blue pigments, as well as various compounds involved in food flavor. Some volatile polyphenols, such as vanillin and eugenol (which is responsible for the characteristic odor of cloves), are extremely potent odorants, but the major flavors associated with polyphenols are bitterness and astringency. Other major polyphenol characteristics include their radical-scavenging capacity, which is involved in antioxidant properties, and their ability to interact with proteins. The latter is responsible for astringency perception (resulting from interactions of tannins with salivary proteins), for formation of haze and precipitates in beverages, and for inhibition of enzymes and reduced digestibility of dietary proteins. Other major polyphenol

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properties, such as the ability to complex with proteins and free radical-scavenging capacity, are primarily related to the number and accessibility of phenol. Properties of polyphenols are also greatly affected by their interactions with other constituents of the food matrix. The astringency of tannins may also be altered by the presence of various molecules, including polysaccharides and proteins. Finally, strong interactions with other constituents of the food matrix are likely to interfere with the metabolism of polyphenols and should be taken into account in bioavailability studies. Indeed, interactions of polyphenols with food proteins and digestive enzymes are well known to reduce protein digestibility and can be expected to alter polyphenol bioavailability similarly. Furthermore, as it was reported recently, domestic processing, such as cooking in boiling water, seems to have a dramatic effect on phenolic content on both kinds of food, and, as a consequence, on antioxidant activity (Gharras et al., 2009).

Caffeine is one of the totalpoly phenol component and the health benefit of Caffeine is a stimulant substance found in coffee, tea, cocoa (chocolate), and kola nuts (cola), soft drinks, energy drinks, and some over-the-counter medications. Coffee is one of the most popular consumed beverages in the world and the most common sources of high caffeine. Even though caffeine contains several chemical components that may provide health benefit in reducing dementia, insulin resistance, type 2 diabetes mellitus, Parkinson disease, cirrhosis and advanced hepatic fibrosis, excess intake is not recommended; specially during pregnancy. This is for the fact that caffeine can cross the placenta into the amniotic fluid and fetus and results in adverse pregnancy outcomes (Aderaw et al., 2018).

2.8. Impact of total polyphenol on health

In contrast to its positive health, a recent study finds coffee does not always have coffee protective benefits. Over drinking 28 cups of coffee or more per week increased a person's probability of dying prematurely by 21 percent was reported. His risk was more than 50 percent higher in adults under 55 years old. Also, heavy coffee consumption associated with higher death risk Excessive coffee intake raises health risks because it increases a person's heart rate and blood pressure and slightly increases peripheral arterial stiffness; distal vascular tone. Recently reported shows that coffee consumption increases the long term risk of coronary heart disease but habitual moderate coffee drinking was associated with a lower risk of coronary heart disease in women. Coffee has

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been around for a long time and blamed for many ills from stunting our growth to causing heart disease. Recent studies have generally found no connection between coffee and an increased risk of heart disease or cancer. In fact, a few studies have found an association between coffee consumption and decreased overall mortality and possibly cardiovascular mortality, although the suggestion is not a fact in young aged people who consume large amounts of coffee. High doses of coffee intake during pregnancy increase the risk of miscarriage, independent of pregnancy related symptom (Wachamo et al., 2017).

2.9. Elemental composition in green coffee

The elemental composition of coffee will vary greatly according to the cultivation location (Szymczycha-Madeja et al., 2012). Environmental conditions, mostly the altitude and the rainfall, soil composition and pH, bean variety, cultivation practices used, significantly affect the elemental composition of coffee. The elemental composition of a plant is generally a reflection of the elemental composition of the soil in which the plant was cultivated. However, the accumulation of an element within the plant depends on the nature of the element, plant species, and prevailing environmental conditions. Plants of the same species that are grown in different geographical locations exhibit varying elemental profiles as a result of the environmental conditions (Mehari et al., 2016).These factors may differ from one locality to another; hence, the concentration of elements may provide a useful tool to determine the geographical origin of coffee beans. The elemental composition of green beans has been used for the discrimination of coffee according to geographical origin ( Mehari et al., 2016).

The elements analyzed, will macro elements; aluminium (Al), calcium (Ca), iron (Fe), magnesium (Mg), sodium (Na), potassium (K), phosphorus (P) and sulphur (S), micro elements; boron (B), copper (Cu), manganese (Mn), nickel (Ni), rubidium (Rb), strontium (Sr) and zinc (Zn), trace elements; beryllium (Be), bismuth (Bi), cerium (Ce), cobalt (Co), chromium (Cr), cesium (Cs), europium (Eu), gallium (Ga), germanium (Ge), hafnium (Hf), holmium (Ho), iridium (Ir), lanthanum (La), lithium (Li), molybdenum (Mo), neodymium (Nd), niobium (Nb), scandium (Sc), selenium (Se), silver (Ag), uranium (U), vanadium (V), yttrium (Y) and zirconium (Zr), and trace toxic elements; arsenic (As), cadmium (Cd), indium (In), mercury (Hg), stannum (Sn), lead(Pb) and thallium (Ti) (Habte et al., 2016).

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The presence of suitable conditions such as favorable altitude, adequate rainfall distribution, suitable temperature and fertile soil, has enabled coffee to grow throughout the country. Likewise, Central Gondar Zone is endowed with suitable agro-ecology and fertile soil for the production of coffee. In addition to the enormous potentials, only a relatively small amount of coffee is produced by farmers scattered in different areas of the Zones. The determination of mineral nutrients that are contained in its great interest due to its large consumption by millions of people around the country and the world. Nowadays many people are interested in healthy food. The analysis of individual components of raw and product is necessary in the coffee industries mainly to control the food safety. As a result, essential and non-essential metals in green coffee are quantitatively studied by using FAAS instrument.

2.10. The health benefits of metals in coffee

As discussed before green coffee has a complex chemical composition. Such as polysaccharides, monosaccharide, lipids, sterols, fatty acids, phenolic acids, polyphenols, alkaloids, proteins, free amino acids, vitamins, and minerals. Additionally, it is a rich source of compounds possessing antioxidant and radical scavenging activities such as chlorogenic acids, hydroxycinnamic acids, caffeine and caffeic acid (Wachamo et al., 2017). Green coffee contains a total of 3–4.5% ash which comprises mostly of K, Na, Ca, Mg, P and S. Numerous trace elements have also been found, including Fe, Al, Cu, I, F, B, and Mn As reported by various researchers, the mineral content of green coffee averages about 4% on dry weight basis, with potassium as its main constituent, at about 40%. The mineral elements: P, Ca, K, S, Na and Cl are sometimes called macronutrients, not because they are more important but because they are necessary in somewhat greater amounts than others. The first five are essential to both plants and animals, and the last two to animals alone. The amount of mineral elements in a plant body varies depending upon their presence in air and soil and other factors such as species, age, root distribution of the plant, physical and chemical nature of the soil, proportions and distributions of the elements and the general climatic conditions. Under most conditions, metallic elements that enter animals are those contained in plants. According to the food and nutrition board, metals are mostly grouped under bone related nutrients (Ca and Mg) and additional trace elements (Cr, Cu, Fe, Mn, Mo, Zn, Ni, and V). On the other hand, heavy metals in foods and beverages are classified into two based upon their essential and toxic nature. For example Fe, Zn, Cu, Mn, Cr, Co, and V are essential while Pb, Cd, Ni, As and

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Hg are toxic at certain levels. Furthermore, based upon the amount that is required in human nutrition, metallic elements are classified as: bulk mineral elements or macro metals; Ca, Mg, K and Na (Recommended Dietary Allowance RDA > 200 mg/day); trace minerals or metals; the most important trace metals (RDA < 200 mg/day) are Cr, Cu, Fe, Mn, Mo, Se, V and Zn. They are used in the enzymatic systems and can be harmful when their injection rate is too high ; other metals that have been not definitively established as essential to human nutrition, which includes Ni, Rb, Sr, Ti, Te, and W. The metallic elements of interest in this study are K, Ca, Mg, Fe, Zn, Mn, Cu, Co, Cr, Pb, Ni and Cd. Trace minerals such as Fe, Cu, Mn, Zn, Ni, Cr and Co are essential micronutrients for both plants and animals ( Gure et al., 2017).

Calcium (Ca) is the most abundant mineral in the body and is essential for a number of vital functions. The body needs adequate dietary calcium (alongside vitamin D and several other nutrients such as vitamin K) to develop and maintain healthy bones and teeth. Calcium also plays a vital role in many systems including intracellular signaling to enable the integration and regulation of metabolic processes, the transmission of information via the nervous system, the control of muscle contraction (including the heart) and blood clotting. Furthermore, it has been suggested that adequate calcium intake (for example from reduced fat dairy products) may help lower high blood pressure and may help protect against colon cancer, although more evidence is needed to fully substantiate these functions. The skeleton contains about 99% of the body’s calcium with approximately 1kg present in adult bones. The major constituents of bone are calcium and phosphate, forming hydroxyapatite, which is associated within a meshwork of collagen fibers to form a rigid structure. The body’s requirement for calcium fluctuates with the rate of bone development, so as well as protecting vital organs, the skeleton acts as a ‘bank’ of minerals from which calcium and phosphorus may be continually withdrawn or deposited to support physiological requirements.

Magnesium (Mg) is an essential mineral present in all human tissues, especially in bone. It has both physiological and biochemical functions and has important interrelationships with calcium, potassium and sodium. It is needed for the activation of many enzymes (for example enzymes concerned with the replication of DNA and the synthesis of RNA) and for parathyroid hormone secretion, which is involved in bone metabolism. It is also needed for muscle and nerve function.

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Iron (Fe) is essential for the formation of haemoglobin in red blood cells; haemoglobin binds oxygen and transports it around the body. Iron is also an essential component in many enzyme reactions and has an important role in the immune system. In addition, it is required for normal energy metabolism and for the metabolism of drugs and foreign substances that need to be removed from the body.

Zinc (Zn) The major function of zinc in human metabolism is as a cofactor for numerous enzymes. Zinc has a key role as a catalyst in a wide range of reactions. It is directly or indirectly involved in the major metabolic pathways concerned with protein, lipid, carbohydrate and energy metabolism and is also essential for cell division and, therefore, for growth and tissue repair and for normal reproductive development. In addition, zinc is required for the functioning of the immune system and in the structure and function of the skin, and hence plays a vital role in wound healing.

Copper (Cu) is the third most abundant dietary trace metal after iron and zinc. It is a component of many enzymes and is needed to produce red and white blood cells. The body also needs copper to utilize iron efficiently and it is thought to be important for infant growth, brain development, and the immune system and for strong bones. Dietary induced copper deficiency is extremely rare due to the plentiful supply in the diet and the high efficiency of absorption. However, a rare genetic condition, known as Menke’s disease, results in the inability to absorb copper and leads to severely impaired mental development, failure to keratinize hair and skeletal and vascular problems.

As with some other minerals, under normal circumstances absorption of copper is tightly controlled so overload of copper is very rare. However, Wilson’s disease, another genetic condition, leads to the inability to excrete excess copper in bile and results in copper accumulation in the body, especially the liver and brain, with consequent pathological damage.

Sources of copper include shellfish, liver, kidney, nuts and wholegrain cereals (about a third of intake in the UK is from cereals).

Manganese (Mn) is required for bone formation and for energy metabolism. It is also a constituent of an antioxidant enzyme, which helps prevent free radical-mediated damage to cells. Manganese deficiency is rarely seen. Manganese toxicity is not a problem because blood levels are carefully controlled. It is present in plant foods such as vegetables, cereals and nuts. Coffee and Tea is also

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a rich source. In the UK, 50% of manganese intake is derived from cereals and cereal products ( Masresha et al., 2018 ).

Various elements are transmitted into living metabolism through food intake. Although some of these elements are beneficial for the human body, some of them may be toxic. Due to the importance of mineral and trace elements present in medical herbs, several studies have been carried out to determine their concentration levels by using atomic absorption spectrometry (AAS), inductively coupled plasma-mass spectrometry (ICP-MS),inductively coupled plasma-atomic emission spectrometry (ICP-AES), neutron activation analysis, X-ray fluorescence, and electrochemical methods (Çam et al., 2008). In addition to this determination of major, minor and trace elements is important to characterize the geographical origin of agricultural commodities as well as to evaluate their dietary importance and possible harmful effects (Endaye et al., 2019).

2.11. Analytical techniques for the determination of total polyphenol

The most widely used methods for the determination of polyphenol in different samples are HPLC, UV/visible spectrophotometry, capillary electrophoresis. Among those methods the simplest and cheapest method for analysis of polyphenol was UV/visible spectrophotometry. UV/visible spectrophotometry is easy to handle and use, is simple to operate, Cost effective instrument, it can be utilized in qualitative and quantitative analysis. However, UV Visible Spectroscopy: Only those molecules are analyzed which have chromophores, the results of the absorption can be affected by pH, temperature, contaminants, and impurities. Only liquid samples are possible to analyze, it takes time to get ready to use it, Cuvette handling can affect the reading of the sample (Rahman et al., 2013)

2.12. Instrumental method for elemental analysis

Analytical methods are keys for the analysis of such complex multi-component mixtures in coffee widely used for identification, quality estimation and quantitative analysis of coffee. Several analytical methods have been used for the determination of major, minor, and trace elements in coffee products, such methods are AAS, ICP-MS, and ICP-OES etc. Flame atomic absorption spectrometry (FAAS) with a deuterium lamp background corrector or intermittently with a Zeeman Effect background corrector. It is quite often used for selective determinations of different major (Ca, K, Mg, Na), minor (Cu, Fe, Mn, Zn) and trace (Cd, Co, Cr, Ni, Pb) elements of coffee.

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the advantage of AAS is inexpensive, high sample throughput, easy to use, high precision, Unfortunately, FAAS is recognized to be not sensitive enough to quantify some important trace elements, only solution can be analyzed, relatively large sample quantities required, problems with refractory elements (Szymczycha-Madeja et al., 2012).

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CHAPTER THREE 3. MATERIALS AND METHODS 3.1. Description of the study area and Coffee Samples

The coffee samples were collected from various districts of Central Gondar Zone (Figure 5) directly from coffee growing districts. A total of 23 samples, three from Takusa, two from Gondar Zuria, eight from Tach Armachiho, and ten from Chilga districts were included in the study. Takusa (12° 14' 60.00" latitude North and 36° 19' 60.00" longitude East, Altitude that ranges from 530 to 1900 meter above sea level), Tach Armachiho ( 13° 00' 0.00" latitude North and 37° 09' 60.00" longitude East, Altitude ranges from 850–920 meter above sea level), Gondar Zuria (12° 39' 59.99" latitude North and 37° 19' 60.00" longitude East, Altitude ranges from 1800-2700 meters above sea level), Chilga (12° 44' 59.99" latitude North and 36° 39' 59.99" longitude East, Altitude from 2146 meters above sea level). All sampling districts are located in Central Gondar Zones.These districts were selected purposefully because of two reasons: higher productivity and geographical location differences. Each sample was 250 g of coffee cherries and was stored in polyethylene plastic bags under room temperature in the laboratory until analysis.

Figure 5: Map of green coffee sample collection districts

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3.2. Coffee Sample Preparation

The coffee cherry samples were dried with sunlight, the husks of the cherries were removed by using mortar and pistil. Coffee beans were washed at first with tap water, followed by rinsing with distilled water to remove soil particles and dust. From each sample 50 g was weighed by using electrical balance and powdered by using an electrical grinder. The coffee powder was then sieved using a 200 μm mesh size sieve. All the samples were collected during the 2019/2020 crop season (December 2019 to February 2020). 3.3. Apparatus and Equipment

UV-Vis double beam spectrophotometer (Abron instruments, ISO 9001:2008 Co), flame atomic absorption spectrometer equipped with deuterium ark background correctors (BUCK scientific, 210 BGP), glass cuvette, nylon centrifuge tube, Whatman No.1 filter paper, digital analytical balance ((ARA520, OHAUS CORP., China)), oven, micropipettes, conical flasks, beakers, magnetic stirrer, volumetric flask and measuring cylinder.

3.4. Chemicals and Reagents

Folin-Ciocalteu (FC) reagent (fine chemicals, India), Gallic acid (fine chemicals, India), sodium carbonate (Blulux Laboratory), methanol 99.8%(fine chemicals, India), sulfuric acid (98%), potassium dichromate, nitric acid (BDH india, ~70%), perchloric acid (45718LR, India, 70%), standard solution of Ca (Blulux, private limited, 45618LR, India), Mg (Blulux, privatelimit, 45621LR, India), Zn (Blulux, private limit, 45622LR, India), Mn (LOVA,CHEMIE, A250N00500, India), Cu (LOVA, Chemie, A160M01000, India) and Fe (Blulux, private limit, 45623LR, India) were used in this study. Distilled water was used throughout the experiment for sample preparation, dilution and rinsing apparatus prior to analysis. Sulfuric acid and potassium dichromate were used for preparing chromate solution for soaking and washing flasks and other glassware before starting digestion to remove metals and other contaminants. 3.5. Extraction of Polyphenols

In this study, acidified 70% acetone in water, 70% ethanol in water, 80% methanol in water and 100% water were tested as solvent to extract phenolic compounds from the powdered green coffee beans. From the tested solvents, the highest efficiency of extraction was obtained with 80%

25

methanol in water. The extraction was performed following a reported procedure (Geremu et al., 2016) with little modification. Briefly, a 0.5 g sample of powdered coffee in a 25 mL conical flask, closed with aluminium foil and macerated with 10 mL of the solvent for 24 h at room temperature. The mixture was then stirred for 30 min by using a magnetic stirrer. Finally the resulting solution was filtered by using what man no.1 filter paper.

3.6. Determination of Total Phenolic Content (TPC)

The TPC of the coffee extract was determined based on spectrophotometric method using Folin- Ciocalteu’s reagent (Asfaw et al., 2019) with some modifications. Briefly, a 0.5 mL of extract was mixed with 0.25 mL of Folin-Ciocalteu reagent and 3 mL distilled water in a test tube. After a while, 1 mL of 7.5% sodium carbonate solution was added, covered by aluminum foil and incubated for 1:30 h in the dark. Blank samples were prepared side by side with the 0.5 mL of 80% methanol instead of the samples. The absorbance was recorded at 760 nm against the blank solution. Quantitation was done using a calibration curve prepared with Gallic acid (GA) as a standard, and the total phenolic content was expressed as milligrams of Gallic acid equivalents per gram dry coffee beans (mg GAE/g).

3.7. Standard Solution Preparation

Stock standard solution of Gallic acid (600 mg/L) was first prepared from accurately weighed 0.075 g of pure Gallic acid in a flask and mixed it with 125 mL of 80% methanol in water. A series of working standards of 300, 200, 100, 50, 25 and 12.5 mg/L were prepared by serial dilution of the stock solution of Gallic acid was taken 12.5, 15.15, 12.5, 6.25, 12.5 and 12.5 mL, respectively.

3.8. Sample Digestion

For elemental analysis, samples were digested with different volumes of nitric acid and per chloric acid on a hot plate digester. A 0.5 g sample was heated to 240 °C for 3 h and 50 min mixed with 4 mL nitric acid and 4 mL per chloric acid as well 2 mL of 1% lanthanum chloride dihydrated, in order to liberate calcium from phosphate because Lantanum phosphate or sulphate are thermally stable compound than calcium or magnesium salphate or phosphate. The digest was filtered into a 50 mL volumetric flask and filled with distilled water to the mark. The sample solution was kept

26

in the refrigerator until analysis by using a flame atomic absorption spectrophotometer. Blank

samples were prepared by taking a mixture of optimized volume ratio of HNO3, HClO4 and treating in a similar procedure to that of the sample.

In order to develop an optimum procedure for the analysis of green coffee beans, different

digestion procedures by varying the volumes of HNO3 and HClO4, temperature and time to produce a clear solution with no residues. The different alternatives procedures tested are given in Table 3. Table 3: Optimization of green coffee bean digestion procedure

Sample mass Reagent volume (mL) T (oC) Time (h) Appearance of solution (g)

HClO4 HNO3(69%) (70%) H2O2 ( 30%)

0.5 3 2 1 240 3 Turbid

0.5 5 1.5 0 240 3 Yellowish

0.5 6 2 0 270 3 Pale yellow and turbid

0.5 4 4 0 240 3:50 Clear and colorless

0.5 4 4 0 240 3:00 Cloud-turbid

Turbid 0.5 4 4 0 240 3:20

The solution burned(Yellowish ) 0.5 4 4 0 270 3

 The bold font indicates optimum digestion time, T (°C) = temperature

3.9. Instrument Calibration for the Analysis of Metals

For the analysis of the samples calibration of the instrument with the known concentration of standards were done for each metal of interest. First intermediate (100 mg/L) standard solutions were prepared from the stock solutions (1000 mg/L). From the intermediate solutions five working standards were prepared by serial dilution for each metal of interest. The working standards were prepared based on the sensitivity of the instrument towards the particular metals and the level of

27

metals to be present in the samples. Standard for Ca 0.5 ppm, 1.5 ppm, 2.5 ppm, 4.5 ppm, 5.5 ppm Standard solution for Mg 0.5 ppm, 2.5 ppm, 3.5 ppm, 4.5 ppm, 5.5 ppm, 6.5 ppm standard solution for Mn 0.1 ppm, 0.2 ppm, 0.3 ppm, 0.5 ppm, 1.5 ppm, 2.5 ppm, 3.5 ppm, standard solution for Fe 15 ppm, 20 ppm, 25 ppm, 30 ppm, 35 ppm, for Cu standard solution 0.5 ppm, 1.5 ppm, 2.5 ppm, 3.5 ppm, 4.5 ppm, and standard solution for Zn 0.5 ppm, 1.5 ppm, 2.5 ppm, 3.5 ppm, 4.5 ppm. Each of the sets of working standards was then aspirated one after the other by their increasing order of concentration into the atomic absorption spectrometry and their absorbance was recorded. Calibration curves were plotted by five points for each of the metals standard.

The instrumental operating conditions used for the determination of metals using FAAS are shown in Table 4.

Table 4: Instrumental operating conditions for determination of metals using FAAS

metals λ(nm) SW(nm) I(mA) Energy(erg) Flame type

Ca 422.7 0.7 2.0 3.96 Air- acetylene

Mg 285.2 0.7 1.0 3.726 Air- acetylene

Cu 342.7 0.7 1.5 3.728 Air- acetylene

Zn 213.9 0.7 2.0 3.022 Air- acetylene

Fe 248.3 0.2 7.0 3.093 Air- acetylene

Mn 279.5 0.7 3.0 4.045 Air- acetylene

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3.10. Method Validation

Precision Precision was expressed as relative standard deviation (RSD) of the three replicate results. Relative standard deviation is the parameter of choice for expressing precision in analytical sciences. The precision determined at each concentration level should not exceed 15% of the relative standard deviations (RSD) (Douglas A. Skoog et al., 2013). Limit of Detection Limit of detection (LOD) is the minimum concentration of analyte that can be detected but not necessarily quantified with an acceptable uncertainty. LOD for each metal was determined from analysis of three replicates of method blanks which were digested in the same digestion procedure as the actual samples(Harvey et al.2000).

Limit of Quantification The limit of quantification (LOQ) which were digested in the same digestion procedure as the actual samples (Harvey et al., 2000).

Recovery In order to ascertain the reliability of the method for the analysis of the samples for metals of interest,the spiking method was adopted. In spike a known amount of analyte was added into the sample matrix and how much we have recovered the amount that was added were determined. It was determined by replicate analysis of samples containing known amounts of the analyte subjected to the same digestion procedure like the actual sample. The acceptable ranges of percentage recovery for the studied metals will be within 80–120% for metal analysis (Harvey et al., 2000).

3.11. Statistical Analysis

All sample measurements were performed in triplicates and results reported as mean ± standard deviation. Data was analyzed statistically using Microsoft Excel 2013 version. One-way ANOVA was used to assess the existence of significant differences among the means due to coffee origin.

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CHAPTER FOUR 4. RESULTS AND DISCUSSION 4.1. Total Polyphenol

4.1.1. Instrument Calibration

The UV-Vis instrument was calibrated using five series of concentrations of Gallic acid (Figure 6).The calibration curve was linear in the studied range with a correlation coefficient of 0.9989, which implies a strong linear relationship between absorbance and concentration.

2 1.8 y = 0.0068x - 0.1799 1.6 R² = 0.9989 1.4 1.2 1 0.8

absorbance 0.6 0.4 0.2 0 0 50 100 150 200 250 300 350 concentration of galic acid in ppm

Figure 6: the calibration curve of Gallic acid

4.1.2. Total Polyphenol Contents of Green Coffee beans

The total phenolic content (TPC) was determined according to the Folin-Ciocalteu colorimetric method, and calculated as gallic acid equivalent (GAE). This is because gallic acid is the commonest organic compound, having a large amount of total polyphenols. The Folin-Ciocalteu reagent is sensitive to reduce polyphenols, and develop a blue color upon reaction which is measured spectrophotometrically at 760 nm. Lastly, the TPCs of methanolic coffee bean extracts

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were calculated as Gallic acid equivalents (GAE) per gram of sample based on the above standard calibration.

The results of TPCs of green coffee bean extracts from each sampling site are summarized in Figure 7 and Table 5. Accordingly, the overall mean values of TPCs in mg GAE/g ranged from 43.1 ± 2.95 (Gondar Zuria district) to 46.73 ± 3.44 (Chilga district). As can be seen in Table 5, individual TPCs (mg GAE/g) values showed that relatively higher values were observed in coffee beans from Kuribas kebele (52.5±5) in Takusa district followed by Eyaho kebele (51.9±4.3) and Bezaho kebele (51.5±8.7) in Chilga district and Kanfenta kebele (50.5 ± 4.6) in Tach Armachiho district, but lower value was recorded for coffee bean samples from Mekonta kebele (31.5 ± 1.9) in Takusa district.

70.0

60.0

50.0

40.0

30.0

20.0 TPC Concentration ) Concentration (mgGAE/gTPC 10.0

0.0 17 19 23 16 15 18 22 14 21 20 6 7 8 9 10 11 12 13 3 4 5 1 2 Chilga Tach Armachiho TakusaGondar Zuria

Sampling sites

Figure 7: The total polyphenol content in each districts of sampling site

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Table 5: The concentration of total polyphenols (TP) determined in green coffee beans grown in different districts of Central Gondar zone

Sampling districts TP(mgGAE/g) STDEVmg GAE/g RSD Chilga 46.73 3.44 7.36 Tach Armachiho 46.53 3.76 8.08 Takusa 43.67 3.53 8.08 Gondar Zuria 43.1 2.95 6.84 Central Gondar zone 45.01 3.42 7.59

The percent relative standard deviation associated with the individual TPCs among the triplicate measurements from the same sampling sites varied in the range 5.6 to 9.8%, which is within the acceptable limit.

In general, the distribution pattern for mean total polyphenol values in green coffee bean samples was in the order: Chilga (46.73 mg GAE/g) > Tach Armachiho (46.54 mg GAE/g) > Takusa (43.67 mg GAE/g) > Gondar Zuria (43.10 mg GAE/g).

4.1.3. Comparison of the Total Phenolic Contents with Coffee from other origins

The determined TPCs of green coffee beans were compared with literature values. Accordingly, the highest mean value obtained in the present study (46.76 ± 3.44 mg GAE/g) and the average TPC of green coffee bean (45.01 ± 3.42 mgGAE/g) from Central Gondar Zone green was lower than those values recorded for Ethiopian coffee beans from Kochere (55.45 ± 1.08 mg GAE/g), Sidamo (49.19 ± 0.70 mg GAE/g), Yirgacheffe (54.53 ± 1.62 mg GAE/g) (Bobková et al., 2020). The variation in the total phenolic contents might be attributed to the geographical factors as well as the different cultivation methods. A previous study had reported that phenolic content was influenced by the origin of the coffee beans and extracting solvents (Daniel et al., 2017).

Moreover, comparison of the results obtained in the current study with those results from other countries revealed that higher TPCs values were reported in Brazil (745.50 ± 10.5 mg GAE/g), Turkey (668.86 ± 11.2 mg GAE/g), and Ethiopia (651.25 ± 13.0 mg GAE/g) (Nassar et al., 2019). In contrast, lower TPCs were reported for Jember (33.21 mg GAE/g), Malang (3.8619 mg GAE/g),

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Bondowso (21.88 mg GAE/g) and Banyuwangi coffee (7.68 mg GAE/g) all from Indonesia than the results observed in our study (Perdani et al., 2019).

Generally, the data indicated above show that the TPCs of green coffee beans from the Central Gondar Zone have lower TPCs than other coffee samples from other regions of Ethiopia, Brazil and Turkey, but higher than those from Indonesia. This shows that variations could occur due to the difference in Climate (temperature, light and water) and altitude (Dechassa et al., 2018).

4.1.4. Statistical comparison (ANOVA) of the total polyphenol contents of coffees from the different sampling districts

The results of one-way ANOVA (Table 6) showed that sources of green coffee beans (sampling sites) had no significant effect (P < 0.05) among the mean total phenol concentrations in coffee beans from the studied four districts in the Central Gondar Zone. Table 6: ANOVA table obtained from the analysis of the variation of mean total polyphenol contents among green coffee beans from the four districts studied. Difference is significant when p < 0.05.

Source of Variation SS df MS F P Fcrit

Between Groups 40.7309 3 13.5769 0.60576 0.61931 3.1273 7 9 5 7 5

Within Groups 425.846 19 22.4129 4 7

4.2. Elemental composition of the coffee beans

4.2.1. Optimal conditions for sample digestion

An optimized procedure was developed for the digestion of green coffee bean samples prior to analyzing the studied metals using FAAS. Applying the optimized digestion procedure, 0.5 g green coffee bean powder was digested with nitric acid (HNO3; 69%) and perchloric acid (HClO4; 70%) mixtures in the ratio 4:4 (v/v) at a digestion temperature of 240 °C for 3.5 h. These optimal conditions were selected based on clarity of digests, minimum reagent volume consumption,

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minimum digestion time, simplicity, and minimum temperature applied for complete digestion of sample.

4.2.2. Instrument calibration

From the calibration curve of standard samples of each of the studied elements, linearity range, regression equation and correlation coefficient are listed in Table 7.

Table 7: linearity range, regression equation and correlation coefficients of the calibration curve

Metal analyzed Linearity range Regression equation* Correlation coefficient (mg/L) Ca 0.5-5.5 A= 0.0045C + 0.0024 R² = 0.998

Mg 0.5-6.5 A= 0.0893C + 0.8061 R² = 0.996

Cu 0.5-4.5 A= 0.0466C - 0.0089 R² = 0.9996

Zn 0.5-4.5 A= 0.1032C + 0.0116 R² = 0.9969

Fe 15-35 A= 0.0013C + 0.0054 R² = 0.9963

Mn 0.1-3.5 A = 0.0367C - 0.0031 R² = 0.9972

*A = absorbance, C = concentration

4.2.3. Method performance

Linearity Calibration curves for all the metals showed good linearity with coefficients of determination (r2) range between 0.996 and 0.9996 which were the acceptable limit for the linearity of the regression line. This showed that there is a good correlation between concentration and absorbance indicating good calibration of the instrument.

Sensitivity The sensitivity of an instrument is a measure of its ability to distinguish between small differences in analyte concentrations. The simplest measure of sensitivity is the slope of the calibration curve in the concentration range of interest (Son et al., 2003). This refers to the calibration sensitivity.

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So Sensitivity indicated that the slope of the regression equation.The high the slope of the regression equation is highly sensitive. So the sensitivity value of the metal in this study was as follows: Zn > Mg> Cu> Mn > Ca > Fe. The slopes of each metal are 0.1032 (Zn), 0.0893 (Mg), 0.0466 (Cu), 0.0367 (Mn), 0.0045 (Ca), 0.0013 (Fe).

Precision The precision of an analytical method describes the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of sample matrix. Precision was expressed as relative standard deviation (RSD) of the three replicate results and the spiked samples were then subjected to the same digestion procedure like the actual sample. In the precision test of the average RSD for selected metals was in the range of 0.25 to 12 in samples from Gondar Zuria sampling site, RSD for selected metals in Takusa district sampling site the range between 0.2 to 9.2, RSD for selected metals in Tach Armachiho district sampling site was between the range 0.5 to 10.8 and RSD for selected metals in Chilga district sampling site was between in the range 0.37 to 10.81. Therefore, the results obtained by this method were in good agreement with each other in each sampling district.

4.2.4. Method detection and quantification limits

In this study after digestion of blank solution containing a mixture of HNO3 & HClO4 triplicate readings were taken for each blank and the standard deviation of the blank was calculated. The method detection limits (LOD) of the metals of interest listed in Table 8. The results show that the values of all the metals were good.

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Table 8: The method detection limits and method of quantification the metals of interest in (mg/kg) for green coffee samples The metal IDL LOQ (mg/kg) LOD(mg/kg) analyzed (mg/L) Ca 0.0034 105 31

Mg 0.01 183 54.9

Cu 0.005 2.3 0.69

Zn 0.014 0.67 0.055

Fe 0.008 3 1.8 Mn 0.003 1.3 0.39

In this study IDL< LOD< LOQ, this result indicates the method was verified and acceptable.

Recovery The accuracy of the optimized procedure was checked by adding different volumes of standard solution (1000 mg/L), 1795.5 µL of of calcium, 424.5 µL of magnesium, 25 µL of copper, 1.65 µL of zinc, 25 µL of mg/L of iron and 110 µL of manganese solution were added to 0.5 g of coffee from Tach Armachiho district, i.e Mahin sample. Triplicate samples were prepared similarly, and digested in the same manner as for original samples. Finally the solution was analyzed by using FAAS. Table 9 shows that the present recovery of metals in the green coffee ranged from 85% to 116.3%. The obtained results are in the accepted range which is mostly no less than 80% and no greater than 120% which revealed that the digestion method and the AAS analysis was reliable (Harvey et al., 2000).

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Table 9: Recoveries of metals in samples

Elements Mean ± SD (Concentration in mg/L) Recovery %

Unspiked concentration Amount added Spiked concentration concentration Ca 33.2±2.18 1.8 34.73± 0.105 85% Mg 8.5 ± 0.01 0.43 9 ±0.033 116.3%

Cu 0.5±0.01 0.025 0.527±0.01 108%

Zn 0.03 ±0.02 0.020 0.049 ± 0.005 95%

Fe 0.5 ±0.0 0.025 0.5231± 0.37 92.4%

Mn 0.2 ±0.01 0.15 0.34± 0.013 93.3%

4.3. The elemental concentration of green coffee beans The results obtained for the mean concentration of metals in green coffee bean samples from each sampling district are presented in Table 10. All six metals analyzed were detected in the green coffee bean collected from all sampling districts in the Central Gondar Zone. Accordingly, the overall mean values (mg/kg) ranged from: 2876.4 ± 22.2 (Gondar Zuria) to 3514.6 ± 66.5 (Takusa) for Ca, 839.4 ± 4.0 (Tach Armachiho) to 877.3 ± 1.5 (Takusa) for Mg, 31.6 ± 1.7 (Gondar Zuria) to 100.2 ± 2.3 (Takusa) for Cu, 10.0 ±1.6 (Tach Armachiho) to 13.3 ± 0.85 (Gondar Zuria) for Zn, 21±1.2 (Tach Armachiho) to 24.2 ± 1.6 (Takusa) for Mn, and 71.8 ± 8.3 (Gondar Zuria) to 140.2 ± 10.4 ( Takusa) for Fe. As can be seen in appendix (A) Table 2, individual metal content (mg/kg), especially Ca values, showed relatively higher values in coffee beans from Genbera kebele (4465.2 ± 105) in Tach Armachiho, followed by Nara-Awudarda kebele (4272.6 ± 71.1) in Chilga district. Magnesium is also the second highest element next to Ca with highest values in coffee bean samples from Dikularba (910.3 ± 1.9) in Takusa district and Eyaho kebele (875.2 ± 3.4) in Chilga district. In contrast, lower Zn (3.0 ± 0.4 mg/kg) and Mn (18.4 ± 1.6 mg/kg) values were recorded, respectively, for coffee bean samples from Mahin kebele and Kanfenta kebele in Tach Armachiho district.

As indicated Table 10, the green coffee bean from all the districts had a higher amount of Ca than other metals under investigated. The levels of Ca (mg/kg) determined in this study can be ordered

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as follows: Takusa (3514.6 ± 66.5) > Tach Armachiho (3249.4 ± 118.6) > Chilga (3020.7 ± 47.0) > Gondar Zuria (2876.4 ± 22.2). The level of Mg (mg/kg) determined in this study can be order as follow: Takusa (839.4 ± 1.5 mg/kg)> Chilga (852.2 ± 3.0 mg/kg)> Gondar Zuria (845.2 ± 2.1 mg/kg) > Tach Armachiho (839.4 ± 4.0 mg/kg). The level of Cu (mg/kg) determined in this study are Takusa (100.2 ± 2.3 mg/kg)> Tach Armachiho (38.8 ± 1.8mg/kg) > Chilga (34.7± 1.7 mg/kg) > Gondar Zuria (31.6 ±1.7 mg/kg). The level of Zn (mg/kg) determined in this study Gondar Zuria (13.3±0.85 mg/kg)> Takusa (12.8±1.2mg/kg) > Chilga (10.7±0.96 mg/kg) > Tach Armachiho (10.0 ± 1.0 mg/kg). The level of Mn (mg/kg) determined in the present study can be order as follow: Takusa (24.2 ±1.6 mg/kg) > Chilga (22.1 ± 1.6 mg/kg) > Gondar Zuria (21.65 ±1.6 mg/kg) and also the level of Fe in this study are Takusa (140.2 ±10.4 mg/kg) > Tach Armachiho (91.1 ±4.5 mg/kg)> Chilga (89.8 ±5.6 mg/kg) > Gondar Zuria (71.8 ±83 mg/kg).

The concentrations of the studied metals in coffee beans from Takusa district were found to be highest compared to their corresponding metal levels from the remaining three districts, except for Zn which was found the highest in Gondar Zuria district.

Table 10:The concentration (Mean ± SD) of metals from sampling districts

Mean ± SD (concentration in mg/kg) of study elements

Sampling district Ca Mg Cu Zn Fe Mn

Gondar Zuria 2876.4 ± 22.2 845.2 ± 2.1 31.6 ± 1.7 13.3 ± 0.85 71.8 ± 8.3 21.6±1.6

Takusa 3515± 66.5 877.3 ±1.5 100.2 12.8 ±1.2 140.2±10.4 24.2±1.6 ±2.3

Tach Armachiho 3249.4± 118.6 839.4 ±4.0 38.8 ±1.8 10.0 ±1.0 91.0 ± 4.5 21±1.2

Chilga 3020.7 ± 47 852.2 ±3.0 34.7 ±1.7 10.7 ± 0.96 89.8 ± 5.6 22.1±1.6

Average 3,163.5 ±65.8 853.5 ±2.65 46 ± 11.1 11.8 ± 1.4 91.75 ± 33.6 22.2 ±1.5

Hence, the elemental contents varied from one sampling district to another, probably due to various factors. As suggested in different literatures, the chemical composition of green coffee beans differs from one site to another as a result of soil fertility, rainfall distribution, geographical location, altitude difference and coffee species (Mehari et al., 2016).

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The general metal concentration patterns in green coffee beans decreased in the order of: Ca>Mg> Fe > Cu >Mn> Zn (Gondar zuria); Ca>Mg> Fe > Cu >Mn> Zn (Takusa); Ca>Mg> Fe > Cu >Mn> Zn (Chilga); and Ca>Mg> Fe > Cu >Mn> Zn (Tach Armachiho).

4.3.1. Comparison of the elemental contents of green coffees with other literature values

There are a number of studies from different countries on the analysis of metal contents in green coffee bean samples. It is important to compare the results obtained from the Central Gondar Zone with other literature values. This comparison helps the researchers to identify the differences in elemental composition of coffee in different origins.

Table 11: Comparison of the elemental contents of green coffee with other literature values

Sampling origins Ca Mg Cu Zn Mn Fe Vietnam(µg/g) 768.22 682.7 17.38 5.97 10.35 42.98 (van Cuong et al., 2014) Bosnia (µg/g) 789 1758 14 3.6 - 60 (Adleret et al., 2019) Sidamo (mg/kg) 880 1670 22.9 21.1 19.0 26.2 (Gure et al., 2017) Wollega (mg/kg) 710 1670 18.3 12.4 15.0 29.0 (Gure et al., 2017) Harar (mg/kg) 900 1670 15.8 14.3 13.0 43.0 (Gure et al., 2017) BenchMaji 1000 1690 15.7 8.3 15.0 28.8 (Gure et al., 2017)) (mg/kg) Kafa (mg/kg) 1250 1690 10.5 3.8 17.0 44.0 (Gure et al., 2017) Central Gondar 3,163.5 853.5 46 11.8 22.2 91.75 Present study Zone (mg/kg)

The level of metals in green coffee beans from Vietnam, Bosnia, Sidamo, Wollega, Harar, BenchMaji, Kafa, and Central Gondar Zone are presented in Tabe 11. The concentrations of the studied metals in coffee beans from Central Gondar Zone were found to be highest compared to their corresponding metal levels from the other study areas, except for Mg which was found the least as compared to other values reported for coffee sample from Bosnia, Sidamo, Wollega, Harar, BenchMaji and Kafa [(van Cuong et al., 2014), (Adleret et al., 2019), (Gure et al., 2017)]. Generally, the level of metals in green coffee from the Central Gondar Zone is relatively higher than other literature values of other origins.

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The one - way ANOVA results from each sampling district are used to compare the significance difference of each metal concentration. The one way ANOVA results are tabulated in Appendix (B) Table 1-7, which indicated that there was no significance difference at 95% of confidence among the means of all metals in four districts, except Cu from Tach Armachiho and Chilga districts.

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5. CONCLUSION

In the present study, the contents of six essential metals and total polyphenols were determined in green coffee grown in four districts of the Central Gondar Zone. There was no significance difference in the concentration of metals and polyphenols from the four districts, except for copper values between Tach Armachiho and Chilga coffee bean samples. With respect to total polyphenol contents, coffee beans from the Central Gondar Zone had higher values compared to most literature values, but lower than for those coffee types from other regions in Ethiopia. The level of metals in the coffee beans from the Central Gondar Zone were, in most cases, found to be higher than literature values. And, metal concentrtions showed the following general trend: Ca>Mg> Fe > Cu >Mn> Zn (Gondar zuria); Ca>Mg> Fe > Cu >Mn> Zn (Takusa); Ca>Mg> Fe > Cu >Mn> Zn (Chilga); and Ca>Mg> Fe > Cu >Mn> Zn (Tach Armachiho). In summary, the coffee bean samples from this study area contained substantial amounts of polyphenols and essential metal compositions that contribute to many health benefits.

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6. RECOMMENDATION

The following recommendations are made as a result of the outcome of this study. o According to the information acquired during the course of this study, there is a high potential of coffee production in Central the Gondar Zone. In addition, in this study area, the total polyphenol content lower than Sidama, Yirgacheffe, and Kochere coffee types from Ethiopia, however compared to other literature reports, coffee beans from Central Gondar Zone contained higher polyphenols than from Arabica coffee types from Indonesia, and the metal content of green coffee from Central Gondar Zone contain relatively higher level of metals to other origins of green coffee beans. From the Central Gondar Zone calcium contents of the coffee beans were found to be much higher than calcium from other coffee producer regions of Ethiopia and other countries. Hence, authorities in the national and regional government positions or nongovernmental organizations should give attention for research in different perspectives. o Further investigations should be made from other districts of the Zone as well as research on the coffee aroma, so that the concerned authority can come up with firm decisions for the production of coffee.

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Sabah, h., al-jaf, Saydam, S. (2019). "comparison of metal content of coffee samples grown in different countries by inductively coupled plasma optical emission spectroscopy " celal bayar university journal of science 15: 35-43. Ayelign, A., and Sabally, K. (2013). "Determination of chlorogenic acids (CGA) in coffee beans using HPLC " American journal of research communication 1: 78-91. Son., J. W. (2003). "Sample preparation technique in analytical chemistry ." department of chemistry and environmental science new jersey institute of technology 3:2-9. Pohl, P., Stelmach, E., Welna, M., Szymczycha-madeja, A. (2012). "Determination of the elemental composition of coffee using instrumental methods." Food anal. methods ) 6:598–613. Kufa,T., Ayano, A., Yilma, A., Kumela,T. and Tefera,W. (2011 ). "The contribution of coffee research for coffee seed development in Ethiopia " Journal of Agricultural Research and Development 1: 9-16. Tefera, M., Chandravanshi, B. S. (2018). "Assessment of metal contents in commercially available Ethiopian red pepper. " international food research journal 25: 989-1000. Solange, I., Mussatto, Ercília, M. S., Machado, Martins, S., José A. Teixeira (2011). "Production, composition, and application of coffee and its industrial residues." food bioprocess technol 4:661–672. Van Cuong, T., Liu HONG, L., Guo kang, Q., jin, S., Shu jie, S., le linh,T., Duc tiep,T. (2014). "Effect of roasting conditions of conconcentration in elements of vietnam robusta coffee." ACTA Universitatis cibiniensis seriese: Food technology 18:19- 34. Kreicbergs,V., Dimins, F., Mikelsone, V., Cinkmanis, I. (2011). "Biologically active compounds in roasted coffee" Foodbalt ID: 11172469: 110-115. Lire, H.,Wachamo.(2017). "Review on health benefits and risks of coffee consumption." Medicinal & Aromatic Plants 6: 1-12. Weldegebreal, B., Redi-Abshiro,M., Singh, B.,Chandravanshi. (2016). "Development of new analytical methods for the determination of caffeine content in aqueous solution of green coffee beans " Chemistry Central Journal 1: 1-9.

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APPENDIXES APPENDIS (A) Table 1: The concentration of total polyphenols (TP) determined in green coffee beans grown in different districts of Central Gondar zone

Sample STDEVmg RSD Sampling site code TP(mgGAE/g) GAE/g Eyaho kese Haile 17 51.9 4.3 8.3 Bezaho Abay Asnakew 19 47.8 3.3 6.9 Nara Awurarda Gudie 23 44.9 2.9 6.5 Eyaho Semalign 16 39.6 2.2 5.6 Walideba Abuhay 15 43.7 3.0 6.9 Bezaho Gola 18 51.5 4.5 8.7 6.2 Nara Awurarda Akal 22 45.4 2.8 7.6 Walideba Abebe 14 47.2 3.6 7.9 Tenbera Worku 21 46.8 3.7

8.5 Tenbera Gashaw 20 48.5 4.1

Chilga Average. 46.73 3.44 7.36 9.1 Kanfenta Gebrie 6 50.5 4.6 6.5 Kanfenta Asmare 7 42.9 2.8 6.8 Kanfenta Mulat 8 47.3 3.2 9.8 Genbera 1 9 49.0 4.8

8.1 Genbera 2 10 42.2 3.4 9.1 Genbera 3 11 48.2 4.4 8.7 Genbera 4 12 43.7 3.8 6.4 Mahin 13 48.5 3.1 Average. 46.53 3.76 8.08

Tach Armachiho Tach 6.0 Mekonta 3 31.5 1.9 7.9 Dikularba 4 47.0 3.7 9.5 Kurabas 5 52.5 5.0 8.08 Average. 43.67 3.53

Takusa Sur Sarwuha 1 43.6 3.1 7.1 Manterno 2 42.6 2.8 6.6 6.84 Average. 43.1 2.95

Gondar Gondar Zuria 7.59 Central Gondar zone 45.01 3.42

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Table 2: The elemental content of each sampling sites from each district of Central Gondar Zone Ca Mg Cu Zn Mn Fe Saple code Av.mg/Kg sd RSD Av.mg/Kg sd RSD Av.mg/Kg sd RSD Av.mg/Kg sd RSD Av.mg/Kg sd RSD Av.mg/Kg sd RSD

14 1909.6 135..8 7.1 808 3.2 0.4 29.8 2.1 7.2 3.3 0.4 12.1 20.3 1.6 7.8 46.2 0 0 15 2568.9 22.2 0.9 842 3 0.4 36 0 0 9.4 1.3 13.8 22.1 0 0 46.2 0 0 16 2998.5 12.8 0.4 875.2 3.4 0.4 37 1.2 3.4 5.9 0.7 11.9 26.6 1.6 5.9 71.8 2.4 3.3 17 3494.8 33.9 1 857.3 3.4 0.4 33.4 3.3 9.8 10.4 1.5 14.2 23 1.6 6.8 46.2 0 0

18 3109.6 89.8 2.9 845.4 4 0.5 33.4 3.3 9.8 9.1 0.7 7.7 20.3 2.1 10.4 46.2 0 0

Chilga 19 2494.8 89.8 2.9 848.7 4 0.5 34.8 1.2 3.6 6.2 0.9 14.5 19.3 2.7 14.1 46.2 0 0 20 3228.1 46.3 1.4 871.1 1.1 0.1 34.1 0 0 11 1 8.8 23 1.6 6.8 46.2 0 0 21 2983.7 12.8 0.4 867.4 1.7 0.2 38.4 3.7 9.7 11 1 8.8 21.2 1.6 7.4 97.4 8.8 9 22 3146.7 44.4 1.4 848.7 2.2 0.3 34.1 2.1 6.3 10.4 1.5 14.4 23 3.1 13.7 46.2 0 0 23 4272.6 71.4 1.7 858 3.9 0.5 36.3 0 0 30.1 0.6 1.9 22.1 0 0 405.1 44.4 11 Average 3020.7 47.0 2.01 852.2 3.0 0.4 34.7 1.7 5.0 10.7 0.96 10.8 22.1 1.6 7.3 89.8 5.6 2.3 6 2791.1 38.5 1.4 821.8 2.2 0.3 34.8 2.5 7.1 8.1 0.7 8.6 18.4 1.6 8.5 46.2 0 0 7 2154.1 33.9 1.6 790.9 2.3 0.3 37 2.5 6.7 12 1 8.1 18.4 1.6 8.5 71.8 4.4 6.1

8 3228.1 33.9 1.1 852.1 1.1 0.1 37.7 1.2 3.3 8.8 1.2 13.7 19.3 0 0 71.8 4.4 6.1 9 3176.3 89.8 2.8 830 2.3 0.3 37.7 1.2 3.2 11.4 1.1 9.8 21.2 1.6 7.6 251.3 10.3 4.1 10 3324.4 200 6 864 14 1.6 38.4 2.1 5.6 6.5 0.9 13.8 22.1 0 0 71.8 9.8 14 11 3531.9 181 5.1 871.9 2.6 0.3 43.4 1.2 2.9 13 1.7 12.9 23.9 3.1 13.2 123.1 6.9 5.6

Tach Armachiho 12 4465.2 105 2.4 837.5 5.6 0.7 34.8 2.5 7.1 16.9 1 5.7 22.1 0 0 46.2 0 0 13 3324.4 266.7 8 847.2 1.7 0.2 46.3 1.2 2.7 3 0.4 13.5 22.2 1.6 7.4 46.2 0 0 Average 3249.4 118.6 3.6 839.4 4.0 0.5 38.8 1.8 4.8 10.0 1.0 10.8 21.0 1.2 5.7 91.1 4.5 4.5 3 2909.6 51.3 1.8 851.7 0.6 0.1 34.1 2.1 6.3 10.1 1 9.6 23.9 1.6 6.6 46.2 0 0

4 3635.6 101.8 2.8 910.3 1.9 0.2 233.7 3.7 1.6 14.6 2 13.8 28.4 1.6 5.5 184.6 13.3 7.2 5 3998.5 46.3 1.2 870 1.9 0.2 32.7 1.2 3.8 13.6 0.6 4.1 20.3 1.6 7.8 189.7 17.8 9.4

Takusa Average 3514.6 66.5 1.9 877.3 1.5 0.2 100.2 2.3 3.9 12.8 1.2 9.2 24.2 1.6 6.6 140.2 10.4 5.5

1 2872.8 22.2 0.8 841.6 2.3 0.3 29.1 1.2 12.1 9.1 1.1 12.1 20.3 1.6 7.8 71.8 8.1 11 2 2880 22.2 0.8 848.7 1.9 0.2 34.1 2.1 6.3 17.5 0.6 3.2 23 1.6 6.8 71.8 8.5 12

Zuria Gondar Average 2876.4 22.2 0.8 845.2 2.1 0.25 31.6 1.7 9.2 13.3 0.85 7.65 21.65 1.6 7.3 71.8 8.3 12

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ANOVA FOR TPC ANALYSIS

Table 1: Comparison of Tach Armachiho total polyphenol with Chilga district

ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.175692 1 0.175692 0.014999 0.904051 4.493998 Within Groups 187.4154 16 11.71346

Total 187.5911 17

Table 2: Comparison of Tach Armachiho total polyphenol with Takusa district

ANOVA

Source of Variation SS Df MS F P-value F crit Between Groups 18.1018 1 18.1018 0.535626 0.482872 5.117355 Within Groups 304.1606 9 33.79563

Table 3: comparison Takusa and Gondar zuria

ANOVA Source of Variation SS df MS F P-value F crit Between Groups 0.331368 1 0.331368 0.004209 0.952353 10.12796 Within Groups 236.1869 3 78.72898

Total 236.5183 4

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Table 4: Comparison Chilga and Takusa

ANOVA Source of Variation SS df MS F P-value F crit Between Groups 21.88054 1 21.88054 0.678654 0.427551 4.844336 Within Groups 354.6521 11 32.2411

Total 376.5326 12

Table 5: Comparison Chilga and Gondar zuria

ANOVA

Source of Variation SS Df MS F P-value F crit Between Groups 21.65651 1 21.65651 1.813144 0.207857 4.964603 Within Groups 119.4417 10 11.94417

Total 141.0982 11

Table 6: Comparison Tach Armachiho and Gondar zuria

ANOVA

Source of Variation SS Df MS F P-value F crit Between Groups 18.56006 1 18.56006 2.153441 0.180432 5.317655 Within Groups 68.95031 8 8.618789

Total 87.51037 9

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Table 7: ANOVA for Central Gondar Zone district

ANOVA Source of Variation SS df MS F P-value F crit Between Groups 40.60282 3 13.53427 0.607058 0.618524 3.12735 Within Groups 423.6024 19 22.29486

Total 464.2052 22

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APPENDIX (B)

2. ANOVA FOR METAL ANALYSIS

Table 1: Comparison calcium in Gondar Zuria, Takusa, Tach Armachiho, and Chilga

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 802917.7 3 267639.2 0.708668 0.558683 3.12735 Within Groups 7175640 19 377665.3

Total 7978558 22

Table 2: Comparison magnesium in Gondar Zuria, Takusa, Tach Armachiho, Chilga

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 3225.495 3 1075.165 2.092025 0.135138 3.12735 Within Groups 9764.768 19 513.9352

Total 12990.26 22

Table 3: Comparison copper in Gondar Zuria, Takusa, Tach Armachiho, and Chilga

ANOVA Source of Variation SS df MS F P-value F crit Between 4861.67 1620.55 2.46445 0.09364 3.1273

Groups 3 3 8 6 4 5 12493.8

Within Groups 7 19 657.572

17355.5 Total 4 22

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Table 4: Comparison zinc in Gondar Zuria, Takusa, Tach Armachiho, and Chilga

ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 27.76955 3 9.256518 0.253294 0.858009 3.12735 Within Groups 694.3479 19 36.54463

Total 722.1175 22

Table 5: Comparison iron in Gondar Zuria, Takusa, Tach Armachiho, and Chilga

ANOVA

Source of Variation SS df MS F P-value F crit

Between 2397.35 799.118 0.09774 0.96031 3.1273 Groups 5 3 3 4 5 5 155336. Within Groups 4 19 8175.6

157733. Total 8 22

Table 6: Comparison manganese in Gondar Zuria, Takusa, Tach Armachiho, and Chilga ANOVA

Source of Variation SS df MS F P-value F crit

Between Groups 25.46721 3 8.48907 1.589045 0.22504 3.12735 Within Groups 101.5027 19 5.342247

Total 126.9699 22

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Table 7: Comparison of Cupper between Tach Armachiho and Chilga

Anova: Single Factor

SUMMARY Groups Count Sum Average Variance 310.157 38.7696 16.3732 Tach_Ar. 8 4 7 8 347.639 34.7639 5.73633 Chilga. 10 5 5 2

ANOVA Source of Variation SS df MS F P-value F crit Between 71.3147 71.3147 6.86378 0.01857 4.49399 Groups 2 1 2 7 5 8 166.239 Within Groups 9 16 10.39

237.554 Total 7 17

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APPENDIX (C)

1. Calibration curve for Ca standard standard for Ca Conce. absorbance 0.5 0.005 1.5 0.009 2.5 0.013 4.5 0.023 5.5 0.027 absorbance 0.03 y = 0.0045x + 0.0024 0.02 R² = 0.998 0.01

0 0 1 2 3 4 5 6

2. Calibration curve for Mg

Concen. absorbance 0.5 0.86 2.5 1.01 3.5 1.12 4.5 1.21 5.5 1.312 6.5 1.379 Chart Title 1.5 y = 0.0893x + 0.8061 R² = 0.996 1

0.5

0 0 1 2 3 4 5 6 7

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3. Calibration curve for Mn standard for Mn Concen. Absorbance. 0.1 0.004 0.2 0.006 0.3 0.008 0.5 0.011 1.5 0.05 2.5 0.088 3.5 0.127 Chart Title 0.14 0.12 y = 0.0367x - 0.0031 0.1 R² = 0.9972 0.08 0.06 0.04 0.02 0 0 0.5 1 1.5 2 2.5 3 3.5 4

4. Calibration curve for Cu standard for Cu Cncen. absorbance 0.5 0.016 1.5 0.059 2.5 0.108 3.5 0.153 4.5 0.202

Chart Title 0.25 y = 0.0466x - 0.0089 0.2 R² = 0.9996 0.15

0.1

0.05

0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

57

5. Calibration curve for Fe standard for iron Conce. Absorbance. 15 0.026 20 0.031 25 0.038 30 0.045 35 0.052 Chart Title 0.06 0.05 y = 0.0013x + 0.0054 0.04 R² = 0.9963 0.03 0.02 0.01 0 0 5 10 15 20 25 30 35 40

6. Calibration curve for Zn standard for Zn Conce. absorbance 0.5 0.053 s1.5 0.176 2.5 0.272 3.5 0.38 4.5 0.467 Chart Title 0.5 0.45 y = 0.1032x + 0.0116 0.4 R² = 0.9969 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

58

APPENDIX (D)

Image A&B Shows that the removal of husks of coffee

A B

Image A&B shows that the coffee sample washed and dried.

A B

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Image A& B indicated that the poly phenol extraction by using magnetic stirrer

A B

Gallic acid standards solution in different series

60

A&B Image indicated that the standard solution and sample solution (for UV reading)

A B

Image indicated digested samples solution

The FAAS Reading

61