Assessment of Quality of Honey Obtained from Different Sources

By:

Banan Abuelgasim AbdElhmied Mahmoud B.Sc. (Honors) in Food Engineering Technology (2008) Faculty of Engineering and Technology University of Gezira

A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science

in Food Engineering Department of Food Science and Technology Faculty of Engineering and Technology University of Gezira

Main supervisor: Prof. Dr. Abdel Moneim Elhadi Sulieman Co-supervisor: Dr. Zakaria Ahmed Salih

August, 2012

Assessment of Quality of Honey Obtained from Different Sources

By:

Banan Abuelgasim AbdElhmied Mahmoud

Approved by: Major Supervisor: Prof. Dr. Abdel Moneim Elhadi Sulieman …………. Co-Supervisor: Dr. Zakaria Ahmed Salih ………….

Assessment of Quality of Honey Obtained from Different Sources

By:

Banan Abuelgasim AbdElhmied Mahmoud

Examination Committee: Name Position Signature Prof. Dr. Abdel Moneim E. Sulieman Chairperson ……………. Prof. Dr. Elnaeim Abdalla Ali External Examiner ……………. Dr. Elzubier Ahmed Salih Internal Examiner …………….

Date of Examination: 12/8/2012

االيّ

)واوحى ربك انى انُحم اٌ اتخذي يٍ انجبال بٍىتا ويٍ انشجز ويًا ٌعزشىٌ ثى كهً يٍ كم انثًزات فاسهكً سبم ربك ذنال ٌخزج يٍ بطىَها شزاب يختهف انىاَه فٍه شفاء نهُاس اٌ فً ذنك الٌه نقىو ٌتفكزوٌ(

ACKNOWLEDGEMENT

Above all, I render my thanks to the merciful "Allah" who offered me health and patience to accomplish this study. I also extend my sincere thanks to my mother, my father and my big family for support and constant encouragement. And all thanks for my fiancé ASHRAF ABDALLA for his advices it was the corner stone. An, special thank and appreciations are extended to DR. MOATAMEN KEHEEL , HASAN ANSARY , MY LOVELY SISTER (RWAN) , ISRAA AHMED and HAJIR ABDALRAHMAN for their encouragement. , special thanks and a very special for all friends and colleagues, especially to BATCH (25) University of Gezira , BATCH(9) & BATCH (10) Master University of Gezira. I take this opportunity to express my sense of gratitude and indebtedness to my supervisor; Prof. Dr. Abdel Moneim Elhadi Sulieman for helping me a lot to complete the project without whose sincere and kind effort. Thanks to my Co- supervisor: Dr. Zakaria Ahmed Salih . I would also like to acknowledge to the contribution of the Department of Food Engineering as a whole, Food Engineering, for having provided us with all the necessary facilities. Fainally, Iam also grateful to all the staff of the college library, laborotary and faculty from the Department of Food Engineering, University of Gezira, their encouragement.

Assessment of Quality of Honey Obtained from Different Sources Banan Abuelgasim AbdElhmied Mahmoud The Degree of Master of Science in food engineering( Augast 2012) Department of Food Science and Technology Faculty of Engineering and Technology University of Gezira Abstract Honey is a food rich in nutrients essential to human life such as sugars, proteins, vitamins and minerals and also honey is used a medicine. Honey is considered as one of the most foods which can be adulteration, therefore this study aimed to assess the quality of different samples of natural honey and Industrial honey (produse from sugar and molasse ) matching the results with the international standards. Chemical and physical methods were used in the study to determine the various chemical and physicochemical characteristics. For determination of sugars, a High Performance Liquid Chromatography (HPLC) was used. The results indicated that there were no significant differences in most of the chemical and physicochemical characteristics of natural and industrial honey. The range of the sugar contents in the natural and industrial honey were: the sucrose (3.48% - 10.7%), fructose (14.74% - 39.01%) and the glucose (14.09% - 35.6%). The pH value and ash, protein, vitamins and minerals percentages were also measured. The results of this study also proved that, there was no significant difference between natural honey and industrial honey in most of their quality aspects. This study recommended the use of ground honey sample for diabetic persons, while industrial honey will be more beneficial for children. Also the study recommended to search for an easy way to detect honey adulteration.

حقييى جىدة انعظم انذي حى انحصىل عهيه يٍ يصادر يخخهفه بُاٌ أبىانقاطى عبذانحًيذ يحًىد ياجظخيز في ُْذطّ االغذيّ اغظطض2102 قظى عهٕو ٔحكُٕنٕجيا االغذيّ كهيّ انُٓذطّ ٔانخكُٕنٕجيا جايعّ انجشيزة

انًهخص انعظم يٍ االغذيّ انغُيّ بانًٕاد انغذائيّ االطاطيّ انضزٔريّ نحياِ االَظاٌ يثم انظكزياث ٔانبزٔحيُاث ٔانفيخاييُاث ٔانًعادٌ ٔيظخخذو انعظم كغذاء ٔدٔاء. يعخبز انعظم يٍ اكثزانظهع انًعزضّ نهغش نذنك اطخٓذفج انذراطت ححهيم جٕدة عيُاث يٍ انعظم انطبيعي ٔانصُاعي )يُخج يٍ انظكز وانًىالص( ويقارَت انُخائج يع انًقاييض انعانًيت. إطخخذيج انطزق انكيًيائيت وانفيشيائيت في هذا انبحث نخحذيذ يخخهف انخىاص انكيًيائيت وانفيشيىكيًيائيت. ونخحذيذ انظكزياث انًخخهفت اطخخذو ٔكاٌ انًذي في انًحخٕي انظكزي HPLCجهاس االطخشزاب انهىَي عاني انجىدة في عيُاث انعظم انطبيعي ٔانصُاعي كاآلحي: انجهكٕس َظبخّ بيٍ )%00.16 - 3..6%( ,انفزكخٕس َظبخّ )00.40% - 66.10%( ٔانظكزٔس )%6.05 - 01.4%(. حى أيضاً قياص قيًت االص انٓيذرٔجيُي, َٔظبت انًحخٕياث يٍ انزياد, انبزٔحيٍ, انفيخاييُاث ٔانًعادٌ. أكذث انُخائج ايضاً اَّ ال يٕجذ فزق يعُٕي بيٍ انعظم انطبيعي ٔانصُاعي في اغهب خٕاص انجٕدة. أٔصج انذراطت باطخخذاو عيُت انعظم االرضي نالشخاص انًصابيٍ بانظكزي بيًُا انعظم انصُاعي يكٌٕ اكثز فائذة نألطفال. ٔكذنك أصج انذراطت بانبحث عٍ طزيقت طٓهت نهكشف عٍ انغش في انعظم انطبيعي.

CONTENTS

Dedications……………………………………………...... i Acknowledgement……………………………………………. ii Abstract ……….……………………………………………… iii Abstract (Arabic)……………………………………………… iv Contents…………………………………………………...... v List of Tables……………………………………………...... ix CHAPTER ONE: INTRODUCTION..……… .…………… 1 1.1. Introduction …………………….…..….………….…. 1 1.2. The main objectives of the study were .…...………… 3 CHAPTER TWO: Literature Review……….…………...... 4 2.1. Introduction: ………………………………...……….. 4 2.2. Formation …………………………………...... 5 2.3. In history, culture, And folklore ………………………. 6 2.4. Ancient times: ………………………………………… 6 2.5. In western culture: ………………………………...... 7 2.6. Religious significance: ……………………………….. 7 2.7. Physical properties: …………………………………. 9 2.8. Acidity: …………………………………...... …….. 11 2.9. Osmotic effect: …………………………………...... 11 2.10. Hydrogen peroxide: ………………………………….. 11 2.11. Methylglyoxal: …………………………………...... 12 2.12. Nutraceutical Effects: ……………………………..….. 12 2.13. Typical Honey Analysis: ………………………….... 12 2.14. Classification: …………………………………...... 13 2.14.1. Floral source: ……………………………… 13 2.14.2. Polyfloral: ………………………………….. 13 2.14.3. Monofloral: ……………………………..….. 13 2.14.4. Honeydew honey: ……………………..…… 14 2.15. Classification by packaging and processing: …..…. 15 2.16. Distinguishing honey: …………………………….... 17 2.16.1. Honey grading: ……………………………... 17 2.17. Indicators of quality: ……………………………….. 18 2.18. Nutrition: ……………………………………...... 19 2.19. Preservation: ……………………………………...... 20 2.20. Uses of honey: …………………………………...... 21 2.20.1. In medicine: ………………………………….. 21 2.20.2. Use for diabetic ulcers: ……………………… 22 2.20.3. Use for sore throats and coughs: …………… 22 2.20.4. Other medical applications: ………………… 22 2.20.5. Modern uses: ……………………...………… 23 2.21. Health Hazards …………………………………...... 24 2.21.1. Botulism: …………………………………...... 24 2.22. Toxic Honey: …………………………………...... 24 2.23. Honey-Producing Countries:…….…………………… 25 2.24. Modern research shows that honey: …………………. 26 2.24.1. Blocks the growth of oral bacteria: …….……. 26 2.24.2. Treatment of burns and skin ulcers: ………..... 26 CHAPTER THREE: MATERIAL AND METHOD……… 29 3.1. Materials: ……………………………………...... 29 3.1.1. Honey Samples: …………………………………….... 29 3.2. Methods: ……………………………………...... 30 3.2.1 Physicochemical characteristic: …………………..…. . 30 3.2.1.1. Acidity ……………………………………...... 30 3.2.1.2. pH Measurement: ………………………..……. ..30 3.2.1.3. Total dissolve Solid (TDS): ……………..……… 30 3.2.2 . Approximate Chemical Analysis of Honey: ..……... . 30 3.2.2.1 Moisture Content: ……………………..………. .. 30 3.2.2.2 Ash Content: …………………………..……….. .. 31 3.2.2.3 Nitrogen and Protein Content: ………..……….. .. 31 3.2.2.4 Sugar Content: ………………………..………... .. 32 3.2.2.5 Ascorbic Acid (Vitamin C)7 …………..…………. . 33 3.2.2.6 mineral content: ………………….…………… 33

CHAPTER FOUR R: ESULT AND DISCUSSION …….. 34 4.1. Physicochemical and approximate chemical characteristics of honey : … ……………………………………...... 35 4.2. Carbohydrate Analysis Using HPLC for Sugar Content:. 37 4.3. Minerals Content: …………………………………….. 39 CHAPTER FIVE: CONCLUSION AND RECOMMENDATION………………………………….… 40 5.1. Conclusions: ……………………...... 40 5.2. Recommendations: …………………………….…….. 39 REFERENCES…………………………………….………. 41 APPENDIX……………………………………….………... 45

LIST OF TABLES

Table Title Page No No Table Name of honey sample and location 29 3.1 Table The physicochemical and chemical characteristics of different 36 4.1 types of honey Table Sugar Content of Different Honey Types 38 4.2 Table The Minerals Content in the Types of Honey 39 4.3

Chapter One Introduction 1.1. Introduction Honey is a natural food, mainly composed of a complex mixture of carbohydrates and other minor substances, such as organic acids, amino acids, proteins, minerals, and vitamins. In almost all honey types, fructose predominates, glucose being the second main sugar. These two account for nearly 85–95% of the honey carbohydrates. More complex sugars made up of two or more molecules of glucose and fructose constitute the remaining carbohydrates, except for a trace of polysaccharide. Honey also contains volatile substances which are responsible for the characteristic flavour. The Codex Alimentarius Standard for honey quality includes several chemical and physical parameters, comprising moisture content, mineral content, acidity, hydroxymethylfurfural (HMF) content, diastase activity, apparent sugar content, and water insoluble solids content. These analyses help the food analyst to determine the ―chemical‖ quality of the honeys analyzed. Moreover, suggest that they may be used in association with multivariate analyses to assign floral origin. The honey analyses conductive pH free acidity and percentages of fructose, glucose and raffinose as variables for the principal component analysis (Finola, 2007). Honey is very hygroscopic, which means that it easily absorbs moisture from the air. Thus, in areas with a very high humidity it can be difficult to produce good quality honey of sufficiently low water content. Honey constitutes a complex food matrix containing small amounts of a high number of floral nectar phytochemicals that are transformed by the bee saliva enzymes and during the honey maturation process in the hive . In addition, it can contain other plant-derived metabolites as is the case of pollen and propolis (plant exudates and resins) constituents. These plant compounds can be used for geographical and floral origin determinations of honey. Honey is color graded into light, amber, and dark categories which do not really have any bearing on quality. Some of the most distinctively and strongly flavored honey varieties, such as basswood, are very light, while very mild and pleasant honeys such as tulip poplar can be quite dark. While it is not an indicator of honey quality and there are exceptions to the rule, generally speaking, the darker color the honey, the higher its mineral contents, the pH readings, and the aroma/flavor levels. Minerals such as potassium, chlorine, sulfur, iron, manganese, magnesium, and sodium have been found to be much higher in darker honeys (Truchado et al, 2011). Honey is a natural food of great interest for its high nutritional value and therapeutic and medicinal properties. Apitheraphy (the medicinal use of honeybee products) has recently gained attention for preventive medicine in several conditions and diseases as well as for promoting health and well-being. Because of its sweetness, color and flavor, honey is also often used in place of sugar and as an ingredient or natural preservative in many manufactured foods. It can prevent oxidative reactions in foods (e.g. lipid oxidation in meat and enzymatic browning of fruits and vegetables. The composition and properties of a particular honey sample depend strongly on the type of flowers the bees visited, on the climatic conditions in which the plants grow and on the beekeeper's contribution. From the chemical point of view, honey is a highly complex, concentrated mixture of sugars with a large pool of minor constituents of different molecular weights (MW) and chemical nature embedded. Many of these components are thought to be responsible for its beneficial properties, from high- MW components (proteins) excreted by honey bees into honey (e.g. glucose oxidase, invertase, saccharase, diastase and catalase) and, to peculiar and particularly abundant plant secondary metabolites (e.g. kynurenic acid (KA) in chestnut honey , cyclohexa-1,3-diene-1-carboxylic acid (CDCA) in lime tree honey, and/or an array of minor compounds (phenolic acid derivatives such as ferulic acid, caffeic acid and coumaric acid and its esters, and flavonoid aglycones) deriving from the original composition of the nectars and sugar-rich materials on which honey bees feed to produce honey (Giangiacomo et al, 2011). In some cases honey is contaminated by the addition of sugar and the search for competitively priced products sometimes drives certain importers to acquire falsified honey (as indicated by the presence of starches and ashes). 1.2. The main objectives of the study were: a. To assess quality of honey samples collected from different sources. b. To compare the quality parameters of honey with the international standards. c. To detect the adulteration of natural honey in trade market.

Chapter Two Literature Review 2.25. Introduction: Honey is a sweet food made by bees using nectar from flowers. The variety produced by honey bees (the genus Apis) is the one most commonly referred to and is the type of honey collected by beekeepers and consumed by humans. Honey produced by other bees and insects has distinctly different properties. Honey bees transform nectar into honey by a process of regurgitation, and store it as a primary food source in wax honeycombs inside the beehive. Beekeeping practices encourage overproduction of honey so the excess can be taken from the colony (Lansing, et.al 1999). Honey gets its sweetness from the monosaccharide’s fructose and glucose, and has approximately the same relative sweetness as that of granulated sugar. It has attractive chemical properties for baking, and a distinctive flavor that leads some people to prefer it over sugar and other sweeteners. Most microorganisms do not grow in honey because of its low water activity of 0.6. However, honey sometimes contains dormant endospores of the bacterium Clostridium botulinum, which can be dangerous to infants, as the endospores can transform into toxin- producing bacteria in the infant's immature intestinal tract, leading to illness and even death (Shapiro, et.al 1998). Honey has a long history of human consumption, and is used in various foods and beverages as a sweetener and flavoring. It also has a role in religion and symbolism. Flavors of honey vary based on the nectar source, and various types and grades of honey are available. It is also used in various medicinal traditions to treat ailments. The study of pollens and spores in raw honey (melissopalynology) can determine floral sources of honey (Vaughn, 2001). Because bees carry an electrostatic charge, and can attract other particles, the same techniques of melissopalynology can be used in area environmental studies of radioactive particles, dust or particulate pollution (Mercuri and Porrini, 1991). 2.26. Formation Honey is produced by bees as a food source. In cold weather or when fresh food sources are scarce, bees use their stored honey as their source of energy. By contriving for bee swarms to nest in artificial hives, people have been able to semi-domesticate the insects, and harvest excess honey. In the hive (or in a wild nest), there are three types of bee in a hive: a single female queen bee, a seasonally variable number of male drone bees to fertilize new queens, and some 20,000 to 40,000 female worker bees. The worker bees raise larvae and collect the nectar that will become honey in the hive. Leaving the hive, they collect sugar-rich flower nectar and return (Val, 2007). In the hive, the bees use their "honey stomachs" to ingest and regurgitate the nectar a number of times until it is partially digested. The bees work together as a group with the regurgitation and digestion until the product reaches a desired quality. It is then stored in honeycomb cells. After the final regurgitation, the honeycomb is left unsealed. However, the nectar is still high in both water content and natural yeasts, which, unchecked, would cause the sugars in the nectar to ferment. The process continues as bees inside the hive fan their wings, creating a strong draft across the honeycomb, which enhances evaporation of much of the water from the nectar. This reduction in water content raises the sugar concentration and prevents fermentation. Ripe honey, as removed from the hive by a beekeeper, has a long shelf life, and will not ferment if properly sealed (Standifer, 2007). 2.27. In history, culture, And folklore: Honey use and production has a long and varied history. In many cultures, honey has associations that go beyond its use as a food. Honey is frequently used as a talisman and symbol of sweetness. 2.28. Ancient times: Honey collection is an ancient activity. Humans apparently began hunting for honey at least 10,000 years ago, as evidenced by a cave painting in Valencia, Spain. The painting is a Mesolithic rock painting, showing two female honey-hunters collecting honey and honeycomb from a wild bee nest. The two women are depicted in the nude, carrying baskets, and using a long, wobbly ladder to reach the wild nest. In ancient Egypt, honey was used to sweeten cakes and biscuits, and was used in many other dishes. Ancient Egyptian and Middle Eastern peoples also used honey for embalming the dead. Pliny the Elder devotes considerable space in his book Naturalis Historia to the bee and honey, and its many uses. The fertility god of Egypt, Min, was offered honey. The art of beekeeping in ancient China has existed since time immemorial and appears to be untraceable to its origin. In the book "Golden Rules of Business Success" written by Fan Li (or Tao Zhu Gong) during the Spring and Autumn Period, there are some parts mentioning the art of beekeeping and the importance of the quality of the wooden box for bee keeping that can affect the quality of its honey. Honey was also cultivated in ancient Mesoamerica. The Maya used honey from the stingless bee for culinary purposes, and continue to do so today. The Maya also regard the bee as sacred. Some cultures believed honey had many practical health uses. It was used as an ointment for rashes and burns, and to help soothe sore throats when no other medicinal practices were available (Tonelli, et.al1990).

2.29. In western culture: The word "honey", along with variations like "honey bun" and the abbreviation "hon", has become a term of endearment in most of the English-speaking world. In some places it is used for loved ones; in others, such as Australia and the Southern United States, including Baltimore, Maryland, it is used when addressing casual acquaintances or even strangers. In many children’s books, bears are depicted as eating honey (e.g., Winnie the Pooh), though most bears actually eat a wide variety of foods, and bears seen at beehives are usually more interested in bee larvae than honey. In some European languages, even the word for bear (e.g. in Russian медведь medvéd, in Polish niedźwiedź, in Czech medvěd, in Serbian медвед medved, in Bosnian medvjed in Croatian medvjed/međed and in Hungarian medve) is coined from the noun meaning honey and the verb meaning to eat, thus "honey eater. Honey is sometimes sold in bear-shaped jars or squeeze bottles (Max and Heidelberg, 1962). 2.30. Religious significance: In Hinduism, honey (Madhu) is one of the five elixirs of immortality (Panchamrita). In temples, honey is poured over the deities in a ritual called Madhu abhisheka. The Vedas and other ancient literature mention the use of honey as a great medicinal and health food. In Jewish tradition, honey is a symbol for the new year, Rosh Hashanah. At the traditional meal for that holiday, apple slices are dipped in honey and eaten to bring a sweet new year. Some Rosh Hashanah greetings show honey and an apple, symbolizing the feast. In some congregations, small straws of honey are given out to usher in the new year. The Hebrew Bible contains many references to honey. In the Book of Judges, Samson found a swarm of bees and honey in the carcass of a lion. The Book of Exodus famously describes the Promised Land as a "land flowing with milk and honey". However, the claim has been advanced that the original Hebrew actually refers to the sweet syrup produced from the juice of dates. Pure honey is considered kosher even though it is produced by a flying insect, a nonkosher creature; other products of nonkosher animals are not kosher. In Buddhism, honey plays an important role in the festival of Madhu Purnima, celebrated in India and Bangladesh. The day commemorates Buddha's making peace among his disciples by retreating into the wilderness. The legend has it that while he was there, a monkey brought him honey to eat. On Madhu Purnima, Buddhists remember this act by giving honey to monks. The monkey's gift is frequently depicted in Buddhist art (Berel, 2005). In the Christian New Testament, Matthew 3:4, John the Baptist is said to have lived for a long period of time in the wilderness on a diet consisting of locusts and wild honey. In Islam, there is an entire Surah in the Qur'an called al-Nahl (the Honey Bee). According to hadith, Prophet Muhammad strongly recommended honey for healing purposes. The Qur'an promotes honey as a nutritious and healthy food. Below is the English translation of those specific verses. And your Lord inspired the bee(s), saying: "Take your habitations in the mountains and in the trees and in what they erect. Then, eat of all fruits, and follow the ways of your Lord made easy (for you)." There comes forth from their bellies, a drink of varying colour wherein is healing for mankind. Verily, in this is indeed a sign for people who think. (Translation of Quran 16:68–69). 2.31. Physical properties: The physical properties of honey vary, depending on water content, the type of flora used to produce it, temperature, and the proportion of the specific sugars it contains. Fresh honey is a supersaturated liquid, containing more sugar than the water can typically dissolve at ambient temperatures. At room temperature, honey is a supercooled liquid, in which the glucose will precipitate into solid granules. This forms a semisolid solution of precipitated sugars in a solution of sugars and other ingredients (Root, 2005). The melting point of crystallized honey is between 40 and 50 °C (104 and 122 °F), depending on its composition. Below this temperature, honey can be either in a metastable state, meaning that it will not crystallize until a seed crystal is added, more often, it is in a "labile" state, being saturated with enough sugars to crystallize spontaneously. The rate of crystallization is affected by the ratio of the main sugars, fructose to glucose, as well as the dextrin content. Temperature also affects the rate of crystallization, which is fastest between 13 and 17 °C (55 and 63 °F). Below 5 °C, the honey will not crystallize and, thus, the original texture and flavor can be preserved indefinitely (Piotr, 2004). Since honey normally exists below its melting point, it is a super cooled liquid. At very low temperatures, honey will not freeze solid. Instead, as the temperatures become colder, the viscosity of honey increases. Like most viscous liquids, the honey will become thick and sluggish with decreasing temperature. While appearing or even feeling solid, it will continue to flow at very slow rates. Honey has a glass transition between -42 and -51 °C (-44 and -60 °F). Below this temperature, honey enters a glassy state and will become a non crystalline amorphous solid (Kántor, et.al 1999). The viscosity of honey is affected greatly by both temperature and water content. The higher the humidity, the easier honey will flow. Above its melting point, however, water has little effect on viscosity. Aside from water content, the composition of honey also has little effect on viscosity, with the exception of a few types. At 25 °C (77 °F), honey with 14% humidity will generally have a viscosity of around 400 poise, while a honey containing 20% humidity will have a viscosity of around 20poise. Viscosity increase due to temperature occurs very slowly at first. A honey containing 16% humidity, at 70 °C (158 °F), will have a viscosity of around 2 poise, while at 30 °C (86 °F), the viscosity will be around 70 poise. As cooling progresses, honey will become more viscous at an increasingly rapid rate, reaching 600 poise around 14 °C (57 °F). However, while honey is very viscous, it has rather low surface tension. A few types of honey have unusual viscous properties. Honey from heather or manuka display thixotropic properties. These types of honey enter a gel-like state when motionless, but then liquify when stirred. Unlike many other liquids, honey has very poor thermal conductivity. Melting crystallized honey can easily result in localized caramelization if the heat source is too hot, or if it is not evenly distributed. However, honey will take substantially longer to liquify when just above the melting point than it will at elevated temperatures. Since honey contains electrolytes, in the form of acids and minerals, it exhibits varying degrees of electrical conductivity. Measurements of the electrical conductivity are used to determine the quality of honey in terms of ash content. The effect honey has on light is useful for determining the type and quality. Variations in the water content alter the refractive index of honey. Water content can easily be measured with a refractometer. Typically, the refractive index for honey will range from 1.504 at 13% humidity, to 1.474 at 25%. Honey also has an effect on polarized light, in that it will rotate the polarization plane. The fructose will give a negative rotation, while the glucose will give a positive one. The overall rotation can be used to measure the ratio of the mixture. Honey has the ability to absorb moisture directly from the air, a phenomenon called hygroscopy. The amount of water the honey will absorb is dependent on the relative humidity of the air. This hygroscopic nature requires that honey be stored in sealed containers to prevent fermentation. Honey will tend to absorb more water in this manner than the individual sugars would allow on their own, which may be due to other ingredients it contains (Root, 2005). 2.32. Acidity: The pH of honey is commonly between (3.2 and 4.5).This relatively acidic pH level prevents the growth of many bacteria. 2.33. Osmotic effect: Honey is primarily a saturated mixture of two monosaccharides, with a low water activity; most of the water molecules are associated with the sugars and few remain available for microorganisms, so it is a poor environment for their growth. If water is mixed with honey, it loses its low water activity, and therefore no longer possesses this antimicrobial property.

2.34. Hydrogen peroxide: Hydrogen peroxide is formed in a slow-release manner by the enzyme glucose oxidase present in honey. It becomes active only when honey is diluted, requires oxygen to be available for the reaction (thus it may not work under wound dressings, in wound cavities or in the gut), is active only when the acidity of honey is neutralized by body fluids, can be destroyed by the protein- digesting enzymes present in wound fluids, and is destroyed when honey is exposed to heat and light. Honey chelates and deactivates free iron, which would otherwise catalyze the formation of oxygen free radicals from hydrogen peroxide, leading to inflammation. Also, the antioxidant constituents in honey help clean up oxygen free radicals present.

C6H12O6 + H2O + O2 → C6H12O7 + H2O2 (glucose oxidase reaction) When honey is used topically (as, for example, a wound dressing), hydrogen peroxide is produced by dilution of the honey with body fluids. As a result, hydrogen peroxide is released slowly and acts as an antiseptic. 2.35. Methylglyoxal: The nonperoxide antibiotic activity is due to methylglyoxal (MGO) and an unidentified synergistic component. Most honeys contain very low levels of MGO, but manuka honey contains very high levels. The presence of the synergist in manuka honey more than doubles MGO antibacterial activity. 2.36. Nutraceutical Effects: Antioxidants in honey have even been associated with reducing the damage done to the colon in colitis in a study involving administering honey enamas to rats, such claims are consistent with its use in many traditions of folk medicine (Bilsel, et.al 2002). 2.37. Typical Honey Analysis: Fructose: 38.2% Glucose: 31.3% Maltose: 7.1% Sucrose: 1.3% Water: 17.2% Higher sugars: 1.5% Ash: 0.2% Other/undetermined: 3.2% Its glycemic index ranges from 31 to 78, depending on the variety. Honey has a density of about 1.36 kilograms per litre (36% denser than water) (Rainer, 1996). Isotope ratio mass spectrometry can be used to detect addition of corn syrup or sugar cane sugars by the carbon isotopic signature. Addition of sugars originating from corn or sugar cane (C4 plants, unlike the plants used by bees, which are predominantly C3 plants) skews the isotopic ratio of sugars present in honey, but does not influence the isotopic ratio of proteins; in an unadulterated honey, the carbon isotopic ratios of sugars and proteins should match. As low as 7% level of addition can be detected. 2.38. Classification: Honey is classified by its floral source, and there are also divisions according to the packaging and processing used. There are also regional honeys.

2.38.1. Floral source: Generally, honey is classified by the floral source of the nectar from which it was made. Honeys can be from specific types of flower nectars, from indeterminate origin, or can be blended after collection. Blended Most commercially available honey is blended, meaning it is a mixture of two or more honeys differing in floral source, color, flavor, density or geographic origin. 2.38.2. Polyfloral: Polyfloral honey, also known as wildflower honey, is derived from the nectar of many types of flowers. The taste may vary from year to year, and the aroma and the flavor can be more or less intense, depending on which bloomings are prevalent. 2.38.3. Monofloral: Monofloral honey is made primarily from the nectar of one type of flower. Different monofloral honeys have a distinctive flavor and color because of differences between their principal nectar sources. To produce monofloral honey, beekeepers keep beehives in an area where the bees have access to only one type of flower. In practice, because of the difficulties in containing bees, a small proportion of any honey will be from additional nectar from other flower types. Typical examples of North American monofloral honeys are clover, orange blossom, blueberry, sage, tupelo, buckwheat, fireweed, and sourwood. Some typical European examples include thyme, thistle, heather, acacia, dandelion, sunflower, honeysuckle, and varieties from lime and chestnut trees. In North Africa, such as Egypt, examples include clover, cotton, and citrus (mainly orange blossoms) (Gounari, 2006). 2.38.4. Honeydew honey: Instead of taking nectar, bees can take honeydew, the sweet secretions of aphids or other plant sap-sucking insects. Honeydew honey is very dark brown in color, with a rich fragrance of stewed fruit or fig jam, and is not sweet like nectar honeys. Germany's Black Forest is a well known source of honeydew-based honeys, as well as some regions in Bulgaria and Northern California in the United States. In Greece, pine honey (a type of honeydew honey) constitutes 60–65% of the annual honey production. Honeydew honey is popular in some areas, but in other areas beekeepers have difficulty selling the stronger flavored product. The production of honeydew honey has some complications and dangers. The honey has a much larger proportion of indigestibles than light floral honeys, thus causing dysentery to the bees, resulting in the death of colonies in areas with cold winters. Good beekeeping management requires the removal of honeydew prior to winter in colder areas. Bees collecting this resource also have to be fed protein supplements, as honeydew lacks the protein- rich pollen accompaniment gathered from flowers (Gounari, 2006). 2.39. Classification by packaging and processing: Generally, honey is bottled in its familiar liquid form. However, honey is sold in other forms, and can be subjected to a variety of processing methods.  Crystallized honey is honey in which some of the glucose content has spontaneously crystallized from solution as the monohydrate. Also called "granulated honey." Honey that has crystallized over time (or commercially purchased crystallized) in the home can be returned to a liquid state if stirred in a container sitting in warm water at 120 °F (approx 49 °C).  Pasteurized honey is honey that has been heated in a pasteurization process (161 °F (71.7 °C) or higher). Pasteurization destroys yeast cells. It also liquefies any microcrystals in the honey, which delays the onset of visible crystallization. However, excessive heat exposure also results in product deterioration, as it increases the level of hydroxymethylfurfural (HMF) and reduces enzyme (e.g. diastase) activity. Heat also affects appearance (darkens the natural honey color), taste, and fragrance.  Raw honey is honey as it exists in the beehive or as obtained by extraction, settling or straining, without adding heat (although some honey that has been "minimally processed" is often labeled as raw honey). Raw honey contains some pollen and may contain small particles of wax. Local raw honey is sought after by allergy sufferers as the pollen impurities are thought to lessen the sensitivity to hay fever.  Strained honey has been passed through a mesh material to remove particulate material (pieces of wax, propolis, other defects) without removing pollen, minerals or enzymes.  Ultrafiltered honey is processed by very fine filtration under high pressure to remove all extraneous solids and pollen grains. The process typically heats honey to 150–170 °F (approx. 65–77 °C) to more easily pass through the fine filter. Ultrafiltered honey is very clear and has a longer shelf life; it crystallizes more slowly because the high temperature breaks down sugar seed crystals, making it preferred by the supermarket trade.  Ultrasonicated honey has been processed by ultrasonication, a nonthermal processing alternative for honey. When honey is exposed to ultrasonication, most of the yeast cells are destroyed. Those cells that survive sonication generally lose their ability to grow, which reduces the rate of honey fermentation substantially. Ultrasonication also eliminates existing crystals and inhibits further crystallization in honey. Ultrasonically aided liquefaction can work at substantially lower temperatures of approximately 95 °F (35 °C) and can reduce liquefaction time to less than 30 seconds (Hielscher, 2011).  Whipped honey, also called creamed honey, spun honey, churned honey, candied honey, honey fondant, and set honey (in the UK), has been processed to control crystallization. Whipped honey contains a large number of small crystals in the honey. The small crystals prevent the formation of larger crystals that can occur in unprocessed honey. The processing also produces a honey with a smooth, spreadable consistency.  Dried honey has the moisture extracted from liquid honey to create completely solid, nonsticky granules. This process may or may not include the use of drying and anticaking agents. Dried honey is commonly used to garnish desserts.  Comb honey is honey still in the honeybees' wax comb. It traditionally is collected by using standard wooden frames in honey supers. The frames are collected and the comb is cut out in chunks before packaging. As an alternative to this labor intensive method, plastic rings or cartridges can be used that do not require manual cutting of the comb, and speed packaging. Comb honey harvested in the traditional manner is also referred to as "cut-comb honey". (Varieties of honey: Polyfloral honey". honey is harvested from forests in bee's natural habitat. It is said that honey will be consumed by the bees on the new moon day, so it is cultivated the day before.  Chunk honey is packed in widemouth containers consisting of one or more pieces of comb honey immersed in extracted liquid honey. 2.40. Distinguishing honey: 2.40.1. Honey grading: In the US, honey grading is performed voluntarily (USDA does offer inspection and grading "as on-line (in-plant) or lot inspection...upon application, on a fee-for-service basis.") based upon USDA standards. Honey is graded based upon a number of factors, including water content, flavor and aroma, absence of defects and clarity. Honey is also classified by color though it is not a factor in the grading scale. The honey grade scale is: Grade Water content Flavor and aroma Absence of defects Clarit Good—has a good, normal flavor and aroma for the predominant floral source and is free from caramelization, smoke, fermentation, chemicals and other odor causes Practically free- practically no defects that affect appearance or edibility Practically free -practically no defects that affect appearance or edibility Reasonably good- practically free from caramelization; free from smoke, fermentation, chemicals, and other causes Reasonably free- do not materially affect appearance or edibility Reasonably clear - may contain air bubbles, pollen grains, or other finely divided particles of suspended material that do not materially affect appearance. Fairly good—reasonably free from caramelization; free from smoke, fermentation, chemicals, and other causes Fairly free—do not seriously affect the appearance or edibility Fairly clear— may contain air bubbles, pollen grains, or other finely divided particles of suspended material that do not seriously affect appearance Substandard Fails Grade C Fails Grade C Fails Grade C other countries may have differing standards on the grading of honey. India, for example, certifies honey grades based on additional factors, such as the Fiehe's test, and other empirical measurements. 2.41. Indicators of quality: High-quality honey can be distinguished by fragrance, taste, and consistency. Ripe, freshly collected, high-quality honey at 20 °C (68 °F) should flow from a knife in a straight stream, without breaking into separate drops. After falling down, the honey should form a bead. The honey, when poured, should form small, temporary layers that disappear fairly quickly, indicating high viscosity. If not, it indicates excessive water content (over 20%) of the product. Honey with excessive water content is not suitable for long-term preservation. In jars, fresh honey should appear as a pure, consistent fluid, and should not set in layers. Within a few weeks to a few months of extraction, many varieties of honey crystallize into a cream- colored solid. Some varieties of honey, including tupelo, acacia, and sage, crystallize less regularly. Honey may be heated during bottling at temperatures of 40–49°C (104–120°F) to delay or inhibit crystallization. Overheating is indicated by change in enzyme levels, for instance, diastase activity, which can be determined with the Schade or the Phadebas methods. A fluffy film on the surface of the honey (like a white foam), or marble- colored or white-spotted crystallization on a containers sides, is formed by air bubbles trapped during the bottling process. A 2008 Italian study determined nuclear magnetic resonance spectroscopy can be used to distinguish between different honey types, and can be used to pinpoint the area where it was produced. Researchers were able to identify differences in acacia and polyfloral honeys by the differing proportions of fructose and sucrose, as well as differing levels of aromatic amino acids phenylalanine and tyrosine. This ability allows greater ease of selecting compatible stocks (Standifer, 2007). 2.42. Nutrition: Honey is a mixture of sugars and other compounds. With respect to carbohydrates, honey is mainly fructose (about 38.5%) and glucose (about 31.0%) making it similar to the synthetically produced inverted sugar syrup, which is approximately 48% fructose, 47% glucose, and 5% sucrose. Honey's remaining carbohydrates include maltose, sucrose, and other complex carbohydrates. As with all nutritive sweeteners, honey is mostly sugars and contains only trace amounts of vitamins or minerals. Honey also contains tiny amounts of several compounds thought to function as antioxidants, including chrysin, pinobanksin, vitamin C, catalase, and pinocembrin. The specific composition of any batch of honey depends on the flowers available to the bees that produced the honey (Martos, et.al 2000). 2.43. Preservation: Because of its unique composition and chemical properties, honey is suitable for long-term storage, and is easily assimilated even after long preservation. Honey and objects immersed in honey, have been preserved for decades and even centuries. The key to preservation is limiting access to humidity. In its cured state, honey has a sufficiently high sugar content to inhibit fermentation. If exposed to moist air, its hydrophilic properties will pull moisture into the honey, eventually diluting it to the point that fermentation can begin. Honey sealed in honeycomb cells by the bees is considered to be the ideal form for preservation. Honey should also be protected from oxidation and temperature degradation. It generally should not be preserved in metal containers because the acids in the honey may promote oxidation of the vessel. Traditionally, honey was stored in ceramic or wooden containers; however, glass and plastic are now the favored materials. Honey stored in wooden containers may be discolored or take on flavors imparted from the vessel. Likewise, honey stored uncovered near other foods may absorb other smells. Excessive heat can have detrimental effects on the nutritional value of honey. Heating up to 37 °C (98.6 °F) causes loss of nearly 200 components, some of which are antibacterial. Heating up to 40 °C (104 °F) destroys invertase, an important enzyme. At 50 °C (122 °F), the honey sugars caramelize. Generally, any large temperature fluctuation causes decay (Halytski Kontrakty" Ukrainian Business Weekly Magazine.).Regardless of preservation, honey may crystallize over time. Crystallization does not affect the flavor, quality or nutritional content of the honey, though it does affect color and texture. The rate is a function of storage temperature, availability of "seed" crystals and the specific mix of sugars and trace compounds in the honey. Tupelo and acacia honeys, for example, are exceptionally slow to crystallize, while goldenrod will often crystallize still in the comb. Most honeys crystallize fastest between about 50 and 70 °F (10 and 21 °C). The crystals can be dissolved by heating the honey (Hagen, 1876). 2.44. Uses of honey: More than 1,400 years ago Allah and His messenger (peace be upon him), told us that honey can heal a variety of medical problems. And the Lord inspired the bee, saying: Take your habitations in the mountains and in the trees and in what they erect. Then, eat of all fruits and follow the ways of your Lord made easy (for you)'. There comes forth from their bellies a drink of varying color wherein is healing for men. Verily in this is indeed a sign for people who think." [Quran 16:68-69]. Traditional uses of honey have included honey mixed with lemon for sore throats and it has also been used for stomach pains and problems, as well as certain eye conditions.

2.44.1. In medicine: Historically, honey has been used by humans to treat a variety of ailments through topical application, but only recently have the antiseptic and antibacterial properties of honey been chemically explained. In Ayurveda, a 4000-year-old medicine originating from India, honey is considered to positively affect all three primitive material imbalances of the body. It has sweetness with added astringent as end taste. It is heavy, dry and cold. It promotes the healing process." Some wound gels which contain antibacterial raw honey and have regulatory approval are now available to help treat drug-resistant strains of bacteria (MRSA). One New Zealand researcher says a particular type of honey (Manuka honey) may be useful in treating MRSA infections (Knox, 2004). As an antimicrobial agent honey may have the potential for treating a variety of ailments. Antibacterial properties of honey are the result of the low water activity causing osmosis, chelation of free Iron, its slow release of hydrogen peroxide. High acidity and the antibacterial activity of methylglyoxal. Honey appears to be effective in killing drug-resistant biofilms which are implicated in chronic rhinosinusitis (Wahdan, 1998). 2.44.2. Use for diabetic ulcers: Topical honey has been used successfully in a comprehensive treatment of diabetic ulcers when the patient cannot use topical antibiotics (Jennifer, 2007). 2.44.3. Use for sore throats and coughs: Honey has also been used for centuries as a treatment for sore throats and coughs and, according to recent research, may be an effective soothing agent for coughs (Randerson, 2007). 2.44.4. Other medical applications: Some studies suggest the topical use of honey may reduce odors, swelling, and scarring when used to treat wounds; it may also prevent the dressing from sticking to the healing wound. Honey has been shown to be an effective treatment for conjunctivitis in rats. Unfiltered, pasteurized honey is widely believed to alleviate allergies, though neither commercially filtered nor raw honey was shown to be more effective than placebo in a controlled study of 36 participants with ocular allergies. Nearly 1 in 3 of the volunteers dropped out of the study because they couldn’t tolerate eating one tablespoon of honey every day due to the overly sweet taste. The official conclusion: "This study does not confirm the widely held belief that honey relieves the symptoms of allergic rhinoconjunctivitis. "A more recent study has shown pollen collected by bees to exert an antiallergenic effect, mediated by an inhibition of Igimmunoglobulin binding to mast cells. This inhibited mast cell degranulation and thus reduced allergic reaction. The risk of experiencing anaphylaxis as an immune system reaction may outweigh any potential allergy relief (Ishikawa, et al. 2008). A review in the Cochrane Library suggests honey could reduce the time it takes for a burn to heal — up to four days sooner in some cases. The review included 19 studies with 2,554 participants. Although the honey treatment healed moderate burns faster than traditional dressings did, the author recommends viewing the findings with caution, since a single researcher performed all of the burn studies. 2.44.5. Modern uses: The main uses of honey are in cooking, baking, as a spread on bread, and as an addition to various beverages, such as tea, and as a sweetener in some commercial beverages. According to the National Honey Board (a USDA-overseen organization), "honey stipulates a pure product that does not allow for the addition of any other substance...this includes, but is not limited to, water or other sweeteners"(Definition of honey and honey products) Honey barbecue and honey mustard are common and popular sauce flavors. Honey is the main ingredient in the alcoholic beverage mead, which is also known as "honey wine" or "honey beer". Historically, the ferment for mead was honey's naturally occurring yeast. Honey is also used as an adjunct in some beers. Honey wine, or Mead, is typically (modern era) made with a honey and water mixture with a pack of yeast added for fermentation. Primary fermentation usually takes 40 days, after which must needs to be racked into a secondary fermentation vessel and left to sit about 35–40 more days. If done properly, fermentation will be finished by this point (though if a sparkling Mead is desired, fermentation can be restarted after bottling by the addition of a small amount of sugar), but most Meads require aging for 6–9 months or more in order to be palatable (Gheldof, et.al 2002). 2.45. Health Hazards 2.45.1. Botulism: Because of the natural presence of botulinum endospores in honey. Children under one year of age should not be given honey. The more-developed digestive system of older children and adults generally destroys the spores. Infants, however, can contract botulism from honey. Medical grade honey can be treated with gamma radiation to reduce the risk of botulinum spores being present. Gamma radiation evidently does not affect honey's antibacterial activity, whether or not the particular honey's antibacterial activity is dependent upon peroxide generation. Infantile botulism shows geographical variation. In the UK, only six cases have been reported between 1976 and 2006, yet the U.S. has much higher rates: 1.9 per 100,000 live births, 47.2% of which are in California. While the risk honey poses to infant health is small, it is recommended not to take the risk (Postmes, et.al 1995). 2.46. Toxic Honey: Honey produced from the flowers of oleanders, rhododendrons, mountain laurels, sheep laurel, and azaleas may cause honey intoxication. Symptoms include dizziness, weakness, excessive perspiration, nausea, and vomiting. Less commonly, low blood pressure, shock, heart rhythm irregularities, and convulsions may occur, with rare cases resulting in death. Honey intoxication is more likely when using "natural" unprocessed honey and honey from farmers who may have a small number of hives. Commercial processing, with pooling of honey from numerous sources, generally dilutes any toxins. Toxic honey may also result when bees are proximate to tutu bushes (Coriaria arborea) and the vine hopper insect (Scolypopa australis). Both are found throughout New Zealand. Bees gather honeydew produced by the vine hopper insects feeding on the tutu plant. This introduces the poison tutin into honey. Only a few areas in New Zealand (Coromandel Peninsula, Eastern Bay of Plenty and the Marlborough Sound) frequently produce toxic honey. Symptoms of tutin poisoning include vomiting, delirium, giddiness, increased excitability, stupor, coma, and violent convulsions. To reduce the risk of tutin poisoning, humans should not eat honey taken from feral hives in the risk areas of New Zealand. Since December 2001, New Zealand beekeepers have been required to reduce the risk of producing toxic honey by closely monitoring tutu, vine hopper, and foraging conditions within 3 km of their apiary (Ishikawa, et al. 2008). 2.47. Honey-Producing Countries: In 2005, China, Argentina, Turkey and the United States were the top producers of natural honey. Significant regional producers of honey include Turkey (ranked third worldwide) and Ukraine (ranked fifth worldwide). Mexico is also an important producer of honey, providing about 10% of the world's supply. Much of this (about one-third) comes from the Yucatán Peninsula. Honey production began there when the Apis mellifera and the A. mellifera ligustica were introduced there early in the 20th century. Most of Mexico's Yucatán producers are small, family operations who use original traditional techniques, moving hives to take advantage of the various tropical and subtropical flowers. Honey is also one of the gourmet products of the French island of Corsica. Corsican honey is certified as to its origin (Appellation d'origine contrôlée) just as are French wines, like Champagne. Homolje honey (eastern Serbia) is certified as to its origin (Lavin and Mariely, 2008).

2.48. Modern research shows that honey: 2.48.1. Blocks the growth of oral bacteria: Coats the throat and reduces throat irritation is effective when used in the treatment of gastric or peptic stomach ulcers is effective in the treatment of various wounds and infections because of its antimicrobial (antibacterial, antiviral and antifungal) properties is also considered an antioxidant. This means it allows the blood to circulate better and provide more oxygen to areas of the body such as the brain. Can also be used externally to promote healing when applied to wounds, even postoperative wounds has also been effective in its use to treat burns contains a variety of sugars and minerals and has been shown to be low in calories and useful as a sweetener for diabetics, people with heart disease or those who are overweight. Researchers are not absolutely sure why honey heals but they are learning new things about honey everyday. "Honey is considered the food of foods, the drink of drinks and the drug of drugs. It is used for creating appetite, strengthening the stomach and eliminating phlegm, as a meat preservative, hair conditioner, eye salve, and mouthwash. The best honey is that produced in the spring, the second best is that of summer, and the least quality is produced in winter. 2.48.2. Treatment of burns and skin ulcers: A study in 1991 by Dr Subrahmanyam compared the results of topical application of honey to burns with conventional Burn treatment, (silver sulfadiazine). The following results were noted and reproduced by Julie McCarthy, a post graduate student at the University of Guelph. "Burn patients of a variety of ages were divided into two treatment groups. The burns of patients in group I were cleaned with a saline solution and pure, undiluted, unprocessed honey was applied daily. Burns of group 2 were cleaned and covered with gauze that was soaked in 5% silver sulfadiazine which was changed daily. Results showed that within 7 days 91 % of the infected wounds treated with honey were free from infection, compared to less than 7% of the silver sulfadiazine treated burns. Within 15 days, 87% of honey treated wounds were healed compared to less than 10% of wounds in group 2. That various types of wounds and skin ulcers which did not respond to conventional methods of treatment such as antibiotics and medicated dressings, responded to treatment with honey. These included:  Fournier's Gangrene Burn wounds Tropical ulcers Bed sores Diabetic ulcers the anti-bacterial activity of honey is based partly on its osmotic effects in the bacteria that cause infection are unable to survive in honey because they become dehydrated. In addition, it was noted that the presence of hydrogen peroxide generated by the enzymatic activity of glucose oxidase in dilute honey contributes to the anti- bacterial activity. As hydrogen peroxide decomposes, it generates highly reactive free radicals that react and kill bacteria.  The Treatment of Gastroenteritis and Stomach Ulcers found that honey treatment shortened the duration of diarrhoea in patients with bacterial gastroenteritis, in that patients treated with honey had a mean recovery of 58 hours compared with 93 hours for control patients. In addition, research conducted in New Zealand has shown that Manuka honey successfully inhibits Helicobacter pylori the organism responsible for upper gastro-intestinal dyspepsia of stomach ulcers. However, it should be noted that research is ongoing in this field, and although the initial results are promising, full clinical trials are yet to be completed. As well as its other therapeutic properties, the presence of potassium, sodium calcium and magnesium means that honey is capable of neutralising acid in the body and thus maintaining the acid-alkaline balance.

Chapter Three 3. Material And Methods 3.1. Materials: 3.1.1. Honey Samples: Nine samples were obtained from local producers. The botanical origin of the honey samples was shown in table (3.1). N Honey name Location o 1 Ground honey(GH) Ethiopia 2 Whitehoney(Alhashab) (WH) Western sudan 3 ALdamazinhoney(AcaciaHoney)(ADH) Damazin (Sudan) 4 Mixed honey(MIH) Damazin (Sudan) 5 Ashab honey (herbs honey)(ASH) Sinnar area (Sudan) 6 Hapashy honey(HH) Ethiopia 7 Alhandal Honey (bitter melon) (AHH) North Sudan 8 Mountains honey(MOH) Southern Blue Nile 9 Industrial Honey(IH) Khartoum (Sudan) Table (3.1) Name of Honey Sample and Location

All the samples were less than 3 months old, as indicated by the producers. 3.2. Methods: 3.2.1. Physicochemical characteristic: 3.2.1.1. Acidity Ten grams of homogenized honey were taken in a glass beaker, 75 ml of water were added to the end point using phenolthalene indicator, and this solution was titrated with carbonate-free 0.10 NaOH until the pH reached 8.5. 3.2.1.2. pH Measurement: The pH of the honey was measured using pH-meter (PHS- 3C Digital) at ambient temperature (ICUMSA, 1998). The pH- meter was adjusted with standard buffer solution. The electrode was rinsed and receptacle with portion of honey sample. A beaker was filled with the honey sample to a depth covering the bulb of the glass electrode. Then the system was allowed to equilibrium and pH was read.

3.2.1.3. Total dissolved Solid (TDS): The (TDS) of honey was measured using Conductivimeter. The Conductivimeter was adjusted with standard buffer solution. 50ml of sample were taken in beaker, and then read directly by conductivimeter (ICUMSA, 1998).

3.2.2. Approximate Chemical Analysis of Honey: 3.2.2.1. Moisture Content: Moisture content of honey was determined according to the (ICUMSA, 1998).

Procedure: An empty open dish was heated for 30 minutes in an oven at 105˚C, then removed from the oven, covered with a lid and placed in desiccators at room temperature then weighted. 5grams of the sample were placed in the dish and weighed with the lid on it, and then placed in the vacuum oven with opened state for three hours at 105˚C. The dish with lid were replaced and removed from desiccators and weighted at room temperature.

Calculation:

W3 × 100 ــ Moisture content (%) = W2

W1 ــ W2 Where:

W1= weight of empty dish in gram.

W2= weight of dish plus sample before drying in gram.

W3= weight of dish plus sample after drying in gram. 3.2.2.2. Ash Content: The ash percentage was determined according to (AOAC, 1984). Two grams of sample were weighed on dry basis and ignited in Heraeues electronic muffle furnace at 550˚C for 5 hours. The ash content was then calculated as percentage as follows:

Ash (%) = W3-W1 × 100%

W2-W1 Where:

W1= weight of the empty crucible

W2= weight of the crucible + the sample

W3= weight of the crucible + Ash

3.2.2.3. Nitrogen and Protein Content: The kjeldahl method was used to determine the amount of nitrogen of the different samples according to( AOAC, 1984) then multiplied by a factor of 6.25. The method was composed of three major steps: a- The digestion: A portion of the prepared sample was weighed out, and

transferred to a Kjeldahl tube. A glass pearl, 20ml H2SO4 and 1:2

g catalyst (15g KSO4 + 1ml CuSO4) were added. The digestion was carried out in a destruction block unit a bright green color appeared, then allowed to cool and 10 ml distilled water were added. b- The distillation: The tube was placed in the distillation equipment and 30 ml NaOH thiosulphate solution was added. The ammonia was distilled into 20ml boric acid indicator solution. c- The titration: Then titrated with 0.05 N HCl (colour: green-purple). The protein content was then calculated as follows:

%N = V × T × 14 × Volume of diluent digest × 100 1000 × G

% protein = % N × 6.25 Where: N: nitrogen content. V: number of ml of the HCl solution T: normality of HCl. G: weight (g) of the sample.

3.3 Sugar Content: High performance liquid chromatography(HPLC) Sample preparation : 1 g of sample was taken and diluted with water, the mixture was introduced to ultra some water bath to aid solubility then the sample was filtered through membrane filter 0.45µm.10µl of filtrate was injected in hplc The sugar content was then calculated as percentage as follows:

concentrate (sample) = c(std) x Area(sample) x dilution factorx100 Area(std)xweight of sample x1000

3.2.4 Ascorbic Acid (Vitamin C): Indophenol method: Reagents: a) Indophenol dye 0.04% 0.2 g 2.6 dicherphenolindophol was dissolved in about 200 ml distilled water. If necessary filter with 4 whatman paper into 500 ml volumetric flask and made up to volume at 20oC and stored in refrigerator. b) oxalic acid 0.4%: 4 g oxalic acid were dissolved in water and diluted to 1000 ml c) 10% oxalic acid: 50 g oxalic acid in 500 ml water d) Ascorbic acid: 50 mg of ascorbic acid were weighed accurately and made up to 250 ml in volumetric flask with 10% oxalic acid. Titration 1) Strength of the dye 5 ml aliquot (Ascorbic acid solution. Reagt. 4) and 5 m1 10% oxalic acid and titrated with the dye solution to a pink color. Number of mg of ascorbic acid equivalent to 1 ml of the dye were calculated by dividing 1 by the number of ml dye. 2) Titration Procedures: 30 g of sample were blended with reasonable amount of 0.4 oxalic acid for 1 minute, aliquot transferred to 500 ml volumetric flask, made up to volume with 0.4 oxalic acid and was filtered through No. 4 whatman filter paper 20 ml aliquot were pipetted and titrated against the dye solution to faint pink end point.

Ascorbic acid (mg/l00g) = Titration x dye strength x dilution Weight of sample

3.2.5. Mineral content:

a) The mineral content percentage was determined according to (AOAC, 1984). Two grams of sample were weighed on dry basis and ignited in Heraeues electronic muffle furnace at 550˚C for 5 hours. The ash content was then calculated as percentage as follows:

Ash (%) = W3-W1 × 100%

W2-W1 Where:

W1= weight of the empty crucible W2= weight of the crucible + the sample

W3= weight of the crucible + Ash b) Moisten the ash with water add 5ml of 33%HNO3 together with the 10ml of HCL before evaporate to dryness on a waterbath .carry out ablank determination .use the solution to determine (Ca ,Fe ,Mn ,P ,Na ,K ,Cl , Zn). c) the minerals were detected automatically using atomic absorption device . One mg of each ash of honey sample was injected to the device.

CHAPTER FOUR 4. RESULTS AND DISCUSSION

4.1 Physicochemical and approximate chemical characteristics of honey: The result of analysis are presented in Table (4.1). The moisture content of Mountain honey, White honey, Ground honey ,Aldamazain honey , Alhandal honey, Mixed honey, Hpashi honey, Ashab honey, Industerial honey samples were ranged between 10.77- 36.9%. These values are within the recommended standards of honey according to USA Standards (beesource.com ,2012) which is 17.2% . The different samples of honey had nearly similar ash values which were ranged between 0.5-2.0%. Values are not compare able with standards of honey of USA (beesource.com ,2012) which is 0.2% . The protein content of honey samples were highly varied, the highest value was that of white honey which is 6.01g and the lowest was that of Ground honey which is 4.37g, the Industerial honey had more protein content (5.16g) which was higher than those of the examined natural honey samples (Ground honey, Alhandal honey , Aldamazain honey (and Mixed honey). The pH and titrable acidity of honey samples (Table 4.1) revealed that had the lowest acidity (0.6%), and the highest pH (4.5%). Ground honey had highest acidity 9.0% and lowest pH and( 3.2%). The results are in agreement with the standards range of USA Standards which indicate that the pH is( 3.96) while the acidity is (29.0%) (Doner, 1977). The highest percentage of vitamin C was found in ground honey (29.4mg), followed by aldamazain honey (6.9mg), mixed honey (5.4mg), alhanal honey (4.4mg). Both industrial honey and hapashi honey had similar values of vitamin C content which was 4.3mg, however, mountain honey had the least value (2.5mg). These values of vitamin C determined in the present study were in agreement the USA Standard (beesource.com ,2012) which is 0.5mg . Vitamin C is used in the treatment of respiratory diseases. This vitamin is very sensitive to temperature and light. Moisture Ash Protein pH Acidity Vitamin C TDS (%) (%) (%) (%) (mg/100g) (%) MOH 14.38 0.5 5.5 3.35 1.7 2.54 80.63 WH 10.77 1.0 6.01 4.58 0.6 3.82 82.86 GH 36.87 1.5 4.37 3.2 9 2.94 38.35 ADH 14.07 1.0 4.6 3.3 7.1 6.94 74.74 AHH 17.8 1.0 4.46 4.04 2.9 4.4 62.23 MIH 14.45 1.0 5.07 4.38 3 5.38 65.62 HH 19.22 1.5 5.86 4.54 1.7 4.26 73.70 ASH 14.76 2.0 5.77 4.54 2 2.98 66.19 IH 10.24 1.5 5.16 4.1 1.1 4.26 69.33 The total dissolved solids (TDS) was very high in damazain honey , industreial honey , alhandal honey which are (62%, 40.1%, 40.3%) respectively. However, ground honey had the least value of TDS (34.5%) that means it has a texture relatively similar to that of water. Table (4.1) The physicochemical and chemical characteristics of different types of honey MOH= Mountain honey WH= White honey GH= Ground honey ADH= Aldamazein honey AHH= Alhandal honey MIA=Mixed honey HH=Hapashy honey ASH=Ashab honey IH=Industrial honey

4.2. Carbohydrate Analysis Using HPLC for Sugar Content: As for the types and quantities of sugar found in honey were analyzed using a device HPLC , the results are shown in Table (4.2). The Mountain honey had the highest percentage of glucose (35.0%), while the highest percentage of fructose was found in Aldamazain honey (39.01%). On the other hand, the Ground honey had the lowest value of both glucose (14.1%) and fructose (14.8%) and contained no any sucrose, therefore, the taste of Ground honey is bitter. The Industrial honey contained the highest percentage of sucrose (10.7%), so this type of honey is not suitable for diabetic patients. Moreover, sucrose was not found in many of the examined types of honey such as Ground honey, Hapashy honey, White honey and Aldamazein honey, these results are higher than the those of the USA Standards (beesource.com , 2012) which indicted that honey contain glucose (31.3%), fructose (38.2%) and the sucrose (1.3%). Generally, three basic types of sugars were found: glucose, sucrose and fructose which were found in varying amounts. However, there are also some other types of sugars (in small amounts) as rafenose , maltose,dextrose (Doner, 1977). See appendix.

Content Glucose (%) Fructose (%) Sucrose (%) MOH 34.97 31.75 3.48 WH 35.6 35.6 Nil GH 14.09 14.74 Nil ADH 26.55 39.01 Nil AHH 26.57 20.64 5.724 MIH 27.84 21.63 5.34 HH 27.167 35.087 Nil ASH 27.161 22.173 5.253 IH 25.31 23.24 10.7 Table (4.2): Sugar Content of Different Honey Types: The different types of honey sugars are presented in Table (4.2). MOH= Mountain honey WH= White honey GH= Ground honey ADH= Aldamazein honey AHH= Alhandal honey MIA=Mixed honey HH=Hapashy honey ASH=Ashab honey IH=Industrial honey

4.3. Minerals Content: Table (4.3) show that, the percentage of Ca ranged between 0.2% - 0.35%, P ranged between 0.2% -0.1%, Na ranged between 0.7% -0.54% , K ranged between 2.3% - 1.2% , Fe ranged between 0.87% - 0.7% , Zn ranged between 0.105% - 0.089% , Cl ranged between 0.205% - 0.11% then Mn ranged between 0.099% - 0.08%. Honey is richer in minerals, that render it unsuitable for winter stores. This result in present study were greater than the finding reported by (Mohammed and Babikr, 2012), who indicated that K (74.6 mg/kg), Na (28.2 mg/kg) , P (204.6) mg/kg and S(131.5) mg/kg , Ca concentration (82.92 mg/kg), Mn (1.019 mg/kg), Fe (2.05 mg/kg), Zn (9.61 mg/kg). Content Ca% P% Na% K% Fe% Zn% Cl% Mn% MOH 0.35 0.20 0.70 2.0 0.87 0.098 0.109 0.086 WH 0.3 0.19 0.69 2.3 0.85 0.096 0.11 0.09 GH 0.25 0.13 0.68 1.2 0.7 0.105 0.205 0.097 ADH 0.2 0.11 0.66 1.5 0.82 0.1 0.111 0.088 AHH 0.23 0.1 0.55 1.9 0.7 0.103 0.12 0.099 MIH 0.3 0.17 0.65 1.6 0.81 0.098 0.105 0.08 HH 0.25 0.18 0.6 2.0 0.73 0.101 0.105 0.086 ASH 0.3 0.17 0.65 1.6 0.81 0.098 0.105 0.08 IH 0.22 0.15 0.54 1.4 0.73 0.089 0.169 0.085 Table (4.3): The Minerals Content in the Types of Honey

MO.H= Mountain honey W.H= White honey G.H= Ground honey AD.H= Aldamazein honey AH.H= Alhandal honey MI.A=Mixed honey H.H=Hapashy honey AS.H=Ashab honey I.H=Industrialhoney

Chapter Five Conclusions and Recommendations 5.3. Conclusions: In this study eight different samples of natural honey were analyzed chemically and physically and compared with the industrial honey. The chemical analysis indicated that ground honey contained the highest contents of moisture, while habashi honey contained the highest contents of ash. On the other hand, the percentage of protein was highly varied, the highest content was found in white honey and the lowest was in the ground honey. It can be mentioned that honey is the acidic in nature since the examined pH of all types of honey was less than 7. The highest percentage of vitamin C was found in ground honey while the highest and lowest TDS% were found in aldamazain honey and the ground honey, respectively. As for the types and quantities of sugars, different types of sugars were found in honey samples. The highest contents of glucose was found in white honey, while the highest contents of fructose was found in aldamazain honey, however, small quantities of sucrose were found in some samples and absent in others. Many minerals were found in the honey samples, such as sodium, potassium, phosphorus, iron, chlorine, manganese, zinc and calcium. Generally, there is no significant differences in most of the chemical components of natural honey and industrial honey. 5.2. Recommendations: 1. Ground honey, contained relatively high quantities of minerals and vitamin C and it was sucrose free, hence, it can be used by the diabetic persons and those with the respiratory diseases. 2. Industrial honey contained relatively more of most important nutritional constituents, hence, it can be specialized for children and those did not suffer of diabetes. 3. An easy, simple, available and cheep ways to detect adulterated honey should be designed. 4. The enzymes within honey samples should be analyzed.

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APPENDIX

Atomic Absorbtion Device

HPLC Device