Physicochemical Characteristics of Bee Honey and Detection of Its Adulteration in Khartoum State

Physicochemical Characteristics of Bee Honey and Detection of its Adulteration in Khartoum State

Hiba Adil Mahmoud Mohammed B. Sc. Agric. (Honours) 2012 Alzaiem Alazhari University

A Dissertation Submitted to the University of Khartoum in Partial Fulfillment of the Requirements for the Degree of Master of Science in Food Science and Technology

Supervisor: Dr : Hassan Ali Mudawi

Department of Food Science and Technology

Faculty of Agriculture

University of Khartoum

Septemper- 2015

Dedication

 To My: Parents Brothers Friends Teachers

With love and respect Hiba Adil

Acknowledgements

I would like to thank ALLAH Whom with his will this work was completed. Thereafter I would like to express my sincere thanks and gratitude to Dr. Hassan A. Mudawi for his kind supervision, advice and discern views throughout my study. And I would like to thank all the staff of Department of Food Science and Technology, Faculty of Agriculture, University of Khartoum. My sincere thanks are to everybody who helped me to make this work successful.

List of contents

Page Dedication………………………………………………………………... I Acknowledgements…………………………………………………….. Ii List of content ………………………………………………………… Iii List of table……………………………………………………………… Vi List of fig………………………………………………………………… Vii Abstract…………………………………………………………………. Viii Abstract Arabic………………………………………………………… Xi CHAPTER ONE: INTRODUCTION………………………..………….…………………. 1 Objectives of the study…………………………………………………. 2 CHAPTER TWO: LITERATURE REVIEW……………..………………………………… 3 2.1 Honey definition…………….……………………………………… 3 2.2 Nectar…………………………………………………………….. 4 2.3 Classification………………………………………………………. 4 2.3.1 Floral source…………………………………………………….. 5 2.3.1.1Blended………………………………………………………. 5 2.3.1.2 Polyfloral……………………………………………………. 5 2.3.1.3 Monoflora…………………………………………………… 5 2.3.1.4 Honeydew honey……………………………………………… 5 2.3.2 Classification by packaging and processing……………………… 6 2.3.2.1 Crystallized honey……………………………………………. 6 2.3.2.2 Pasteurized honey……………………………………………. 6 2.3.2.3 Raw honey …………………………………………………… 6 2.3.2.4 Strained honey……………………………………………….. 7 2.3.2.5 Filtered honey……………………………………………….. 7 2.3.2.6 Creamed honey……………………………………………. 7 2.3.2.7 Dried honey………………………………………………….. 7 2.3.2.8 Chunk honey………………………...………………………… 8 2.4 Physical and chemical properties…………………………………. 8 2.4.1 Viscosity………………………………………………………… 8 2.4.2 Specific gravity…………………………………………………. 8 2.4.3 Color……………………………………………………………. 9 2.4.4 Refractive index………………………………………………… 9 2.4.5 Hygroscopy and fermentation…………………………………... 9 2.4.5.1Hygroscopicity…………………………………………………. 9 2.4.5.2 Fermentation………………………………………………… 10

iii

2.4.6 Thermal characteristics………………………………………….. 10 2. 5 Chemical properties of bee honeys…………………..…………… 11 2. 5.1 Moisture content……………………………………….……….. 11 2. 5.2 Ash and minerals content…………………………..………….. 11 2. 5.3 Carbohydrates…………………………………………………. 12 2. 5.4 Protein content…………………………………………………. 13 2. 5.4.1 Amino acids………………………………………………….. 13 2.5.5 Acidity and pH………………………………………………… 15 2.5.6 Hydroxymethylfurfuraldehyde (HMF)………………………… 15 2.5.7 Enzymes…………………………………………………………. 17 2.5.8 Vitamins………………………………………………………… 18 2.5.9 Aroma and flavor……………………………………………… 19 2.6 The antimicrobial activity of bee honey…………………………. 19 2.7 As Antioxidant…………………………………………………… 20 2.8 Religious significance…………………………………………… 20 2.9 Modern uses……………………………………………………. 20 2.9.1 As a food and in cooking……………………………………… 20 2.9.2 .Health effects………………………………………………….. 20 2.10 Health hazards…………………………………………………… 21 2.10.1 Toxic honey………………………………………………….. 21 2.11 Adulteration………………………………………………………. 21 2.11.1 Adulteration detection……………..………………………….. 23 2.12 Indicators of quality………………..…………………………… 23 CHAPTER THREE MATERIALS AND METHODS………………………………………… 26 3.1Materials………………………………………………………….. 26 3.2 Physical properties………………………………………………... 26 3.2.1 Refractive index………………………………………………… 26 3.2.2 pH value………………………………………………………… 26 3.3 Chemical composition…………………………………………….. 27 3.3.1 Moisture content………………………..……………………….. 27 3.3.2 Ash content……………………………………………..……….. 27 3.3.3 Free acidity, lactone and total acidity values…………………… 29 3.4 Sugar analysis…………………………………………………… 30 3.4.1 Determination of sugars by HPLC……………………………. 30 3.4.2 Preparation of standard sugars solutions………………………..... 30 3.5 Statistical analysis.….………………………………………….. 32 CHAPTER FOUR RESULTS AND DISCUSSION………………………………..……… 33 4.1 Physical properties……………….………………………….……… 33

4.1.1 Refractive index……..…………………………………………….. 33 4.1.2 pH value…………………………………………………………… 33 4.2 Chemical composition of bee honey……………………….…….. 36 4.2.1 Moisture content………………………………………….……. 36 4.2.2 Ash content……………………………………………………….. 36 4.2.3 Free acidity, lactone and total acidity values….………………….. 37 4.2.4 Hydroxymethylfurfural (HMF)…..……………………………….. 37 4.2.5 Sugar content…………………….………………………………. 43 4.2.5.1 Fructose content………………………………………………. 43 4.2.5.2 Glucose content………………………………….…………….. 43 4.2.5.3 Sucrose content……………………………………………….. 43 4.2.4.4 Maltose content………………………………………………. 44 4.2.5.5 Fructose : glucose ratio(F: G)……………………………….. 44 CHAPTER FIVE CONCLUSIONS AND RECOMMENDATIONS…………………….. 49 5.1 Conclusions…………………………………………………………. 49 5.2 Recommendations……..…………………………………………… 49 References…………………………………………………………….. 50

LIST OF TABLES

No. of Title Page table (2.1) Specification of Sudanese Honey 25

(4.1) Physical Properties of Honey Samples 34

(4.2) Chemical Composition of Honey Samples 39

(4.3) Sugar Composition of Honey Samples 46

LIST OF FIGUERS

No. of Page No Fig 4.1 pH values of bee honey samples 35

4.2 Moisture content of bee honey samples 40

4.3 Ash content of bee honey samples 40

4.4 Free acidity values of bee honey samples 41

4.5 Lactone values of bee honey samples 41

4.6 Total acidity values of bee honey samples 42

4.7 Fructose content of bee honey samples 47

4.8 Glucose content of bee honey samples 47

4.9 Sucrose content of bee honey samples 48

4.10 Maltose content of bee honey samples 48

vii

Physicochemical Characteristics of Bee Honey and Detection of its Adulteration in Khartoum State

M. Sc. in Food Science and Technology

Hiba Adil Mahmoud Mohammed

Abstract: The Objective of this study was to determine the physicochemical properties of bee honey and identify adulterated bee honey in Khartoum State. Fifteen samples of honey were collected from local markets in Khartoum, Bahri and Omdurman . 5 samples from each locality. The 15 samples were analyzed for refractive index, pH, moisture, ash, free acidity, lactone, total acidity, fructose, glucose, sucrose, maltose and fructose: glucose ratio (f:g).The results showed that: the refractive index for all samples of honey was in the range 1.451-1.504. All honey samples were slightly acidic and gave pH of 4.13- 4.56. The average of moisture content for the three areas of Khartoum, Bahri and Omdurman were 15.17 %, 15.95 % and 28.30 %, respectively , the samples obtained from Omdurman disagreed with the standard of Sudan SSMO (2007) which stated that Sudanese honey should not contain moisture more than 20%. The average of ash content for the three areas of Khartoum, Bahri and Omdurman were 0.40 %, 0.30 % and 0.35 %, respectively, all samples were in agreement with the SSMO (2007) which stated that Sudanese honey should not contain ash more than 1%. The free acidity for all samples of honey was in the range 7.38 - 15.17 meq/kg. The lactonic was in the range 0.83 - 3.40 meq/kg. The average of total acidity for the three areas of Khartoum, Bahri and Omdurman were 12.76 meq/kg, 11.09 meq/kg and 12.21 meq/kg, respectively, all samples were in agreement with the SSMO (2007) which stated that Sudanese honey should not contain acid more than 50 milliequiv acid/kg. The results of hydroxymethylfurfural (HMF) was (+ve) in five samples (two

viii samples from Khartoum, two samples from Bahri and one sample from Omdurman). The fructose content for all honey samples was in the range 33.20 %-53.84 %.The glucose content was in the range 23.25 % -34.66 %. The average of sucrose content for the three areas of Khartoum, Bahri and Omdurman were 3.70 %, 3.50 % and 1.59 %, respectively, all of samples were in agreement with the SSMO (2007) which stated that Sudanese honey should not contain sucrose more than 10%. The maltose content was in the range 0.71%- 4.99 %. Fructose:glucose ratio (f:g) was in the range 1.05-1.46. Analysis of physicochemical properties of honey samples revealed highly significant (P‹0.01) differences. Based on the obtained results it can be concluded that the honey samples were slightly acidic, in the pH range 4.13- 4.56. The ash content, lactonic , total acidity, glucose and sucrose were higher in the samples obtained from Khartoum. While the free acidity, fructose, maltose and fructose: glucose ratio were higher in the samples obtained from Bahri. The moisture content was higher in the samples obtained from Omdurman as it was above the limits of standards of Sudan. The study recommends the establishment of efficient management system during the processing, collection, storage and marketing of honey to avoid adulteration. Also there is need for improvement and development of new analytical techniques to detect adulteration of honey.

المستخلص: الهدف من الدراسه هو تقدير الخواص الفيزوكيميائيه للعسل و التعرف على عسل النحل المغشوش في والية الخرطوم. تم جمع 51 عينه من العسل من األسواق المحلية الخرطوم، بحري وأمدرمان، خمسة من كل محلية. شملت التحاليل معامل اإلنكسار، األس الهيدروجيني، الرطوبه، الرماد، الحموضه الحرة، الالكتون، الحموضة الكلية، الفركتوز، الجلكوز، السكروز، المالتوز ونسبة الفركتوز: الجلكوز. أوضحت النتائج :أن معامل اإلنكسار في كل عينات العسل في المدى 5.415- 5.1.4. جميع عينات العسل كانت ذات حمضية قليله وأعطت pH في المدى 4.54-4.14. محتوى الرطوبة كان في المدى % ...5-% 8.73.. متوسط محتوى الرطوبة للثالثة مناطق الخرطوم، بحري وامدرمان كان 51.71%، 51.11% و ...37% على التوالي . العينات المتحصل عليها من أمدرمان التتفق مع الهيئه السودانية للمواصفات والمقاييس )8..3(م التي تنص على أن العسل السوداني يجب أن ال يحتوي على رطوبة أكثر من .3%. متوسط محتوى الرماد للثالثة مناطق الخرطوم، بحري وامدرمان كان .4..% ، ....% و1...% على التوالي ، جميع العينات إتفقت مع الهيئه السودانية للمواصفات والمقاييس )8..3(م التي تنص على أن العسل السوداني يجب أن ال يحتوي على رماد أكثر من 5%. الحموضة الحرة لجميع عينات العسل كانت في المدى 7..8-.3..5 ميللي مكافئ /كجم. الالكتون كان في المدى .7..-.4.. ميللي مكافئ/كجم. متوسط الحموضة الكلية للثالثة مناطق الخرطوم، بحري وامدرمان كان 53.84 ، 1..55 و 53.35 ميللي مكافئ/كجم على التوالي ، جميع العينات إتفقت مع الهيئه السودانية للمواصفات والمقاييس )8..3(م التي تنص على أن العسل السوداني يجب أن اليحتوي على حمض أكثر من .1 ميللي مكافئ/كجم. نتائج الهيدروكسي ميثايل فورفيورال كانت إيجابية في 1 عينات )عينتان من الخرطوم، عينتان من بحري وعينه من أمدرمان(. الفركتوز في كل عينات العسل كان في المدى .3...- 74..1 %. الجلكوز كان في المدى 31..3-4.44. %. متوسط السكروز للثالثة مناطق الخرطوم، بحري وامدرمان كان 85.. % ، 15..% و 5.11% على التوالي ، جميع العينات إتفقت مع الهيئة السودانية للمواصفات والمقاييس )8..3(م التي تنص على أن العسل السوداني يجب أن اليحتوي على

سكروز أكثر من .5%. المالتوز كان في المدى 85..-4.11 %. نسبة الفركتوز: الجلكوز كان في المدى 1..5-5.44 % . أوضحت نتائج التحليل الفيزوكيميائية لعينات العسل إختالفا معنويا عاليا. خلصت الدراسة الى أن عينات العسل قليلة الحموضه حيث أن األس الهيدروجيني كان في المدى .-4.5 4.14. الرماد ، الالكتون ، الحموضة الكلية ، الجلكوز و السكروز كانت أعلى في العينات المتحصل عليها من الخرطوم ، بينما الحموضة الحرة ، الفركتوز ، المالتوز ونسبة الفركتوز: الجلكوز كانت أعلى في العينات المتحصل عليها من بحري. محتوى الرطوبة كان أعلى في العينات المتحصل عليها من أمدرمان التي كانت أعلى من حدود المواصفة السودانية، لذا فإن العينات المتحصل عليها من امدرمان غير مطابقة للمواصفات القياسية السودانية. توصي الدراسة بإنشاء نظام إدارة فعال أثناء التصنيع، تجميع ، تخزين وتسويق العسل .أيضاهناك حوجة الى تحسين وتطوير تقنيات تحليلية جديده للكشف عن غش العسل.

CHAPTER ONE INTRODUCTION

Honey is that natural sweet substance produced by honey bees(Codex Alimentarius,2001). Since ancient time honey is discribed as a most valuable food and in most cases has enhanced its consumption by its medical characteristics. It consists mainly of sugars but also contains acids, nitrogenous compounds phenolic contents, hydroxymethylefurfural (HMF), minerals and water . Honey get its sweetness from monosaccharides fructose and glucose and has approximately the same relative sweetness of granulated sugar(FAO,2012).

Honey may be designated according to floral or plant source if it comes wholly or mainly from that particular source and has the organoleptic ,phsicochemical and microscopic properties corresponding to that origin. The honey types produced in a certain country or area represent the floral or nectar source in that place , whose presence solely depends on the climate, topography and agricultural pattern of that area(Mohammed,2006).

Total natural honey production in the world is 1,260,229 metric tons. The main top producers of natural honey in the world in 2012were China, Turkey and Ukraine(FAO,2012). Significant regional producers of honey include United States of America(ranked fourth world wide) and Russia (ranked fifth worldwide). Mexico is also an important producer of honey, providing more than 4% of the world’s supply (FAO,2013).

Honey consumption per capita per year exceeds one kilogram in some countries like Australia, Germany and Switzerland. (FAO,2013).

The problem to be discussed in this study is that there are adulteration in honey trade, which has negative effect on curing of humans folk medicine. So adultrated honey will lead to hazard specially for diabetics . Adulteration negatively influence market growth by damaging customer confidence. So some honey bees may be mixed with water, sucrose, gum, glucose….etc, by unhonestable peoples that leads fraud of honey.

Objectives of the study

- To determine the physicochemical properties of honey.

- To identify adulterated bee honey in Khartoum state.

CHAPTER TWO LITERATURE REVIEW

2.1 Honey definition

Honey is the substance made when the nectar and sweet deposits from plants are gathered, modified and stored in the honeycomb by honey bees. The definition of 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 ( National Honey Board,2003).

Mature honey bees collect nectar from plant blossoms. Nectar is 80 to 95 percent water and 5 to 20 percent sucrose. As the bee transports the nectar back to the hive, a protein enzyme in its stomach, called invertase, breaks the sucrose down into the two simple sugars, fructose and glucose ( National Honey Board,2003).

Young bees remove water from the sugar solution using two methods. They pass the nectar from bee to bee and 'drink' the water out of the nectar by absorbing it through their stomach wall. They also create heat and air flow in the hive by vibrating their wings and flight muscles, thus evaporating water out of the nectar which has been stored in open cells ( National Honey Board,2003).

When most of the sucrose has been converted to fructose and glucose and enough water has been dehydrated out of the mixture to bring it approximately 17.8% water content, a delicious sticky mixture, called honey! After honey is made, bees cap it with bees wax to maintain the low moisture content ( National Honey Board,2003).

Honey contains mainly sugars (National Honey Board, 2008). It contains more carbohydrates (82.3%) than does any other animal product. A unique property of honey is its predominant sugar, fructose. This sugar is primarily responsible for its sweet taste. Honey contains a large amount of glucose but is low in sucrose (<8%) (USDA, 2008). At normal temperatures, honey is frequently saturated with respect to glucose and exists as clear syrup mainly preferred by consumers. The chief sources of micro-organisms in honey are the nectar of the flowers and the honey bee. Yeasts have been shown to come from the nectar and from the intestinal content of the bee (Frazier and Westhoff, 1994).

2.2 Nectar

Nectar, a liquid high in sucrose, is produced in plant glands known as nectaries. It is an important energy resource for honey bees and plays a significant role in foraging economics and evolutionary differentiation between different subspecies. It was proposed through an experiment conducted with the African honey bee, Apis mellifera.scutellata, that nectar temperature impacts the foraging decisions of honey bees(Fewell and Susan ,2002).

2.3 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. In the USA honey is also graded on its color and optical density by United States Department of Agriculture (USDA) standards, graded on a scale called the Pfund scale, which ranges from 0 for "water white" honey to more than 114 for "dark amber" honey (FAO,2011).

2.3.1 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 or can be blended after collection. The pollen in honey is traceable to floral source and therefore region of origin. The rheological & melissopalynological properties of honey can be used to identify the major plant nectar source used in its production (Nicola, 2009).

2.3.1.1Blended

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. (National Honey Issac Board,2011).

2.3.1.2 Polyfloral

Polyfloral honey, also known as wildflower honey is derived from the nectar of many types of flowers (National Honey Board ,2011)

2.3.1.3 Monoflora

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(Nicola, 2009).

2.3.1.4 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 as sweet as nectar honeys. Germany's Black Forest is a well known source of honeydew-based honeys, as well as some regions in Bulgaria, Tara

(mountain) in Serbia and Northern California in the United States. In Greece, pine honey (a type of honeydew honey) constitutes 60–65% of the annual honey production (Gounari, 2006).

2.3.2 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 (FAO,2011).

2.3.2.1 Crystallized honey

It honey in which some of the glucose content has spontaneously crystallized from solution as monohydrate. Also called "granulated honey" or "candied honey." Honey that has crystallized (or commercially purchased crystallized) can be returned to a liquid state by warming (Flottum ,2010).

2.3.2.2 Pasteurized honey

It honey that has been heated in a pasteurization process which requires temperatures of 161 °F (72 °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 ( (Subramanian et al.,2007).

2.3.2.3 Raw honey

Is honey as it exists in the beehive or as obtained by extraction, settling or straining, without being heated (although some honey that has been "minimally processed" is often labeled as raw honey). Raw honey contains

6 some pollen and may contain small particles of wax. Some allergy sufferers wrongly believe that raw, local honey can help build tolerance to the pollen in the air (Nicola, 2009).

2.3.2.4 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(Nicola, 2009).

2.3.2.5 Filtered honey

Is honey of any type that has been filtered to the extent that all or most of the fine particles, pollen grains, air bubbles, or other materials normally found in suspension, have been removed ( USDA, 2012). The process is typically heating honey to 150-170 °F (66-77 °C) to more easily pass through the filter. Damerow(2011) reported that the filtered honey is very clear and will not crystallize quickly.

2.3.2.6 Creamed honey

Also called whipped honey, spun honey, churned honey, honey fondant, and (in the UK) set honey, has been processed to control crystallization. Creamed honey contains a large number of small crystals, which prevent the formation of larger crystals that can occur in unprocessed honey. The processing also produces a honey with a smooth, spreadable consistency (Sharma ,2005).

2.3.2.7 Dried honey

Has the moisture extracted from liquid honey to create completely solid, non sticky granules. This process may or may not include the use of drying and anti caking agents. Dried honey is used in baked goods, and to garnish desserts. (Krell ,1996).

2.3.2.8 Chunk honey

Is packed in wide mouth containers consisting of one or more pieces of comb honey immersed in extracted liquid honey (Flottum ,2010).

2.4 Physical and chemical properties 2.4.1 Viscosity The viscosity of a material is its resistance to flow. Beekeeper calls it body. Viscosity of honey depends upon the composition of honey,especially its moisture content. Honey viscosity is of great practical importance to the beekeeper and processor(White,1975).

Thixotrophy is the decrease of viscosity following stirring or agitation which then returns on standing. Heather (Europe) and manuka (Newzealand) honeys are markedly thixotopic while the American honeys are not(White,1975). Pryce-Jones (1953) found a protein in honey to be responsible of this property.

Bogdanov (2009) reported that the viscosity increase due to temperature occur very slowly at first. A honey containing 16% water, at 70° C (158° F), will have a viscosity around 2 poise, while at 30 °C (86 °F), the viscosity is around 70 poise. As cooling progresses, honey becomes 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.

2.4.2 Specific gravity

Is the ratio of the mass of a volume of given substance (at a stated temperature) to the mass of the same volume of water (at stated temperature) (White,1979). Wedmore(1955) modification of Chataway(1935) Table shows

8 that the specific gravity of bee honey decreases with the increase in moisture content of honey.

2.4.3 Color

Colors of honey form a continuous range from very pale yellow through ambers to a dark reddish amber to nearly black, greenish casts are fairly common. Color ranges are generally characteristic of floral types(White,1979).

2.4.4 Refractive index

The refactive index of a substance is the ratio of the velocity of light in the substance to that in air (White,1975). The primary interest in this property of honey is to provide a rapid, accurate and simple methods of determining the moisture content of honey. By using the refractometer, moisture in honey can be determined with ease, compared with other method. Chataway (1935) Table which was modified by Wedmore(1955) can be used ; the Table relates the refractive index value of honey measured at(20oC) to its corresponding moisture content values.

2.4.5 Hygroscopy and fermentation 2.4.5.1Hygroscopicity Honey, especially when rich in fructose, is very hygroscopic i.e. it absorbs moisture from the air when the container is not closed. This may lead to an increase in water content and possible fermentation. For this reason it is important that honey is always stored in containers with tight fitting lids(Nicola, 2009).

2.4.5.2 Fermentation

Fermentation of honey is caused by the action of sugar-tolerant yeasts upon the sugars dextrose and levulose, resulting in the formation of ethyl alcohol and carbon dioxide. The alcohol in the presence of oxygen then may be broken down into acetic acid and water. As a result, honey that has fermented may taste sour (White and Doner,1980).

Fermentation of honey is sometimes a problem. The main factors causing fermentation are:

- High moisture content (above 20 percent);

- High temperature; and

-A high yeast count (>10/gram).

Uneven granulation of honey within a container can lead to small pockets with high levels of water, and this may result in fermentation. Honey that has begun to ferment can be used for making into fermented products like beer, wine or vinegar (Nicola, 2009).

2.4.6 Thermal characteristics

Like all sugar compounds, honey will caramelize if heated sufficiently, becoming darker in color, and eventually burn. However, honey contains fructose, which caramelizes at lower temperatures than the glucose(Hans- Dieter Belitz et al., 2004).The temperature at which caramelization begins varies, depending on the composition, but is typically between 70 and 110° C (158 and 230° F). Honey also contains acids, which act as catalysts, decreasing the caramelization temperature even more. Of these acids, the amino acids, which occur in very small amounts, play an important role in the darkening of honey. The amino acids form darkened compounds called

10 melanoidins, during a Maillard reaction. The Maillard reaction will occur slowly at room temperature, taking from a few to several months to show visible darkening, but will speed-up dramatically with increasing temperatures. However, the reaction can also be slowed by storing the honey at colder temperatures (Zdzisław,2007).

2. 5 Chemical properties of bee honeys 2. 5.1 Moisture content The moisture content of honey is an important factor contributing to its stability against fermentation and granulation during storage. Moisture content was affected by climate, season and moisture content of original plant nectar. Honey moisture is the quality criterion that determines the capability of honey to remain stable and to resist spoilage by yeast fermentation : the higher the moisture, the higher the probability that honey will ferment upon storage. The water content is that value determined from the refractive index of honey by reference to a standard Table. White et al.(1962) reported that the moisture content of 490 American bee honey samples ranged from 13.4- 22.9% and averaged 17.2%.while Al-Sarrag(1977) reported that the moisture content of seven Sudanese bee honey samples ranged from 14.8 to 27.0%.

Ibrahim (1985) reported that the moisture content of Sudanese bee honey samples ranged from 13.01-26.08% and averaged 19.09%.

2. 5.2 Ash and minerals content

Nicola ( 2009) reported that the ‘ash’ content of honey is mainly mineral trace elements. Minerals present are calcium, copper,iron, magnesium, manganese, potassium, sodium, and chlorides, phosphates, silicates and sulphates. Dark honeys are often very rich in minerals, but variation in the

11 mineral content of different honeys is great. These trace amounts of minerals may be important for human nutrition.

The average ash of American bee honeys as found by White et al. (1962) was 0.17% and ranged from 0.02% to 1.028%. Ismaeil(1972) found that the average ash content of Egyptian honeys analyzed was 0.17% . Iron content was 6.6 mg/kg.

Mclenllan(1975) mentioned that Ca, Mg, K, and Na were found in honey samples, K was found in higher amount compared to the other minerals.

While Ibrahim (1985) reported a range of 0.088-1.975% and an average ash content of 0.374% in Sudanese honeys. Ibrahim (1985) reported the following range of values for minerals in bee honey(ppm): Na(73-697) ,K(25-1987), Zn(1-103), Cu(0-85), Fe(35-544), and Pb(0-37).

2. 5.3 Carbohydrates

Carbohydrates represent the largest portion in honey.The sugars are responsible for much of the physical nature of honey such as viscosity, hygroscopicity, granulation properties and energy values.

Fructose and glucose together account for 85- 95% of honey carbohydrates, the proposed Codex (1969) standard for honey requires a minimum reducing - sugar content of 65% for flower honeys. Both fructose and glucose are defined as reducing sugars. Other sugars present in significant amounts are disaccharide sucrose(cane sugar), maximum sucrose content of 5% is required by proposed Codex (1969). Sucrose is defined as non reducing, which is hydrolysed either by mineral acids or by enzyme invertase. Each molecule combines with a molecule of water, and fructose and glucose are formed in equal quantities (Walker, 1917). According to White et al.(1962) fructose,

12 glucose and sucrose were ranged between 27.2 and 44.3%, 22.0 and 40.7% then 0.2 and 7.6% respectively. Ibrahim(1985) studied the total sugars, reducing sugars and sucrose and reported values of 60.6 -79.4% ,57.8 -75.7% and 0.6 -10.5% respectively. Lazzaridou et al,(2004) found that the range of fructose and glucose were 22.1-41.3% and 13.5-36.3% respectively. Moreover , Serrano et al,(2004) noted the range of glucose 10.69 -45.25% , fructose 13.55 - 45.0%, sucrose 0.14 - 11.49% and total sugars 08.0 - 83.5%. While Shin and Ustunol (2005) reported values of 37.7%fructose, 35.3% glucose and 2.5% sucrose. Recently Ischayek and Kern (2006) working on four varieties of honey in the United States, found that the averages of total sugar, glucose ,sucrose , fructose content were 85.9%, 31.3%, 2.7% and 36.7% respectively. Also Ouchemoukh et al,(2007) reported ranges of total sugars 71.25- 84.25% , reducing sugars 67.83 - 80.25%, sucrose 0.08 - 5.31% and glucose 29.4-42.0% While Finola et al,(2007) reported 24- 39.7% glucose and 33 - 48.4% fructose.

2. 5.4 Protein content

The level of protein is dependant on the type of flora and thus it is variable(Saxena et al,2010). Juszczak et al.,(2009) reported that the amount of nitrogenous compounds of different honeys are at the range of:199- 13100(ug/g) for protein content. White et al.,(1962) reported that the nitrogen content in American bee honeys ranged from 0-0.138% and averaged 0.041%. Ibrahim (1985) reported a range of 0.077-1.378% and average of 0.53% protein in Sudanese bee honeys.

2. 5.4.1 Amino acids

Krell (1996) reported the presence of different amino acids in honey of bee and plant origin. Krell(1996) reported that the following free amino acids

13 exist in honey: proline, lysine, histidine, arginine,aspartic acid, threonine, serine, glutamic acid, glycine, alanine,cystine, valine, methionine, isoleucine, tyrosine, phenylalanine, and tryptophan. Komamine(1960) reported the preponderance of proline(45% in finnish honey and 80% in an imported honey) of the overall amino acids content. The next most abundant amino acid reported in the finnish honey was glutamic acid and imported honey was leucine. Baugmerten and Mockesch (1956) analyzed 15 honey types for their amino acid content ,they reported that the free amino acids originate from the bee, arguing that the relatively uniform occurrence over widely varying group of honey types markes it unlikely that they could originate from pollen or nectar. Bergner and Koromi(1968) reported that stores from sugar feeding of bees contained the same 19 amino acids found in mixed samples of honey. White and Rudyj(1978b) determined proline content of 740 American honey samples, for 482 samples of the season (1956-1957) the range of proline was (14.8-139mg/100g) and the average was 48.3 mg/100g , for the other 258 samples of the season (1956-1967) the range was (16.9-148 mg/100g) and the average was 54.1 mg/100g. Davies(1975) suggested the use of amino acids analysis of bee honeys as an alternative to pollen analysis for determining the geographical origin of honey. Davies(1978) reported that honeybees by using proline equlize the osmotic pressure and thus facilitate the transfer of enzymes from the hypopharyngeal glands to raw nectar. Costa et al.(1999) analyzed 74 samples of honey from different floral sources that were collected from four regions in Brazil, the samples showed a variation in their proline content(389-520 ppm). Joshi et al.(2000) reported that the average proline content(ppm) of honeys from Apis mellifera, Apis cerana and Apis dorsata are 610.19, 323.0 and 875.82 respectively. The proline content was found to be significantly different between the three groups.

2.5.5 Acidity and pH

The acidity of honey is due to the presence of organic acids, particularly the gluconic acid, in equilibrium with their lactones or esters and inorganic ions such as phosphate and chloride (Echingo and Takenaka,1974). The variation in acidity among different honey types may be attributed to variation in these constituents due to extraction season(Perez-Arquillue et al.,1994). Organic acids comprise most of the acids in honey, accounting for 0.17–1.17% of the mixture. Gluconic acid is the most prevalent. Gluconic acid is formed by the actions of an enzyme called glucose oxidase. Other organic acids are minor, consisting of formic, acetic, butyric, citric, lactic, malic, pyroglutamic, propionic, valeric, capronic, palmitic, and succinic, among many others. (Wilkins and Yinrong 1995). White et al. (1962) reported an average pH value of 3.91 and a range of (3.42-6.10) for American honey samples. While Ismaeil(1972) reported an average pH value of 3.9 for Egyptian honey samples analyzed. Ibrahim(1985) mentioned a range of (3.3-4.3) and average of (3.8) for the pH of Sudanese honey samples.

2.5.6 Hydroxymethylfurfuraldehyde (HMF)

HMF is hydroxymethylfufural, a breakdown product of fructose (one of the main sugars in honey) that is formed slowly and naturally during the storage of honey, and much more quickly when honey is heated. The amount of HMF present in honey is the reference used as a guide to the amount of heating that has taken place: the higher the HMF value, the lower the quality of the honey is considered to be. Some countries set an HMF limit for imported honey (sometimes 40 miligrams per kilogram), and honey with an HMF value higher than this limit will not be accepted. However, some honeys have a naturally high HMF level. HMF is measured by laboratory tests (Nicola, 2009).

Duisberg and Hadorn(1966) examined over 1600 samples of honey for (HMF); they reported most honeys to contain less than 10 ppm of HMF. Simonyan(1972) examined 20 Russian honeys and found the HMF content was less than 10mg/kg. Fini and Sabatini (1972) found that 400 Italian unprocessed honeys cotained an average of 13 mg/kg of HMF , while processed commercial samples averaged 59 mg/kg and 27% were above the codex regional standard limit. Thrasyvoulou(1986) in his study on fresh Greek honeys, concluded that fresh samples of honey vary widely in their HMF content. He reported that the HMF content in fresh Greek honey samples varied (0-21.0) ppm. If the content of the 5-hydroxymethylfurfural HMF increased and the diastase activity is reduced beyond the given limits it is considered that overheating of honey has taken place or the honey sample is adulterated. High levels of HMF were originally taken to indicate adulteration with invert sugar but honey from tropical areas may naturally contain over 40 mg/kg. If it is desired to confirm that a honey is adulterated rather than that it merely has a high HMF level, a more extensive analysis is necessary

(FAO,1986).The Codex Alimentarius Commission (1997) has proposed a maximum limit of 80 ppm for HMF in honey after processing and plending. The use of excessive heat or liquefaction or pasteurization of honey has adverse effects on honey quality, i.e. loss of volatile compounds, accumulation of HMF and reduction of diastase and invertase activities. Quantification of HMF content and enzyme activities are useful tools in detecting heat induced defects in honey but cannot be used for the determination of botanical or geographical origin (Ruoff and Bogdanov, 2004).

2.5.7 Enzymes

The levels of enzymes present in honey are sometimes assayed and used as a guide to honey quality. The enzymes in honey (invertase, glucose oxidase, amylase, etc.) come from the bees, or from the plant where the bee foraged. They are present in very small quantities, but may still have a nutritional importance in the human diet. The enzymes are very sensitive to overheating (above 35 °C) or storage at too high temperature. Because they are destroyed by heating, a low enzyme level may mean that honey has been heated, but many honeys of good quality are naturally low in enzyme content. (Nicola, 2009).

White and Doner (1980) reported that some of the most important honey enzymes are invertase, diastase, and glucose oxidase. Invertase, also known as sucrase or saccharase splits sucrose into its constitutent simple sugars, dextrose, and levulose. Other more complex sugars have been found recently to form in small amounts during this action and in part explain the complexity of the minor sugars of honey. Although the work of invertase is completed when honey is ripened, the enzyme remains in the honey and retains its activity for some time. Even so, the sucrose content of honey never reaches zero. Since the enzyme also synthesizes sucrose, perhaps the final low value for the sucrose content of honey represents an equilibrium between splitting and forming sucrose(White and Doner, 1980) Diastase (amylase) digests starch to simpler compounds but no starch is found in nectar. What its function is in honey is not clear. Diastase appears to be present in varying amounts in nearly all honey and it can be measured. It has probably had the greatest attention in the past, because it has been used as a measure of honey quality in several European countries(White and Doner,1980). Glucose

17 oxidase converts dextrose to a related material, a gulconolactone, which in turn forms gluconic acid, the principal acid in honey. Since this enzyme previously was shown to be in the pharyngeal gland of the honey bee, this is probably the source. Here, as with other enzymes, the amount varies in different honeys. In addition to gluconolactone, glucose oxidase forms hydrogen peroxide during its action on dextrose, which has been shown to be the basis of the heat-sensitive antibacterial activity of honey. Other enzymes are reported to be present in honey, including catalase and an acid phosphatase. All the honey enzymes can be destroyed or weakened by heat ( White and Doner,1980).

2.5.8 Vitamins

Honey also contains wide range of vitamins like A, B, C, D, E, and K.

To process such a complex mixture for consistent product quality, retaining most of the nutritional value and imparting a better product appeal, is a real challenge for the processing industries. Studies on honey proved its wide spread usage other than its nutritional value (Lagrange,1991). Kalimi and 3mg/100g vitaminsــSohonie (1965) using a micro-chemical method, found 2 C for one°30ــin four Indian honeys, with one fifth lost after storage at 28 ــ year. Rahmanian et al. (1970) have found high ascorbic acids values 118 240 mg/100g in three samples of honey unknown sources from mountainous Damavand area in Iran. A sample containing 118 mg/100g was assayed with guinea pigs. Weight gains of animals fed daily 5 mg ascorbic acid or 4 g honey did not differ significantly. They suggested the possibility of encouraging the use of honey from the region as a means of helping to relieve the marginal vitamin C deficiency often found in Iran.

2.5.9 Aroma and flavor

Maeda et al, (1962) ascribed the taste of honey to the sugars, gluconic acid and proline content. But it is evident to anyone who examines a variety of honeys that, although there seems to be a chacteristic honey flavor, almost an infinite number of aroma flavor variations can exist. It is realized that the various sugars, amino and other acids, tannins and minor non volatile substances all contribute to honey flavor. Further, additional contribution to flavor effects of certain honeys may be due to glucoside or alkaloidal compounds specific to the plant source.(White,1979). Bicchi et al,(1983) by using chomatographic techniques, reported the persence of 52 volatile components in Pidemontese honeys that are responsible about their flavor. It is probable that the compounds that are and will be identified in honey groups will be of value in the comparison and identification of floral types of honey as with (Methyl anthranilate- MA) which is a minor constituent of citrus honey and contributes to its distinctive aroma (Bouseta et al.,1992). Krell(1996) reported the persence of more than 45 aroma constituents in bee honey that can be classified into different groups (esters, ketones, aldehydes and alcohols).

2.6 The antimicrobial activity of bee honey

Bee honey was reported by many researchers to possess antimicrobial activity (antibacterial and antifungal). Honey was found by some workers to possess higher antimicrobial activity than commonly used antibiotics.

Honey has been suggested by the World Health Forum(1981) for use in enhancing the healing of wounds and pressure sores and was reported to be anti bacterial to maney Gram(+), Gram(-) bacteria and the fungi(candida

19 albicans)(Smith et al.,1969;Ibrahim(1981);Obsaeiki-Ebor and Afonya(1984); Al-somal et al,(1994) and Nzeako and Hamdi(2000).

2.7 As Antioxidant

Honey also contains tiny amounts of several compounds thought to function as antioxidants, including chrysin, pinobanksin, vitamin C, catalase, and pinocembrin. (Martos et al,2000) and( Gheldof et al.,2002) .

2.8 Religious significance

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 chapter (Surah) in the Qur'an called an-Nahl (the Bee). According to his teachings (hadith), Muhammad strongly recommended honey for healing purposes. The Qur'an promotes honey as a nutritious and healthy food.

2.9 Modern uses 2.9.1 As a food and in cooking 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". Honey barbecue and honey mustard are common and popular sauce flavors(USDA,2012).

2.9.2 .Health effects

Historically, honey has been used by humans both orally and topically to treat various ailments including gastric disturbances, ulcers, wounds, and burns.

Honey was used medicinally by ancient Greeks and Egyptians and has been traditionally used in Ayurveda in India and in Traditional Chinese Medicine. (Pećanac et al.,2013) and ( Altman, 2011). The Quran mentions honey as a medicine. ( Altman, 2011) Medieval Islamic and Christian scholars described the use of honey in medicine of that period ( Altman, 2011).

2.10 Health hazards 2.10.1 Toxic honey Honey produced from flowers of 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, is thought to dilute any toxins (Wayback, 2010).

2.11 Adulteration

Adulteration of honey is the addition of other sugars, syrups or compounds into honey to change its flavor, viscosity, make it cheaper to produce, or to increase the fructose content in order to stave off crystallization. According to the Codex Alimentarius of the United Nations, any product labeled as honey or pure honey must be a wholly natural product, although different nations have their own laws concerning labeling. Adulteration of honey is sometimes used as a method of deception when buyers are led to believe that the honey is pure. The practice was common dating back to ancient times, when crystallized honey was often mixed with flour or other fillers, hiding the

21 adulteration from buyers until the honey was liquefied. In modern times the most common adulteration-ingredient became clear, almost-flavorless corn syrup, which, when mixed with honey, is often very difficult to distinguish from unadulterated honey(Martin,2004). Pilizota and Nedic( 2009) reported that honey adulteration appeared on the world market in the 1970s when high- fructose corn syrup was introduced by the industry. As the sugars (60.7- 77.8%) are the major components of honey and the most dominant are the monosaccharides fructose and glucose (accounting for 85-95%), the actual proportion of glucose to fructose in any particular honey depends largely on the source of the nectar. The average ratio of fructose to glucose is 1.2:1. The amount of glucose in honey is usually at a supersaturated level at normal temperatures. With reduction in temperature or water content, the glucose can crystallize out. Saccharose (sucrose) is present in honey at approximately 1% of its dry weight. Normally, honey contains 12.4-24.5% moisture. Unless the moisture content is below 17%, no fermentation takes place (Pilizota and Nedic, 2009). The processing of honey includes controlled heating to destroy yeast and dissolve dextrose crystals, combined with fine straining or pressure filtration. Most honey will crystallize during some period of time unless action is taken to prevent it. Generally, when honey is stored below 10 °C, crystallization can be prevented or delayed (Pilizota and Nedic, 2009). Honey is usually warmed to a temperature of 32±40 °C to lower its viscosity, which facilitates extraction, straining or filtration. This temperature is similar to that in beehives and does not affect the honey very much during the relatively short processing period. However, some honeys are heated to a higher temperature for liquefaction or pasteurization reasons (Pilizota and Nedic, 2009).

2.11.1 Adulteration detection

All food products targeted for adulteration are high-value commercial products, including honey. The detection of adulteration can pose a technical problem. The quality of honey is mainly determined by its sensorial, chemical, physical and microbiological characteristics. Analytical methods applied to honey generally deal with different topics: determination of botanical or geographical origin, quality control according to the current standards and detection of adulteration or chemical residues (Pilizota and Nedic, 2009). At present, a variety of analytical techniques have been developed to detect adulteration of honey, such as isotopic (stable isotope methodology), chromatographic, spectroscopic, trace elements techniques and thermal analysis. Some of these methods are time-consuming, and some are expensive. Although there are powerful methods to prove honey adulteration, they have to be further improved in order to ensure honey quality (Pilizota and Nedic, 2009). Due to the limitations of classical analytical methods, which measure chemical parameters to detect adulteration, many experiments have been carried out using new indicators derived from physical analysis, such as thermal analysis (Pilizota and Nedic, 2009).

2.12 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 ( Bogdanov, 2008) 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%) (Bogdanov, 2008) of the product. Honey with excessive water content is not suitable for long-term preservation (Allan, 2008).

Anupama et al, (2003) reported that moisture content varied from 17 to 22.6%, Brix from 76 to 81%, pH from 3.62 to 5.46, apparent viscosity from 1.79 to 13.8 Pascal sec, acidity from 0.03 to 0.15%, total reducing sugars from 61.3 to 72.6% and sucrose from 1.2 to 5.7%.

The sensory properties such as color, aroma and taste of honey vary according to the geographical and seasonal conditions as well as the floral source. Commercial honey in India is generally of assorted type as it is collected from regions having different climatic conditions and vary often from forests or wild flower sources(Anupama et al.,2003).

Table.2.1Specification of Sudanese honey

Composition Range

Moisture Not more than 20%

Reducing sugars Not more than 60%

Sucrose Not more than10%

Ash Not more than1%

Acid 50mellimeque/kg

Distress enzymes 8

HMF 1%

TS (total solid) Not more than0.1% Source: SSMO (2007).

CHAPTER THREE MATERIAL AND METHODS

3.1Materials

Fifteen samples of honey( Zizyphus spina-christi) were collected from Khartoum, Bahri and Omdurman local markets. (5 samples from each city). The honey samples were stored in clean bottles and immediately transferred to the laboratory for analysis.

Market Sample code

Khartoum A Bahri B Omdurman C

3.2 Physical properties

3.2.1 Refractive index

The refractive index was measured at 23.5o C using an Abbe refractometer (Hilger, 27137). Determinations made were corrected by adding 0.00023 per °C for difference in temperature 20o C according to IHC (2002) which states that (to temperatures above 20o C: add 0.00023 per °C and temperatures below20o C subtract 0.00023 per °C).

3.2.2 pH value

Ten grams of honey were added to 75ml of distilled water into 250 ml beaker. Stirring was done using a magnetic stirrer. The electrodes of the PH meter were immersed in the solution and pH value was recorded ( AOAC,2003).

3.3 Chemical composition 3.3.1 Moisture content The method is based on the refractometric examination of honey. Moisture content was determined using Wedmore(1955) modification of Chataway (1935) method. The method correlates the refractive index of honey measured at 200 C to its moisture content.

Procedure:

The refractive index of the honey samples was determined using an Abbe refractometer (Hilger,27137) at temperature of 32° C. The refractive index readings were convereted to moisture content values, using Chataway (1935) Table which was modified by Wedmore (1955). The Table relates the refractive index of honey to its moisture content. Determinations made at temperatures other than 20°C should be corrected (temperatures above 20°C: add 0.00023 per °C and tempertures below 20° C subtract 0.00023 per °C).

3.3.2 Ash content

The ash content of honey samples was determined using the method described by the AOAC. (2003).

Procedure:

Two grams of honey were weighed into previously weighed crucibles, and then dried at 1050 C in a drying oven . After removing the crucibles from the oven the crucibles were placed over a bunsen flam to do an initial carbonization. The crucibles were then transferred to the muffle furnace and was ignited at 550° C for about eight hours until free of carbon (residue appears white).

Calculation:

Ash%= (B – C) x100 A A: sample weight in grams.

B: weight in grams of dish and contents after ashing.

C: weight in grams of empty dish.

3.3.3 Free acidity, lactone and total acidity values

Principle:

The sample is titrated with sodium hydroxide to obtain the free acidity. Excess sodium hydroxide is added to hydrolyze any lactose present and immediately back titrated with hydrochloric acid. The total acidity is calculated as free acidity plus lactone in milli equivalents per kilogram (AOAC,2003).

Apparatus:

pH meter.

Reagents:

1. 0.05 N NaOH 2. 0.05 N HCL

Procedure:

Ten grams of honey were added to 75 ml of distilled water in a 250 ml beaker. Stirring was done using a magnetic stirrer. The electrodes of the pH meter were immersed in the solution and the pH was recorded. The honey solution was titrated against 0.05 N NaOH at a rate of 5 ml/minute until the pH reached 8.5 and the burette reading was recorded. Immediately 10 ml of

0.05 N NaOH was added to the honey solution and was immediately back titrated with 0.05 N HCL from a 10 ml burette until the pH reached 8.3. A reagent blank was also made.

Calculation:

Free acidity = (ml of 0.05N NaOH to bring solution to PH 8.5 - blank)x 0.05x1000/10.

Lactone =(10- titre of 0.05 HCL in ml)x 0.05x1000/10.

Total acidity = free acidity + lactone(all results being expressed as milli- equivalents of acid per kg of honey).

3.3.4. Detection of technical invert sugar

Principle:

Fiehe’s test for detection of invert sugar in honey depends on the formation of red or pink color when the HMF reacts with resorcinol(Pearson, 1970).

Reagents:

1.Diethylether

2. Resorcinol indicater

3. 0.1N HCL

Procedure:

Five grams of honey were weighed and dissolved in 5ml cold water in a 100 ml beaker. Diethylether 20 ml was added to the solution and was put in a porcelain dish to evaporate at room temperature. The residue was dissolved in 10 ml diethylether.

1% a fresh resorcinol solution (1g resorcinol dissolved in conc. hydrochloric acid 0.1N) was added to the solution and shake was done. After 10 min the pink color appeared in the acid layer.

3.4 Sugar analysis 3.4.1 Determination of sugars by HPLC This method is based on the originally published method by Bogdanov and Baumann(1988). After filtration of the solution , the sugar content was determined by High Performance Liquid Chromatography (HPLC) with refractive index (RI)-detection. Peaks were identified on the basis of their retention times. Quantitation was performed according to the external standard method on peak areas or peak heights.

Reagents:

Acetonitrile HPLC-grade

Eluent solution for the HPLC: 75 volumes of acetonitrile with 25 volume of water. Degassed and filtered with membrane filter prior to use.

The standard sugars were: fructose, glucose, sucrose and maltose.

3.4.2 Preparation of standard sugars solutions

Fructose, glucose, sucrose and maltose(1g, 0.75g, 0.127g and 0.175g) were weighed and dissolved in approximately 50 ml deionized water and transfered quantitatively to 50 ml volumetric flask and was filled to the mark with deionized water.

Procedure:

Preparation of the sample solution:

About 2.5g of honey were weighed in a100 ml beaker and dissolved in 50 ml water. Then the honey solution was transferred quantitatively to the flask. The flask was filled to the mark with distilled water. The solution was poured and passed through a membrane filter and collected in sample vials.

High performance Liquid Chromatography (HPLC):

HPLC: prominence system controller CBM- 20A

Column: shim-pack CLC-NH2(M) 250mm *4.7mm

Detection:RID-10A

Mobile phase: Acetonitrile: Water(75:25,v/v)

Flow rate: 1.3 ml/min

Sample volume: 10 ul

Promanence Auto Sampler: SIL – 20 AC

Bump: LC - 10 AB

Colum oven: CTO -20 AC

Calculation and expression of results

The honey sugars were identified and quantified by comparison of the retention times and the peak area of the honey sugars with those of the standard sugars by using LC solution system package release 1- 24 SP1.

The mass percentage of the sugars (w) to be determined for fructose, glucose, sucrose and maltose in g/ 100g was calculated according to the following formula (external standard procedure):

W=A1 xV1 x m1x100 / A2 x V2 x m0

Where:

A1 = Peak areas of the given sugar compound in the sample solution.

A2 =Peak areas of the given sugar compound in the standard solution.

V1 =Total volume of the sample solution in ml.

V2 =Total volume of the standard solution in ml. m1= Mass amount of the sugar in grams in the total volume of standard (V1).

m0 = Sample weight in g.

3.5 Statistical analysis

Data generated were subjected to SAS. Single factor (Completely Randomized Design - CRD) with 3 replication, and then means were separated using LSD.

CHAPTER FOUR RESULTS AND DISCUSSION

4.1 Physical properties 4.1.1 Refractive index The refractive index of honey samples falls within the range of 1.504- 1.451 (Table 4.1) . The refractive index of honey samples are approximately in the same range of the Codex standards of 1.4000 -1.9000. The refractive index of honey is rapid, accurate and simple measure of its moisture content (White,1975).

4.1.2 pH value

Table 4.1 Fig 4.1show pH values of the samples was ranged from 4.13 to 4.56 . The differences between the pH values of the samples was high significant p<.0001. pH values of honey samples fall within the range of established Codex Standard of honey (3.6 -5.6). These values are comparable to the pH values (3.4 -6.1) of honey of the United State of America (White,1975).

Table 4.1: Physical Properties of Honey Samples: Samples PH Refractive Index A 4.14(±0.005)e 1.492(±0.000)bc A1 4.22(±0.025)d 1.504(±0.000)a A2 4.13(±0.005)e 1.486(±0.000)c A3 4.22(±0.025)d 1.499(±0.000)a A4 4.15(±0.001)ed 1.503(±0.001)a B 4.15(±0.0.001)ed 1.504(±0.000)a B1 4.14(±0.005)e 1.474(±0.000)d B2 4.15(±0.011)ed 1.486(±0.001)c B3 4.50(±0.020)ab 1.478(±0.000)d B4 4.48(±0.112)ab 1.504(±0.001)a C 4.47(±0.112)b 1.498(±0.001)ab C1 4.45(±0.035)bc 1.458(±0.001)e C2 4.56(±0.062)a 1.461(±0.001)e C3 4.50(±0.020)ab 1.459(±0.001)e C4 4.38(±0.017)c 1.451(±0.001)f Values are means(±standard deviation). - Means with the same letter are not significantly different. Samples A-A4 From Khartoum local markets. Samples B-B4 From Bahri local markets. Samples C-C4 From Omdurman local markets.

PH values

4.6 4.5 4.4 4.3 PH values 4.2 4.1 4 3.9 A A1 A2 A3 A4 B B1 B2 B3 B4 C C1 C2 C3 C4

Fig 4.1 pH values of bee honey samples

4.2 Chemical composition of bee honey 4.2.1 Moisture content Table 4.2 and Fig4.2. show the moisture content of the samples analyzed. The moisture was ranging from 13.00% to 37.82%.The moisture was highest in the samples C4, C3, C1, C2, B2, B1and A2 which were 37.83%, 30.77%, 30.43%, 27.43%, 25.67%, 25.07% and 20.17% respectively . These samples were higher than the maximum limit quoted, for moisture content in honey by the Codex Standards. The SSMO (2007) standards stated that Sudanese honey should not contain moisture more than 20%. Usually the moisture content of honey depends on the floral source, rainfall ,relative humidity, temperature and methods of processing and storage .There was a great variation in moisture among the samples. This variation in moisture could be attributed to climate, method of processing, or may be due to adulteration by adding water. Generally most of the samples ranged from 13.00 to 17.80% and are similar to those of other workers. White et al.(1962) and Ibrahim (1985) reported values of 13.4-22.9%. The moisture content of honey is most important in assessment of ripeness and shelf life.

4.2.2 Ash content

Table 4.2 and Fig4.3.show the ash content of the honey samples. The ash content was highest in sample A2 (1.07%) and lowest in samples C and A4(0.05% and 0.06%) . The difference between the ash content of the honey samples were highly significant. The ash content of the most of samples achieved were within the range reported by White et al.(1962a) (0.02- 1.028%) and Ibrahim (1985) (0.088-1.975%). Ismaeil (1972) reported a lower average value (0.17%) for the Egyptian honeys. The ash content values for

36 most of samples except the samples(A2, B3, C4, C1). were in agreement with the proposed value for ash content given by the Codex Alimentarius Commission(1997) which states that it shouldn’t exceed 0.6%.

4.2.3 Free acidity, lactone and total acidity values

Table 4.2 shows the results of the free acidity, lactone and total acidity. The free acidity, lactone and total acidity values were highest in samples C1, B2 ,C1 , the values obtained were (15.17, 3.40, 17.98) meq/kg respectively. The differences between the free acidity, lactone and total acidity values were highly significant (p<0.01). The values obtained for the free acidity of the honey samples fall in range of the values 6.75-47.19 meq/kg reported by White et al. (1962a),and less than the average value 21.90 meq/kg reported by Ismaeil(1972). The values obtained for lactone of honey samples were similar to the values obtained by White et al.(1962a) which is 0-18.76 meq/kg and less than values obtained by Ismaeil(1972) 8.69 meq/kg. The total acidity values of the honey samples also fall in range of values (8.68-59.49 meq/kg) for American honey samples reported by White et al. (1962a). The free acidity values obtained for all fifteen samples fell within the permitted range proposed by Codex Alimentarius (2001) of not more than 50 milliequiv acid/kg. The free acidity of honey samples in this study ranged from 7.38to 15.17milliequiv acid/kg . The SSMO (2007) standards stated that for Sudanese honey should not contain acid more than 50 milliequiv acid/kg.

4.2.4 Hydroxymethylfurfural (HMF)

Table 4.2. shows H M F. The results were positive (+) in five samples (A, A3, B2, B3 and C2 ) . That indicted appearance of invert sugar which could be attributed to adulteration by Sugar cane syrup or may be due to unripped honey. HMF is formed slowly and naturally during the storage of honey and

37 much more quickly when honey heated. The higher the HMF value, the lower the quality of honey. The SSMO (2007) standards stated that for Sudanese honey should not contain HMF more than 1%.

Table 4. 2: Chemical Composition of Honey Samples: Samples Moisture% Ash% Free Acid Lactone Total H M F (meq/kg) (meq/kg) Acidity(meq/kg) A 17.80(±0.000)h 0.38(±0.028)c 11.39(±0.028)c 2.23 (±0.115)d 13.62(±0.144)d + A1 13.03(±0.057)k 0.38(±0.028)c 8.55(±0.202)e 2.23 (±0.202)d 10.79(±0.000)f - A2 20.17(±0.057)g 1.07(±0.152)a 13.20(±0.632)b 2.83 (±0.057)c 16.03(±0.635)b + A3 15.00(±0.000)i 0.10(±0.000)ef 8.53(±0.202)e 2.22 (±0.202)d 10.75(±0.000)f - A4 13.33(±0.028)j 0.06(±0.038)f 10.43(±0.028)d 2.17(±0.028)d 12.60(±0.000)e - B 13.00(±0.000)k 0.10(±0.000)ef 11.39(±0.028)c 2.24(±0.115)d 13.62(±0.144)d - B1 13.00(±0.000)k 0.37(±0.028)c 11.37(±0.028)c 2.22 (±0.115)d 13.57(±0.115)d - B2 25.67(±0.028)e 0.10(±0.000)ef 11.85(±0.039)c 3.40(±0.173)a 15.25(±0.866)c + B3 13.00(±0.000)k 0.72(±0.57)b 8.33(±0.115)e 2.02(±0.144)ed 10.35(±0.259)f + B4 15.07(±0.011)I 0.22(±0.028)d 11.83(±0.680)c 0.83(±0.230)g 12.67(±0.837)e - C 15.07(±0.011)I 0.05(±0.000)f 10.52(±0.104)d 1.75(±0.132)f 12.18(±0.202)e - C1 30.43(±0.011)c 0.63(±0.057)b 15.17(±0.225)a 2.82 (±0.104)c 17.98(±0.275)a - C2 27.43(±0.011)d 0.18(±0.028)e 7.38(±0.076)f 2.17(±0.076)d 9.55(±0.150)g + C3 30.77(±0.057)b 0.20(±0.000)d 7.48(±0.076)f 1.78(±0.175)ef 9.27(±0.236)g - C4 37.82(±0.011)a 0.67(±0.057)b 8.93(±0.028)e 3.12 (±0.028)b 12.05(±0.050)e - - Values are means(±standard deviation). - Means with the same letter are not significantly different. Samples A-A4 From Khartoum local markets. Samples B-B4 From Bahri local markets. Samples C-C4 From Omdurman local markets. H M F= Hydroxymethylfurfural.

Moisture content

40 35 30 25

20 Moisture content 15 10 5 0 A A1 A2 A3 A4 B B1 B2 B3 B4 C C1 C2 C3 C4

Fig 4.2 Moisture content of bee honey samples

Ash content

1.2

0.8

0.6 Ash content 0.4

0.2

0 A A1 A2 A3 A4 B B1 B2 B3 B4 C C1 C2 C3 C4

Fig 4.3:Ash content of bee honey samples

Free acid

16 14 12 10 8 Free acid 6 4 2 0 A A1 A2 A3 A4 B B1 B2 B3 B4 C C1 C2 C3 C4

Fig 4.4 Free acidity values of bee honey samples

Lactone values

3.5 3 2.5 2 Lactone values 1.5 1 0.5 0 A A1 A2 A3 A4 B B1 B2 B3 B4 C C1 C2 C3 C4

Fig 4.5 :Lactone values of bee honey samples

Total acidity

18 16 14 12 10 8 Total acidity 6 4 2 0 A A1 A2 A3 A4 B B1 B2 B3 B4 C C1 C2 C3 C4

Fig 4.6 Total acidity values of bee honey samples

4.2.5 Sugar content

Table4.3 Shows the results for the sugar content of the bee honey samples. Sugars are the largest constituents of honey.

4.2.5.1 Fructose content

Table 4.3 and Fig 4.7 show that the fructose content of the honey samples was in the range of 33.20%-53.84%. The fructose content was highest in sample B3(53.84%). Statistical analysis indicated that the difference between the fructose content of the samples was significant((p<0.05). The results of the most of samples obtained were similar to the values reported by Al- Arrify(2002)(43.19%), Joshi et al,(2000)(45.93%), Ismaeil(1972) (42.81%) and White et al,(1962)(38.19).

4.2.5.2 Glucose content

From Table 4.3 and Fig4.8 the glucose of the honey samples were in the range of 23.25% -34.66%.The glucose content was highest in sample B(34.66%) and the differences between samples were highly significant (p<0.01). This results comply with that found by White et al.(1962)the range (22.03% - 40.75%) and disagreement with Al-Arrify(2002) who reported that the average value of glucose content(39.5%).

4.2.5.3 Sucrose content

Table 4.3 and Fig 4.9 show that the sucrose is the most important disaccharide present in honey. Its determination is considered as an important quality criterion. The sucrose content of the honey samples were in the range of 1.15% - 8.84%. sucrose content of the samples B2, A2 and A were slightly high 8.84, 8.18, 5.40% respectively. This could be due to unripped honey which usually contain high Sucrose or may be due to the adulteration with

43 inexpensive sweeteners such as sugar cane syrups. The sucrose content of the samples were in the range of 1.15% - 8.84 %. Statistical analysis indicated that the difference between the sucrose content of the samples were highly significant(p<0.01) . Generally the results of sucrose content in this study are in consistence with the results obtained by White et al.(1962)(0.25-7.57%) except for samples(B2(8.84%) and A2(8.18%)). Ibrahim (1985) recorded (0.6%-10.5%) for Sudanese honey. The Codex Alimentarius Commission (2001) stated that the sucrose content should not be more than 5%. The SSMO (2007) standards stated that Sudanese honey should not contain sucrose more than 10%.

4.2.4.4 Maltose content

The maltose content of the honey samples are showen in Table4.3 and Fig 4.10. The maltose content was in the range of 0.7%- 4.99%. The differences between samples were highly significant(p<0.01). The samples results achieved were lower than the range reported by White and Doner (1980)( 5.42%- 11.33%).

4.2.5.5 Fructose : glucose ratio(F: G)

Fructose in bee honey is found in larger proportions than glucose as reported by (White et al, 1962a; Serra Bonvehi and Coll (1995); Joshi et al, 2000 and Al- Arrify, 2002), leading to the fructose: glucose ratio higher than 1.

The F:G ratio is presented in Table 4.3. The F: G ratio was highest in sample B2(1.68). The differences between the honey samples F: G ratio was highly significant(P< 0.01). The values obtained were higher than the values reported by A- Arrify (2002) which was 1.09 and Joshi et al. (2000) for Nepalese honeys which was 1.1 except sample B1(1.05) and is a closer to the values

44 obtained by White et al.(1962) (1.22), Serra Bonvehi and Ventura Coll(1995) (1.19). and less than the value obtained by Ismaeil (1972) which was 1.75.

Table 4.3 : Sugar Composition of Honey Samples:

Samples Fructose% Glucose% Sucrose % Maltose % Fructose: Glucose Ratio A 38.96(±0.011)abc 26.93(±0.028)j 5.40(±0.000)c 1.82(±0.115)l 1.44(±0.023)cd A1 44.38(±0.020)abcd 30.64(0.011)g 2.51(±0.057)e 4.99(±0.202)a 1.44(±0.011)c A2 37.61(±0.028)bcd 26.47(±0.020)l 8.18(±0.000)b 2.91(±0.692)g 1.42(±0.017)f A3 44.13(±0.020)abcd 34.26(±0.115)c 1.17(±0.000)m 3.93(±0.115)e 1.28(±0.115)j A4 44.75(±0.011)abc 34.44(±0.011)b 1.27(±0.028)k 4.16(±0.028)c 1.29(±0.115)g B 44.81(±0.011)abc 34.66(±0.020)a 1.23(±0.000)l 4.11(±0.028)d 1.29(±0.000)h B1 33.23(±0.000)d 31.48(±0.000)f 2.54(±0.011)d 4.83(±0.028)b 1.05(±0.000)m B2 39.64(±0.011)bcd 23.59(±0.011)n 8.84(±0.028)a 2.64(±0.039)h 1.68(±0.346)a B3 53.84(±0.020)a 26.44(±0.020)m 2.38(±0.011)f 2.35(±0.115)k 1.46(± 0.023)b B4 46.45(±0.011)ab 32.13(±0.028)e 2.54(±0.011)d 4.83(±0.068)b 1.44(±0.017)d C 44.14(±0.020)abcd 34.21(±0.005)d 1.15(±0.011)n 3.84(±0.104)f 1.29(±0.115)i C1 37.99(±0.020)abd 26.74(1.42(±0.017)±0.0k 1.82(±0.011)g 1.04(±0.225)m 1.42(±0.017)f C2 36.81(±0.011)cbd 29.42(±0.042)h 1.80(±0.011)h 2.48(±0.076)I 1.25(±0.005)l C3 34.94(±0.011)cd 27.93(± 0.011)i 1.68(±0.057)I 2.37(±0.076)j 1.25(±0.461)k C4 33.20(±0.020)d 23.25(±0.020)o 1.50(±0.011)j 0.71(±0.028)n 1.42(±0.017)e -Values are means(±standard deviation). - Means with the same letter are not significantly different. Samples A-A4 From Khartoum local markets. Samples B-B4 From Bahri local markets. Samples C-C4 From Omdurman local markets.

Fructose content

30 Fructose content 20

0 A A1 A2 A3 A4 B B1 B2 B3 B4 C C1 C2 C3 C4

Fig 4.7 Fructose content of bee honey samples

Glucose content

35 30 25 20 Glucose content 15 10 5 0 A A1 A2 A3 A4 B B1 B2 B3 B4 C C1 C2 C3 C4

Fig 4.8 Glucose content of bee honey samples

Sucrose content

9 8 7 6 5 4 Sucrose content 3 2 1 0 A A1 A2 A3 A4 B B1 B2 B3 B4 C C1 C2 C3 C4

Fig 4.9 Sucrose content of bee honey samples

Maltose content

3 Maltose content 2

0 A A1 A2 A3 A4 B B1 B2 B3 B4 C C1 C2 C3 C4

Fig 4.10 Maltose content of bee honey samples

CHAPTER FIVE CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions

1. Honey samples differ in quality on account of various factors such as season, the origin of honey, the activity of the bee, the food of the bee, the period and technique of extraction of honey , conditions of storage and the freshness of honey.

2. The refractive index was low in samples obtained from Omdurman.

3. All honey samples were slightly acidic, in the pH range 4.13-4.56.

4. The ash content, lactonic , total acidity, glucose and sucrose were higher in the samples obtained from Khartoum. While the free acidity, fructose, maltose and fructose: glucose ratio were higher in the samples obtained from Bahri. The moisture content was higher in the samples obtained from Omdurman as it was above the limits of standards of Sudan.

5.2 Recommendations

1. The need for efficient management system during the processing, collection, storage and selling of honey to avoid adulteration.

2. The need for improvement and development of new analytical techniques to detect adulteration of honey.

3. The need for updating the Sudanese honeys standards and specifications .

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