ABSTRACT A protocol for the determination of two important monosaccharide sugars (fructose and glucose) in honey was established

in the current study by using normal phase partition liquid chromatography and 15% combined working standard of glucose, fructose and sucrose and using HPLC analysis and Lane-Eynon titrimetric method

CHAPTER ONE INTRODUCTION Honey is the natural sweet substance produced by honey bees from the nectar of plants or from secretions of living parts of plants or excretions of plant sucking insects on the living parts of plants, which the bees collect, transform by combining with specific substances of their own, deposit, dehydrate, store and leave in the honey comb to ripen and mature. 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, as it 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 evaporation. They store it as a primary food source in wax honeycombs inside the beehive. Honey gets its sweetness from the monosaccharides 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 (L. Prescott et al, 1999) 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 infants' immature intestinal tracts, 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). Bees carry an electrostatic charge whereby they attract other particles in addition to pollen, which become incorporated into their honey; the honey can be analysed by the techniques of melissopalynology in area environmental studies of radioactive particles, dust and particulate pollution.

USES OF HONEY The most popular uses of honey include the following Antibacterial Solution Antiseptic Blood Flow Improver Burn Remedy Cancer and Heart Disease Prevention Colon Damage Prevention Diabetic Ulcer Remedy Dry Elbow Softener Energy Booster Facial Scrub Fruit Preserver Hair Conditioner Lip Balm Parasite Remover Relaxant for Anxiety and Nervousness Skin Moisturizer Sore Throat Treatment Sugar Substitute Water Softener Vitamin A Enhancement Zit Zapper

CHAPTER TWO LITERATURE REVIEW HISTORY OF HONEY 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. Honey collection is an ancient activity. Humans apparently began hunting for honey at least 8,000 years ago, as evidenced by a cave painting in Valencia, Spain (Crane, 1983). The painting is a Mesolithic rock painting, showing two honey-hunters collecting honey and honeycomb from a wild bee nest. The figures are depicted carrying baskets or gourds, and using a ladder or series of ropes to reach the wild nest. The Greater Honeyguide bird guides humans to wild bee hives and this behavior may have evolved with early hominids So far, the oldest remains of honey have been found in Georgia. Archaeologists have found honey remains on the inner surface of clay vessels unearthed an ancient tomb, dating back to some 4,7005,500 years ago. In ancient Georgia, honey was packed for people's journeys into the afterlife. And more than one type, too along for the trip were linden, berry, and a meadowflower variety. 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. The fertility god of Egypt, Min, was offered honey. Pliny the Elder devotes considerable space in his book Naturalis Historia to the bee and honey, and its many uses. In the absence of sugar, Honey was an integral sweetening ingredient in Roman recipes, and references to its use in food can be found in the work of many Roman authors including Athenaeus, Cato and Bassus. Some of these are collected in the book Roman cookery. 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 (see Mayan stingless bees of Central America). 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 practices were available.

FORMS OF HONEY Honey can be found in a variety of forms. This include liquid honey, comb honey, crme honey and cut comb honey

Liquid Honey Free of visible crystals, liquid honey is extracted from the honey comb by centrifugal force, gravity or straining. Because liquid honey mixes easily into a variety of foods, it's especially convenient for cooking and baking. Most of the honey produced in the United States is sold in the liquid form. Creme (or Spun) Honey While all honey will crystallize in time, creme honey (also known as spun honey, sugared honey or whipped honey) is brought to market in a crystallized state. The crystallization is controlled so that, at room temperature, the honey can be spread like butter. In many countries around the world, creme honey is preferred to the liquid form. Comb Honey Comb honey is honey that comes as it was produced--in the honey bees' wax comb. The comb, as well as the honey, is edible! Cut Comb Cut comb honey is honey that has been packaged with chunks of the honey comb.

CLASSIFICATION OF HONEY Honey is classified by its floral source, and there are also divisions according to the packaging and processing used. There are also regional honeys. Honey is also graded on its color and optical density by 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.( Tonelli D,1990). 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 & mellisopalynological properties of honey can be used to identify the major plant nectar source used in its production.

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. 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. 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, mesquite and sourwood. Some typical European examples include thyme, thistle, heather, acacia, dandelion, sunflower, honeysuckle, and varieties from lime and chestnut trees.[citation needed] In North Africa (e.g. Egypt) examples include clover, cotton, and citrus (mainly orange blossoms). 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 6065% 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

TYPES OF HONEY BASED ON PROCESSING TECHNIQUES Blended honey is a combination of honey from different floral sourcese.g. alfalfa, wildflowers, clover. Blending is done with the more commonly available and less distinctly flavored honeys to create a common denominator flavor profile for mass-merchandising. The result tends to be sweet and honey without any other flavor characteristic. The opposite of blended honey is varietal honey (see below). Raw honey is unprocessed. It is honey as it exists in the beehive or as obtained by extraction, settling or straining without adding heat (pasteurization). Raw honey contains some pollen and may contain small particles of wax. Raw honey comes out of the comb and go into the bottle: it is one of the purest foods on the table. Strained honey or filtered honey has been passed through a mesh filter to remove particles (pieces of wax, e.g.) without removing pollen. It has a cloudy appearance due to the included pollen, and tends to crystallize more quickly than ultra-filtered honey. Popular with health food buyers. Ultra filtered honey is processed by very fine filtration under high pressure to remove all extraneous solids and pollen grains. Ultra filtered honey is very clear and has a longer shelf life, because it crystallizes more slowly. Preferred by the supermarket trade. Pasteurized honey. Supermarket honey is often pasteurized to help prevent crystallization on the shelf. Crystallization is not harmful; the crystals will dissolve in the microwave (heat for 30 seconds) or in a pan of hot water (10 to 15 minutes). Varietal or monofloral honey comes from a single flower, e.g. orange blossom, lavender, sage. In addition to the flavor of the honey, it will express secondary flavor characteristics of the

lavender, sage, raspberry, et al, and the better honeys will have complex tertiary flavors as well. The opposite of varietal honey is blended honey.

CHEMICAL CHARACTERISTICS OF HONEY pH and Acids pH 3.9 (3.4-6.1) 0.57% (0.17-1.17%)

Acids

(primarily gluconic acid)

Proteins, Amino acids, Isoelectric point Protein Nitrogen Amino acids 0.266% 0.043% 0.05-0.1%

Isoelectric point 4.3 Analysis: Honey is chemically compatible

with a wide variety of products. Its pH and isoelectric point are in a range common to many food systems.

COMPOSITION OF HONEY Honey is essentially a highly concentrated water solution of two sugars, dextrose and levulose, with small amounts of at least 22 other more complex sugars. Many other substances also occur

in honey, but the sugars are by far the major components. The principal physical characteristics and behavior of honey are due to its sugars, but the minor constituents such as flavoring materials, pigments, acids, and minerals are largely responsible for the differences among individual honey types. Honey, as it is found in the hive, is a truly remarkable material, elaborated by bees with floral nectar, and less often with honeydew. Nectar is a thin, easily spoiled sweet liquid that is changed (ripened) by the honey bee to a stable, high-density, high-energy food. The earlier U.S. Food and Drug Act defined honey as the nectar and saccharine exudation of plants, gathered, modified, and stored in the comb by honey bees (Apis mellifera and A. dorsata); is levorotatory; contains not more than 25% water, not more than 0.25% ash, and not more than 8% sucrose. The limits established in this definition were largely based on a survey published in 1908. Today, this definition has an advisory status only, but is not totally correct, as it allows too high a content of water and sucrose, is too low in ash, and makes no mention of honeydew. Colors of honey form a continuous range from very pale yellow through ambers to a darkish red amber to nearly black. The variations are almost entirely due to the plant source of the honey, although climate may modify the color somewhat through the darkening action of heat. The flavor and aroma of honey vary even more than the color. Although there seems to be a characteristic honey flavor, almost an infinite number of aroma and flavor variations can exist. As with color, the variations appear to be governed by the floral source. In general, light-colored honey is mild in flavor and a darker honey has a more pronounced flavor. Exceptions to the rule sometimes endow a light honey with very definite specific flavors. Since flavor and aroma judgments are personal, individual preference will vary, but with the tremendous variety available, everyone should be able to find a favorite honey. By far, the largest portion of the dry matter in honey consists of the sugars. This very concentrated solution of several sugars results in the characteristic physical properties of honey high viscosity, stickiness, high density, granulation tendencies, tendency to absorb moisture from the air, and immunity from some types of spoilage. Because of its unique character and its considerable difference from other sweeteners, chemists have long been interested in its composition and food technologists sometimes have been frustrated in attempts to include honey in prepared food formulas or products. Limitations of methods available to earlier researchers made their results only approximate in regard to the true sugar composition of honey. Although recent research has greatly improved analytical procedures for sugars, even now some

compromises are required to make possible accurate analysis of large numbers of honey samples for sugars.

NUTRIENT: Water Carbohydrates (total) ....Fructose ....Glucose ....Maltose ....Sucrose Proteins, amino acids,

Average in 100g of honey 17.1 g 82.4 g 38.5 g 31.0 g 7.20 g 1.50 g 0.50 g 304 Kcal Amount 100g of honey < 0.006 mg < 0.06 mg < 0.36 mg < 0.11 mg < 0.32 mg 2.2 -2.4 mg

amount

Range (12.2-22.9 g)

(25.2-44.4 g) (24.6-36.9 g) (1.70-11.8 g) (0 50-2.90 g)

vitamins and minerals Energy Vitamins ....Thiamin ....Riboflavin ....Niacin ....Pantothenic acid ....Pyridoxine (B6) ....Ascorbic Acid (C) Minerals ....Calcium ....Copper ....Iron ....Magnesium ....Manganese ....Phoshorus ....Potassium ....Sodium ....Zinc

in

U.S. RDI 1.5 mg 1.7 mg 20.0 mg 10.0 mg 2.0 mg 60.0 mg

4.4-9.20 mg 0.003-0.10 mg 0.06-1.5 mg 1.2-3.50 mg 0.02-0.4 mg 1.9-6.30 mg 13.2-16.8 mg 0.0-7.60 mg 0.03-0.4 mg

1000.0 mg or 1 g 2.0 mg 18.0 mg 400.0 mg

1000.0 mg or 1 g

15.0 mg

CHAPTER THREE MATERIALS AND METHODS 3.1. DETERMINATION OF FRUCTOSE AND GLUCOSE CONTENT IN HONEY BY HPLC ANALYSIS.

Reagents and Apparatus

1. HPLC : Agilent 1200 Series - RID detector - Column: Grace-Davison Prevail CarbohydateES5 of 150mm x 4.6mm i.d. 2. Acetonitrile LC Grade 3. Glucose 4. Fructose 5. Sucrose 6. PTFE and cellulose-acetate membrane filters 7 Honey samples A 75% :25% acetonitrile:ultrapure water mobile phase was prepared at a volume of 1L at a time. The freshly prepared mobile phase was filtered and degassed by vacuum filtration through a 0.45m PTFE membrane filter. A standard stock sucrose solution of approximately 8g/L was prepared and aliquots of this solution were used to make up the required sucrose concentration in the standard solutions. The mixed calibration standards were prepared in the concentration ratios of 1:1:0.2 g/L, 2:2:0.4g/L and 5:5:1 g/L of fructose:glucose:sucrose respectively, by accurately weighing appropriate masses of fructose and glucose and adding accurately pipetted volumes of sucrose stock solution. For the determination of the fructose and glucose content of honey samples solutions of approximately 10g/L were prepared. For recovery check analyses solutions of approximately 5g/L were prepared by accurately weighing approximately 2.5g of honey, adding an accurately weighed mass of glucose of approximately 0.5g and making up to 500ml.

All samples were filtered and degassed by vacuum filtration through cellulose-acetate membrane filters. Before injection onto the column all syringes were fitted with syringe filters. Standards were injected into the HPLC at 0.6ml/min flow rate and 0.9ml/min flow rate to observe the effect Once optimum flow conditions had been established repeated injections of standards were performed to obtain reproducible elution times and peak heights. The average peak heights were used to construct calibration graphs for fructose and glucose. Using the same conditions samples and recovery check samples were injected and calibration graphs were used to determine the fructose and glucose content of the honey samples.

3.2. LANE-EYNON TITRIMETRIC METHOD FOR THE DETERMINATION OF SUGARS: Reagents and Apparatus 1. Fehlings solutions A and B 2. Honey samples 3. Glucose standard solution (0.5%) 4. 0.2% methylene blue indicator A 50ml burette was filled with an accurately prepared standard glucose (0.5%) solution. Fehlings solutions A and B, 200ml of each, were mixed in a beaker. It was calculated that this would be sufficient for the entire analysis to avoid standardization of a fresh batch. A 25ml aliquot of mixed Fehlings was pipetted into a 150ml conical flask and 22ml glucose solution from the burette was added. The mixture was heated to boiling whilst stirring with a magnetic stirrer and maintained at boiling for 2 minutes. Methylene-blue indicator solution (2-3 drops) was added during this time. The titration was completed within a total boiling time of 3 minutes by adding standard glucose, drop-wise, to decolourisation of the indicator. The titrations were repeated until successive titrations agreed within 0.4ml. Approximately 3.37g of each honey sample was made up to 250ml with water in a volumetric flask. This gives a solution of about 10g/L of reducing sugars using the information that honey is around 74% carbohydrate. Pipette 10ml of the sample into a conical flask and add 25ml of the mixed Fehlings solution. The mixture was heated to boiling whilst stirring and maintained at boiling for 2 minutes adding methylene-blue indicator solution. The titration was completed within the

total boiling time of 3 minutes by drop-wise addition of standard glucose solution from the burette. 3.3 LIQUID CHEOMATOGRAPHY Manual syringe (>100 L) was used for injecting the sample on Rheodyne injector with 20 L loop on the system. Column was Waters Radial-Pak silica (7 100 mm) while Cartridges was RCM 8 10 Cartridge Holder. A radial compression column was used in this LC system for sugar analysis, which has several advantageous such as minimize channeling, fluid velocity front due to flexible wall moldable around the packing media and cheaper to replace cartridge then the whole column. Moreover, dynamically close-up voids characteristic of radial compression can be explained due to its decreased void spaces capacity within the bed and by this way eliminates wall effects, which cause channeling and loss of efficiency.

CHAPTER FOUR RECOMMENDATIONS AND CONCLUSION CONCLUSION

RECOMMENDATION Remember that the quality of honey is a function of the plants and environment from which pollen, saps, nectars and resins were gathered. Other substances found in the environmentincluding traces of heavy metals, pesticides, and antibioticshave been shown to appear in honey. The amount varies greatly. Do not feed honey-containing products or use honey as a flavoring for infants under one year of age; honey may contain Clostridium botulinum spores and toxins that can cause infant botulism, a life-threatening paralytic disease. Honey is safe for children older than 12 months and adults. Although our food ranking system did not qualify honey as a dense source of traditional nutrients, it did emerge as a source of vitamin B2, vitamin B6, iron and manganese.

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