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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 1–5% combined working standard of glucose, fructose and sucrose and using HPLC analysis and Lane-Eynon titrimetric method

6 (L. which can be dangerous to infants. the honey can be analysed by the techniques of melissopalynology in area environmental studies of radioactive particles. transform by combining with specific substances of their own. They store it as a primary food source in wax honeycombs inside the beehive. Prescott et al. . Honey gets its sweetness from the monosaccharides fructose and glucose. Honey bees transform nectar into honey by a process of regurgitation and evaporation. Honey produced by other bees and insects has distinctly different properties. The study of pollens and spores in raw honey (melissopalynology) can determine floral sources of honey (Vaughn. Honey is a sweet food made by bees using nectar from flowers.( Shapiro et al. and various types and grades of honey are available. It has attractive chemical properties for baking and a distinctive flavor that leads some people to prefer it over sugar and other sweeteners. and is used in various foods and beverages as a sweetener and flavoring. honey sometimes contains dormant endospores of the bacterium Clostridium botulinum. 1999) However. dehydrate. as the endospores can transform into toxin-producing bacteria in infants' immature intestinal tracts. Bees carry an electrostatic charge whereby they attract other particles in addition to pollen. dust and particulate pollution. Flavors of honey vary based on the nectar source. store and leave in the honey comb to ripen and mature. The variety produced by honey bees (the genus Apis) is the one most commonly referred to. and has approximately the same relative sweetness as that of granulated sugar. Most microorganisms do not grow in honey because of its low water activity of 0. which the bees collect. It also has a role in religion and symbolism. It is also used in various medicinal traditions to treat ailments. leading to illness and even death. deposit. 2001). as it is the type of honey collected by beekeepers and consumed by humans.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 become incorporated into their honey. 1998) Honey has a long history of human consumption.

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 .

too – along for the trip were linden. Honey is frequently used as a talisman and symbol of sweetness. showing two honey-hunters collecting honey and honeycomb from a wild bee nest.CHAPTER TWO LITERATURE REVIEW HISTORY OF HONEY Honey use and production has a long and varied history. and references to its use in food can be found in the work of many Roman authors including Athenaeus. In many cultures. berry. Pliny the Elder devotes considerable space in his book Naturalis Historia to the bee and honey. Cato and Bassus. honey has associations that go beyond its use as a food. the oldest remains of honey have been found in Georgia. The painting is a Mesolithic rock painting. was offered honey. In the book "Golden Rules of Business Success" written by Fan Li (or Tao Zhu Gong) during the Spring and Autumn Period. The art of beekeeping in ancient China has existed since time immemorial and appears to be untraceable to its origin. Min.700–5. The fertility god of Egypt. In ancient Egypt. In ancient Georgia. Humans apparently began hunting for honey at least 8. 1983).500 years ago. Honey was an integral sweetening ingredient in Roman recipes. honey was used to sweeten cakes and biscuits. and was used in many other dishes.000 years ago. Archaeologists have found honey remains on the inner surface of clay vessels unearthed an ancient tomb. Spain (Crane. dating back to some 4. Some of these are collected in the book Roman cookery. and using a ladder or series of ropes to reach the wild nest. and a meadowflower variety. In the absence of sugar. The Greater Honeyguide bird guides humans to wild bee hives and this behavior may have evolved with early hominids So far. . as evidenced by a cave painting in Valencia. Honey collection is an ancient activity. and its many uses. The figures are depicted carrying baskets or gourds. And more than one type. 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 packed for people's journeys into the afterlife. Ancient Egyptian and Middle Eastern peoples also used honey for embalming the dead.

creme honey (also known as spun honey.1990). FORMS OF HONEY Honey can be found in a variety of forms. In many countries around the world. Most of the honey produced in the United States is sold in the liquid form. is edible! Cut Comb Cut comb honey is honey that has been packaged with chunks of the honey comb. It was used as an ointment for rashes and burns. This include liquid honey. crème honey and cut comb honey Liquid Honey Free of visible crystals. Honey is also graded on its color and optical density by USDA standards.( Tonelli D. and to help soothe sore throats when no other practices were available. Creme (or Spun) Honey While all honey will crystallize in time. Floral source . graded on a scale called the Pfund scale. liquid honey is extracted from the honey comb by centrifugal force. The comb. Comb Honey Comb honey is honey that comes as it was produced--in the honey bees' wax comb. The crystallization is controlled so that. The Maya also regard the bee as sacred (see Mayan stingless bees of Central America). which ranges from 0 for "water white" honey to more than 114 for "dark amber" honey. Some cultures believed honey had many practical health uses. it's especially convenient for cooking and baking. There are also regional honeys. gravity or straining. comb honey. CLASSIFICATION OF HONEY Honey is classified by its floral source. Because liquid honey mixes easily into a variety of foods. and continue to do so today. as well as the honey.Honey was also cultivated in ancient Mesoamerica. sugared honey or whipped honey) is brought to market in a crystallized state. at room temperature. creme honey is preferred to the liquid form. The Maya used honey from the stingless bee for culinary purposes. the honey can be spread like butter. and there are also divisions according to the packaging and processing used.

Generally. Honeydew honey is very dark brown in color. mesquite and sourwood. Some typical European examples include thyme. blueberry. heather. To produce monofloral honey. thistle. Honeydew honey is . In Greece. Honeys can be from specific types of flower nectars or can be blended after collection. a small proportion of any honey will be from additional nectar from other flower types. as well as some regions in Bulgaria. because of the difficulties in containing bees.[citation needed] In North Africa (e. also known as wildflower honey. buckwheat. The pollen in honey is traceable to floral source and therefore region of origin. The taste may vary from year to year. fireweed. the sweet secretions of aphids or other plant sap-sucking insects. bees can take honeydew. honey is classified by the floral source of the nectar from which it was made. orange blossom. Egypt) examples include clover. is derived from the nectar of many types of flowers. flavor. density or geographic origin. and the aroma and the flavor can be more or less intense. Different monofloral honeys have a distinctive flavor and color because of differences between their principal nectar sources.g. depending on which bloomings are prevalent. Typical examples of North American monofloral honeys are clover. and is not as sweet as nectar honeys. In practice. The rheological & mellisopalynological properties of honey can be used to identify the major plant nectar source used in its production. with a rich fragrance of stewed fruit or fig jam. Honeydew honey Instead of taking nectar. Germany's Black Forest is a well known source of honeydew-based honeys. Blended Most commercially available honey is blended. honeysuckle. beekeepers keep beehives in an area where the bees have access to only one type of flower. color. cotton. and citrus (mainly orange blossoms). Polyfloral Polyfloral honey. pine honey (a type of honeydew honey) constitutes 60–65% of the annual honey production. tupelo. Monofloral Monofloral honey is made primarily from the nectar of one type of flower. acacia. Tara (mountain) in Serbia and Northern California in the United States. dandelion. and varieties from lime and chestnut trees. sage. meaning it is a mixture of two or more honeys differing in floral source. sunflower.

Bees collecting this resource also have to be fed protein supplements. Varietal or monofloral honey comes from a single flower.) without removing pollen. because it crystallizes more slowly. Preferred by the supermarket trade. The opposite of blended honey is varietal honey (see below). It has a cloudy appearance due to the included pollen. e. and tends to crystallize more quickly than ultra-filtered honey. resulting in the death of colonies in areas with cold winters. Strained honey or filtered honey has been passed through a mesh filter to remove particles (pieces of wax. Ultra filtered honey is very clear and has a longer shelf life. clover. alfalfa. The honey has a much larger proportion of indigestibles than light floral honeys. the crystals will dissolve in the microwave (heat for 30 seconds) or in a pan of hot water (10 to 15 minutes). Ultra filtered honey is processed by very fine filtration under high pressure to remove all extraneous solids and pollen grains. Popular with health food buyers. but in other areas beekeepers have difficulty selling the stronger flavored product. lavender. In addition to the flavor of the honey. Good beekeeping management requires the removal of honeydew prior to winter in colder areas. sage. it will express secondary flavor characteristics of the . Crystallization is not harmful. Raw honey comes out of the comb and go into the bottle: it is one of the purest foods on the table. wildflowers. 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 sources—e. Pasteurized honey. Supermarket honey is often pasteurized to help prevent crystallization on the shelf.g. orange blossom. Blending is done with the more commonly available and less distinctly flavored honeys to create a common denominator flavor profile for mass-merchandising. settling or straining without adding heat (pasteurization). Raw honey is unprocessed. Raw honey contains some pollen and may contain small particles of wax.g. e. thus causing dysentery to the bees.g.popular in some areas. The production of honeydew honey has some complications and dangers. The result tends to be ―sweet‖ and ―honey‖ without any other flavor characteristic. It is honey as it exists in the beehive or as obtained by extraction.

043% 0. CHEMICAL CHARACTERISTICS OF HONEY pH and Acids pH 3. et al.1) 0. The opposite of varietal honey is blended honey.9 (3.05-0. with small amounts of at least 22 other more complex sugars. Its pH and isoelectric point are in a range common to many food systems.57% (0.17-1. dextrose and levulose.4-6. sage.3 Analysis: Honey is chemically compatible with a wide variety of products. Amino acids. and the better honeys will have complex tertiary flavors as well. COMPOSITION OF HONEY Honey is essentially a highly concentrated water solution of two sugars. Isoelectric point Protein Nitrogen Amino acids 0.1% Isoelectric point 4.lavender. Many other substances also occur .17%) Acids (primarily gluconic acid) Proteins. raspberry.266% 0.

easily spoiled sweet liquid that is changed (―ripened‖) by the honey bee to a stable.25% ash. although climate may modify the color somewhat through the darkening action of heat. Although recent research has greatly improved analytical procedures for sugars. modified. individual preference will vary. This very concentrated solution of several sugars results in the characteristic physical properties of honey – high viscosity. and stored in the comb by honey bees (Apis mellifera and A. contains not more than 25% water. Food and Drug Act defined honey as ―the nectar and saccharine exudation of plants. is levorotatory. and immunity from some types of spoilage. and less often with honeydew. tendency to absorb moisture from the air. this definition has an advisory status only. Colors of honey form a continuous range from very pale yellow through ambers to a darkish red amber to nearly black. everyone should be able to find a favorite honey. Limitations of methods available to earlier researchers made their results only approximate in regard to the true sugar composition of honey. high-density. acids. 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.‖ almost an infinite number of aroma and flavor variations can exist. pigments. Exceptions to the rule sometimes endow a light honey with very definite specific flavors. The flavor and aroma of honey vary even more than the color.‖ high density.S. light-colored honey is mild in flavor and a darker honey has a more pronounced flavor. By far. but the minor constituents – such as flavoring materials. but with the tremendous variety available. elaborated by bees with floral nectar. gathered. and makes no mention of honeydew. is too low in ash. The principal physical characteristics and behavior of honey are due to its sugars.in honey. as it is found in the hive.‖ The limits established in this definition were largely based on a survey published in 1908. even now some . Because of its unique character and its considerable difference from other sweeteners. but is not totally correct. Since flavor and aroma judgments are personal. In general. As with color. Honey. not more than 0. The earlier U. the largest portion of the dry matter in honey consists of the sugars. Nectar is a thin. is a truly remarkable material. high-energy food. as it allows too high a content of water and sucrose. ―stickiness. Today. but the sugars are by far the major components. dorsata). and minerals – are largely responsible for the differences among individual honey types. and not more than 8% sucrose. the variations appear to be governed by the floral source. granulation tendencies. Although there seems to be a characteristic ―honey flavor. The variations are almost entirely due to the plant source of the honey.

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

11 mg < 0.003-0. RDI 1.0 g 7..32 mg 2.20 mg 0...Phoshorus ...2-22.Pantothenic acid .50 mg 0.50 g 304 Kcal Amount 100g of honey < 0.7 mg 20.5 mg 1..2-44.06 mg < 0.2 -2...8 mg 0.Glucose .4 mg 1000.8 g) (0 50-2...20 g 1.10 mg 0....Riboflavin ...006 mg < 0.Calcium ..90 g) vitamins and minerals Energy Vitamins .0 mg 60.....Potassium .....Manganese .4 mg 1.Zinc in U.9 g) (1..0 mg or 1 g 2.02-0.Pyridoxine (B6) ..Sodium .4 mg amount Range (12...0 mg or 1 g 15..4 g 38....6-36..60 mg 0.0 mg ..1 g 82.Iron .Niacin ...30 mg 13...Ascorbic Acid (C) Minerals .0 mg 1000..5 g 31..0 mg 2......9 g) (25.Magnesium . Average in 100g of honey 17...NUTRIENT: Water Carbohydrates (total) .0 mg 4.S.50 g 0.70-11. amino acids..Thiamin .06-1.4-9..36 mg < 0.Fructose .0-7.0 mg 10.5 mg 1....0 mg 18.4 g) (24.03-0.Maltose ..Sucrose Proteins.Copper .0 mg 400.2-16.9-6...2-3...

Fructose 5. Acetonitrile LC Grade 3. The mixed calibration standards were prepared in the concentration ratios of 1:1:0. Sucrose 6. .1. For the determination of the fructose and glucose content of honey samples solutions of approximately 10g/L were prepared.5g and making up to 500ml.d.45μm PTFE membrane filter. by accurately weighing appropriate masses of fructose and glucose and adding accurately pipetted volumes of sucrose stock solution. For recovery check analyses solutions of approximately 5g/L were prepared by accurately weighing approximately 2. HPLC : Agilent 1200 Series . 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 0.4g/L and 5:5:1 g/L of fructose:glucose:sucrose respectively.CHAPTER THREE MATERIALS AND METHODS 3. adding an accurately weighed mass of glucose of approximately 0. 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. DETERMINATION OF FRUCTOSE AND GLUCOSE CONTENT IN HONEY BY HPLC ANALYSIS.RID detector . Reagents and Apparatus 1.5g of honey.2 g/L. 2. 2:2:0.Column: Grace-Davison Prevail CarbohydateES5μ of 150mm x 4.6mm i. Glucose 4.

The titrations were repeated until successive titrations agreed within 0. Pipette 10ml of the sample into a conical flask and add 25ml of the mixed Fehling’s solution.5%) solution. LANE-EYNON TITRIMETRIC METHOD FOR THE DETERMINATION OF SUGARS: Reagents and Apparatus 1. Methylene-blue indicator solution (2-3 drops) was added during this time. to decolourisation of the indicator.37g of each honey sample was made up to 250ml with water in a volumetric flask. The mixture was heated to boiling whilst stirring with a magnetic stirrer and maintained at boiling for 2 minutes. 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. Approximately 3. were mixed in a beaker.4ml. Glucose standard solution (0.All samples were filtered and degassed by vacuum filtration through cellulose-acetate membrane filters.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. Honey samples 3. Before injection onto the column all syringes were fitted with syringe filters.2. Fehling’s solutions A and B. Fehling’s solutions A and B 2. The average peak heights were used to construct calibration graphs for fructose and glucose. Standards were injected into the HPLC at 0. The titration was completed within a total boiling time of 3 minutes by adding standard glucose. 3. It was calculated that this would be sufficient for the entire analysis to avoid standardization of a fresh batch.2% methylene blue indicator A 50ml burette was filled with an accurately prepared standard glucose (0. The titration was completed within the .5%) 4.6ml/min flow rate and 0. This gives a solution of about 10g/L of reducing sugars using the information that honey is around 74% carbohydrate. drop-wise. 0. A 25ml aliquot of mixed Fehling’s was pipetted into a 150ml conical flask and 22ml glucose solution from the burette was added. The mixture was heated to boiling whilst stirring and maintained at boiling for 2 minutes adding methylene-blue indicator solution. 200ml of each.

Moreover. which cause channeling and loss of efficiency. . which has several advantageous such as minimize channeling. A radial compression column was used in this LC system for sugar analysis.total boiling time of 3 minutes by drop-wise addition of standard glucose solution from the burette. 3. 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‖.3 LIQUID CHEOMATOGRAPHY Manual syringe (>100 μL) was used for injecting the sample on Rheodyne injector with 20 μL loop on the system. Column was Waters Radial-Pak silica (7 × 100 mm) while Cartridges was RCM 8 × 10 Cartridge Holder. fluid velocity front due to flexible wall moldable around the packing media and cheaper to replace cartridge then the whole column.

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

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