2.2 HONEY THE SIMPLE SUGAR

Transcription

1 PRACTICAL 2 HONEY Structure 2.1 Introduction 2.2 Honey The Simple Sugar 2.3 Honey Characteristics, Specifications and Requirements 2.4 Physical Criteria for Quality 2.5 Marking Activity 1: Determination of Total Reducing Sugar Activity 2: Determination of Sucrose Content Activity 3: Detection of Adulteration Activity 4: Determination of Fructose to Glucose Ratio Activity 5: Determination of Acidity 2.1 INTRODUCTION The first Unit in the theory course (MFN-008) focused on simple sugars. In the unit, you may recall studying that foods made from sugar, as well as, corn syrup, honey and molasses are simple carbohydrates. The second practical in this manual focuses on the study of honey, which is a simple sugar. Honey comes under the purview of Prevention of Food Adulteration Act (PFA). We shall learn about the characteristics of honey and the physical criteria for quality of honey. This practical will help you check the conformance of any sample of honey to the standards. Objectives After studying this practical and undertaking the activities included in this practical, you will be able to: enlist the honey characteristics which are basic for a good quality honey, estimate the reducing and non-reducing sugars present in honey, determine the fructose to glucose ratio in honey, detect adulteration of honey with commercial sugars, and check the conformance of a honey sample to the standard. 2.2 HONEY THE SIMPLE SUGAR Honey is the oldest sweet food known to man. Although honey is a natural sweetener, it is considered a refined sugar because 96% of dry matter is simple sugars: fructose, glucose and sucrose. Honey has the highest calorie content of all sugars with 65 calories/tablespoon, compared to the 48 calories/tablespoon found in table sugar. Honey is produced by honey bees from nectar of plants, as well as, from honey dew. Among the components of honey (sugars, water, organic acids, enzymes, amino acids, wax, pigments and pollen etc.), some of the components are derived from plants, some are added by the bees and some are due to maturation of honey. The honey marketed in India falls under two categories: 1) Extracted Apiary Honey 2) Squeezed Honey Bulk of squeezed honey is obtained from Apis Dorsata, the rock bee, and bulk of the extracted honey is obtained by Apis Serana Indica (Honey bee). We shall get to know about the basic characteristics, specifications and requirements of honey next. 20

2 2.3 HONEY CHARACTERISTICS, SPECIFICATIONS AND REQUIREMENTS Honey comes under the purview of Prevention of Food Adulteration Act (PFA). This Indian Standard was adopted by ISI on 30 th September, 1974 after the draft finalized by the Apiary Industry Sectional Committee had been approved by the Agricultural and Food Product Division Council (AFPDC). Due to its limited production and high cost, honey is prone to adulteration by cane sugar, invert syrup and high fructose glucose syrup. While preparing this specification, the committee recognized the need to market squeezed and apiary honey separately so that there can be growth of consumer demand of apiary honey, which is reared scientifically and processed hygienically. The present revision of the standard incorporates a number of modifications, which include: a) The scope of the standard has been restricted to extracted honey. b) Limit of total reducing sugar has been increased for standard grade. c) Limit of sucrose content has been reduced for A grades and standard grades. d) Method of glucose fructose ratio content has been modified to make it more precise. So far, no separate standard has been issued on squeezed honey. While compiling the revision, the Apiary Industry Sectional Committee (AISC) felt that squeezed honey collected from jungles is being sold at a much lower price, further it was possible to distinguish it from apiary honey which has much less number of pollens. The Central Bee Keeping Research Institute is conducting investigation on the method of quantitative estimation for pollens and its limits in Apiary and squeezed honey. Subsequently, a separate standard would be issued and has been issued on squeezed honey, while framing these specification consideration have been given to the prevailing trade practices and the different grades prescribed by the Agricultural marketing Advisor to the Government of India for incorporation of Agmark rules framed under the Agricultural procedure for grading and marketing. This standard is also subject to the restriction imposed under the Prevention of Food Adulteration Act 1954 and rules framed there under whenever applicable. This standard prescribes the requirements and methods of sampling and testing for extracted in Apiary honey obtained from Apis Serana indica. Honey shall be of the following grades: a) Special b) Grade A c) Standard Table 2.1 gives the characteristics and specifications for honey. Table 2.1: Characteristics and specifications of honey S.No Characteristics ISI-4941, 1974 PFA Act, rule A07.03d Grade Special A Standard 1. Specific gravity at 27º C minimum Moisture % by mass (maximum) Total reducing sugar % by mass (minimum) 4. Sucrose % by mass (maximum) Fructose glucose ratio (minimum) Ash % by mass (maximum) Acidity expressed as formic acid, % by mass (maximum) 8. Fiehe s Test -ve -ve -ve -ve 9 Aniline chloride Test -ve -ve -ve - Honey 21

3 Principles of Food Science According to PFA Act, 1954 and the rules in 1955, honey has been defined as the food derived entirely from the work of bees operating upon the nectar of flowers and other sweet exudation of plants. IS specifications have been laid down as given herewith to prescribe the requirements of honey, which are as follows: A) General Characteristics 1) It should be a well-ripened natural product. 2) It should be clear and visually transparent. 3) It should be extracted with the help of an extractor. 4) It should free from objectionable flavour due to over heating, fermentation and smoke. 5) It should have been strained clean through double thickness of cheese cloth about 150 µ at a temperature not exceeding 70º C. 6) Freedom from foreign matter: When visually inspected honey shall be free from foreign matter such as dirt, mould, scum, piece of beeswax, fragments of bees and other insects and from any other extraneous matter. 7) Colour: Colour of honey shall be uniform throughout and may vary from light to dark brown. 8) Packaging and Marketing: Honey shall be packed in hygienically clean wide mouthed glass containers, or in acid-resistant lacquered tin containers or in suitable polyethylene containers. The screwed saps of glass containers shall be non-corrosive and non-reacting material to honey and shall be provided with cork washes to avoid spilling. Having studied about the characteristics, specifications and requirements, let us next look at the physical criteria for quality of honey. 2.4 PHYSICAL CRITERIA FOR QUALITY Colour, crystallization, ph, acidity, water content are some of the criteria used for analysis of honey. These criteria are described next. A) Colour The colour of the honey varies from straw-yellow to nearly black according to its botanical source and to conditions of processing and storage it has undergone. Light coloured honey typically has a mild flavour, while dark coloured honey is usually stronger in flavour. Blended honey is normally graded by colour, the lighter the colour, the higher the quality and value. B) Crystallization At normal temperatures honey exists as clear syrup preferred by consumers. However, on storage, coarse granulation or crystallization can occur, which is a natural process that occurs in honey. Honey is a supersaturated sugar solution out of which the glucose tends to crystallize. The tendency of honey to granulate depends on glucose/water ratio. At a ratio of 2:1, it granulates rapidly, whereas, honeys with ratio of 1.7:1 or less tend to remain liquid. Crystallization is most rapid at 14º C (57º F) and can be revered by heating. C) Sugars 22 The major sugars present in honey are fructose, glucose, followed by lower concentration of sucrose and maltose. 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 1.1:1. Other sugars found in small amounts in honey are isomaltose, nigerose, kojibiose, turanose, gentibiose and laminaribose.

4 To assess the quality of honey, total reducing sugars and sucrose content is most commonly determined. Lane and Eynon method is used for determination of sugars. The total reducing sugars are estimated by titration using Fehling A and B solutions, whereas, sucrose is determined indirectly by calculating difference in total reducing sugar before and after inversion of sugars in honey. Invert sugar reduces the copper in Fehling s solution to red, insoluble cuprous oxide. The sugar content of the food sample is estimated by determining the volume of the unknown sugar solution required to completely reduce a measured volume of Fehling s solution. However, more recently this method is replaced by measurement of specific sugars by high performance liquid chromatography equipped with refractive index director. As per the Indian standards, honey should not contain reducing sugars less than 65% and sucrose more than 5%. Value of sucrose higher than this indicates bees might be fed artificially with sugar or direct addition of sugar, to earn more profits. Its is interesting to note that enzymes present in honey cause changes in the proportions of the original sugars present and the sucrose may disappear completely during prolonged storage. Honey The principle behind the Lane and Eynon method is discussed next. Lane and Eynon method: Principle Reducing sugars are those, which have free sugar groups (e.g. glucose, fructose etc.) and hence may be estimated directly by titrating the solution of the sample with Fehling s solution. You may recall reading about this property of sugars in unit 1 in the Nutritional Biochemistry Thoery Course (MFN-002). Total sugars include both reducing and non-reducing sugars. Non-reducing sugars (e.g. maltose, lactose, sucrose etc.) do not contain free sugar groups and cannot reduce Fehling solution. Hence, non-reducing sugars must be hydrolyzed to monosaccharides by heating with acid before titration. Reducing sugars are acted upon by the alkali of the Fehling solution to form enediols. These enediols are very unstable and reactive and they reduce Cu² + ions to Cu + ions. These Cu + ions combine with hydroxyl groups to form cuprous hydroxide, which on heating gives red precipitate of cuprous oxide. To get a sharp end point, methylene blue is added which is reduced to a colourless leuco compound restoring the red colour of the solution. Sodium potassium tartarate keeps the Cu² + ions in the solution, thus ensuring a continuous supply of Cu² + ions for reduction. Reactions involved in the estimation of Reducing sugars and Non-Reducing sugars are illustrated next. Reaction for Reducing Sugars 23

5 Principles of Food Science Reaction for Non-Reducing Sugars 24 D) Total Solids and Water Most genuine honeys contain per cent of water. The total solids or moisture can be estimated by drying in a vacuum oven at 70ºC. Alternatively, it can also be determined by measuring refractive index at 40ºC or by measuring the specific gravity of 20% (m/v) solution of honey. Tables relating the refractive index of honey with water content are available in literature. E) Ash Standards allow ash content in honey up to 0.5 per cent, but the ash of genuine honey seldom exceeds 0.35 per cent. It is determined by charring, preferably under an infrared lamp followed by ashing at 600ºC and very recently, this measurement is replaced by measurement of electric conductivity. This measurement depends on the ash and acid content of honey the higher their content, the higher the resulting conductivity. Extensive conductivity data published on thousands of commercial honeys suggest that blossom honeys, mixtures of blossom and honey dew honeys should have conductivity less than 0.8 milli Siemens/cm. F) ph and Acidity The ph of natural honey ranges from 3.4 to 6.1. Acidity of honey is primarily due to presence of acids such as gluconic acid, pyruvic acid, malic acid, citric acid and succinic acid. Acidity of fresh honey is usually very low, 13 to 35 meq/kg. Honey with acidity more than 40 meq/kg is considered as poor in quality. Acidity is determined by titration of a known weight of honey with 0.1M NaOH. Finally, let us look at the marking regulations for honey. 2.5 MARKING The marking regulations indicate that each container of honey shall be suitably marked so as to give suitable information as follows:

6 a) Name of the material and grade designation Honey b) Name of the packer c) Batch or code no. d) Net weight Honey is the nectar of flowers that is collected, modified and concentrated by honey bee. It contains 75 % glucose and fructose and 2% or more sucrose. As defined by the Food and Drug Administration, honey may not contain more than 8% sucrose - a higher % is taken as an indication of adulteration by added sucrose. Honey is one material food product, which contains more fructose than glucose. The relative amounts being 40.5% of fructose and 34.5% of glucose. Flavours of honey differ according to the characteristics esters present in the nectar of different flowers. Honeys also come from orange and other citrus blossom, wild sage, cultivated buckwheat and tulip tree. The colour of honey may vary from white to amber or darker graded or qualities of honey are independent of colour but darker coloured honey generally has a stronger flavour than the light coloured ones. It is also more acid, which has some significance in the use of soda to neutralize the acidity of honey used as a partial substitute for sugar in flavour mixture. A process has been developed for producing dried honey. The product has colour and flavour quite close to that of original honey. It has granular form, is free flowing and has a longer shelf life. It may have sucrose added for the purpose of raising the temperature at which the dried product will soften thus making it more resistant to caking at high temperature. With the basic understanding about the characteristics, specifications, requirements and criteria of quality for honey, let us now carry out the activities 1-5 given herewith. 25

7 Principles of Food Science ACTIVITY 1 DETERMINATION OF TOTAL REDUCING SUGAR Date:. Aim: To determine total reducing sugars in the given sample of honey. Objectives This activity will help you to: estimate reducing sugars in a sample of honey, check the conformance or non-conformance of the samples to the standards, and detect adulteration of honey with commercial sugars. Principle Now, write the principle regarding the estimation of reducing sugars by Lane and Eynon method as studied in section 2.4 above. Reaction Involved (Write the reaction given by reducing sugar in the space provided). 26

8 Reagents Required 1) Standard invert sugar solution: Weigh accurately g of sucrose and dissolve in 500 ml of water. Add 2 ml of concentrated H 2 SO 4. Boil gently for 30 minutes and keep aside for 24 hours. Neutralize this with Na 2 CO 3 and make the final volume to 1000 ml. 30 ml of this solution contains 0.05 g of invert sugar. 2) Fehling A and Fehling B 3) Methylene blue indicator. Materials Required Sample of honey Burette Pipette Conical flask Beaker Distilled water Procedure You will be carrying out the procedure in two steps using Lane Eynon Method. Step 1 It involves the standardization of copper sulphate solution. The procedure for this standardization can be conducted in the underlined manner as: 1) Pipette accurately 5 ml of Solution A and Solution B in conical flask of 250 ml capacity. 2) Heat this mixture to boiling on an asbestos gauge and add standard invert sugar solution from the burette about 1ml less than the expected volume which will reduce the Fehling solution of say, 48 ml. 3) Add 1 ml methylene blue indicator. 4) Carry out the titration and complete it within 3 minutes. 5) The change in blue to reddish brown colour due to cuprous oxide formation is taken as the end point. 6) From the volume of the invert sugar solution used, the strength of CuSO 4 is calculated by multiplying the titrated value with (mg/ml of the standard invert sugar solution). This is known as Fehling factor. Note: Carry out the titration till you get consecutive titre value. This means you may have to repeat the titration 3-4 times till you get the same result. Step 2 It involves the titration of the sample honey solution wherein the steps can be followed as given below: 1) Place accurately 1 gm of honey solution in 250 ml of volumetric flask. 2) Dilute with about 150 ml of water. 3) Mix thoroughly contents of glass and make the volume to 250 ml. 4) In another conical flask, add 5 ml of Fehling A + 5 ml of Fehling B. 5) Heat to boiling with 20 ml of water. 6) From burette add honey solution (approx 40 ml) and boil. 7) Add methylene blue indicator do the titration within 3 minutes. 8) Carry out the titration till blue colour changes to red. 9) Now calculate the reducing sugar using the calculation given next. Honey 27

9 Principles of Food Science Calculations: Reducing sugar = where, S H M S = Fehling s factor (as obtained from standardization procedure of CuSO4 undertaken in step 1 of the procedure earlier). Strength of CuSO4 solution / Fehling factor (S) is calculated as: Titre value of standard invert sugar solution H = Volume of honey solution required (burette reading) M = Mass of honey Precautions 1) Each titration should be completed within three minutes. 2) Maintain continuous evolution of steam to prevent reoxidation of Cu² + ions. Results and Observations Record your observations in the format below according to the procedure you followed in step 1. Standard invert sugar solution Burette reading (ml) S. No. Initial Final Difference Pilot Strength of CuSO4 solution / Fehling factor (S) is: Titre value 0.001=.. Solution of Honey Titre value =. Record your observations in the format below according to the procedure you followed in step 1. Burette reading (ml) S. No. Initial Final Difference Pilot Titre value =.. 28

10 H (volume of honey solution required) =.. ml Honey M (mass of honey taken for preparation of the solution) = g Putting the values in the formula, we get Reducing sugar = S H M Inference and Conclusion Total reducing sugar in given honey sample (% by mass) was found to be The given sample of honey according to BIS falls under grade*. * (Look up the specifications given in Table 2.1 for comparison). Submit the activity for evaluation. Counsellor Signature 29

11 Principles of Food Science ACTIVITY 2 DETERMINATION OF SUCROSE CONTENT Date:. Aim: To determine the sucrose content in the given sample of honey. Objectives This activity will help you to: estimate the non-reducing sugars and total sugars in a sample of honey, check the conformance or non-conformance of the samples to the standards, and detect adulteration of honey with commercial sugars. Principle Write down the principle in the space provided herewith. You have already studied the principle in section 2.4. Reaction Involved (Write the reaction of non-reducing sugars in the space provided) 30

12 Reagents Required 1) Standard invert sugar solution: Weigh accurately g of sucrose and dissolve in 500 ml of water. Add 2 ml of conc. H 2 SO 4. Boil gently for 30 minutes and keep aside for 24 hours. Neutralize this with Na 2 CO 3 and make the final volume to 1000 ml. 30 ml of this solution contains 0.05 g of invert sugar. 2) Fehling A and Fehling B 3) Methylene blue indicator. 4) 0.4 N HCl 5) 0.6 N NaOH Materials Required Burette Pipette Conical flask Beaker Distilled water Procedure The procedure (method of estimation is Lane Eynon method as studied in section 2.4) involving the estimation of non-reducing sugars will be carried out in 3 steps. Step 1: Acid Hydrolysis of Non-Reducing Sugar It involves the acid hydrolysis of non-reducing sugars to reducing sugars. It is also known as the process of inversion. Acid hydrolysis results in breakdown of sucrose into glucose and fructose. The process of inversion can be carried out in the following manner: 1) Place accurately 1 gm of honey solution in 250 ml of volumetric flask. 2) Dilute with about 150 ml of water. 3) Mix thoroughly contents of glass and make the volume to 250 ml. 4) Take 100 ml of this solution and add 6 ml of 0.04 N HCl. 5) Heat the solution to boiling. 6) Keep for ½ an hour. 7) Neutralize this inverted honey solution with 0.6N NaOH. Step 2: Standardization of Copper Sulphate Solution It involves the standardization of copper sulphate solution. This is same as done in the previous activity. 1) Pipette accurately 5 ml of Solution A and Solution B in conical flask of 250 ml capacity. 2) Heat this mixture to boiling on an asbestos gauge and add standard invert sugar solution from the burette about 1 ml less than the expected volume which will reduce the Fehling solution of say, 48 ml. 3) Add 1 ml methylene blue indicator. 4) Carry out the titration and complete it within 3 minutes. 5) The change in blue to reddish brown colour due to cuprous oxide formation is taken as the end point. 6) From the volume of the invert sugar solution used, the strength of CuSO4 is calculated by multiplying the titrated value with (mg / ml of the standard invert sugar solution). This is known as Fehling factor. Honey 31

13 Principles of Food Science Note: carry out the titration till you get consecutive titre value. This means you may have to repeat the titration 3-4 times till you get the same result. Step 3: Titration of inverted honey solution It involves the titration of the inverted honey solution (sample obtained after carrying out step 1). Carry out the titration in the following manner. 1) In a conical flask take 5 ml of Fehling A+5 ml of Fehling B. 2) From burette add the inverted honey solution (approx 10 ml) and boil. 3) Add methylene blue indicator do the titration within 3 minutes. 4) Carry out the titration till blue colour changes to red. 5) Calculate total sugar using the formula given next. Calculations Total sugars = S H M where, S = Fehling s factor (as obtained from standardization procedure of CuSO4 undertaken in step 2 of the procedure earlier). Fehling factor (S) is calculated as: Titre value of standard invert sugar solution H = Volume of inverted honey solution required (burette reading) M = Mass of honey Sucrose (% by mass) = (Total sugars reducing sugar) sucrose factor where, sucrose factor is 0.95 and reducing sugar value is taken as calculated in Activity 1. Precautions 1) Each titration should be completed within three minutes 2) Maintain continuous evolution of steam to prevent reoxidation of Cu 2+ ions. Results and Observations Record your observations in the format below according to the procedure you followed in step 2 above. Standard invert sugar solution Burette reading (ml) S. No. Initial Final Difference Pilot Strength of CuSO 4 solution / Fehling factor (S) is Titre value 0.001= Solution of Honey (inverted) Titre value =. 32 Record your observations in the format below according to the procedure you followed in step 3.

14 Burette reading (ml) Honey S. No. Initial Final Difference Pilot Honey solution required = H (volume of honey solution required) =... ml M (mass of honey taken for preparation of the solution) =.. g Putting the values in the formula, we get Total sugars = S H M Sucrose (% by mass) = (Total sugars reducing sugar) sucrose factor where, sucrose factor is 0.95 and take reducing sugar value as calculated in Activity 1. Sucrose (% by mass) for the given honey sample is... Inference Total reducing sugar in given honey sample (% by mass) was found to be.. Total sugars in the given sample of honey was found to be. Sucrose percentage by mass was found to be... The given sample of honey conforms / does not conform to the specification laid down and falls under. grade. Submit the activity for evaluation. Counsellor Signature 33

15 Principles of Food Science ACTIVITY 3 DETECTION OF ADULTERATION Date:. 34 Aim: To determine the adulteration in the given honey sample by Fiehe s test and Aniline chloride test. Objectives This activity will help you to: check the given samples of honey for any adulteration with commercial sugars, and check the conformance or non-conformance of the samples to the standards. Principle The major quality factor in honey is the indicator of honey freshness and overheating. Hydroxy Methyl Furfural (HMF) occurs in honey due to acid-catalyzed dehydration of hexose sugars. Its value in natural fresh honey varies from 10 to 14 mg/ kg, but it increases upon storage, depending on the ph of honey and on the storage temperature. HMF content of honey also increases upon its adulteration by invert sugars. Presence of invert sugar in honey is assessed by Fiehe s test where HMF reacts with resorcinol and gives a red coloured complex. According to PFA Act, Fiehe s test should be negative for honey, whereas Codex Standards states that HMF content of honey should not be more than 60 mg/kg. Materials Required 1) Sample of honey 2) Resorcinol solution 3) Ether 4) Aniline chloride solution 5) Pestle and Mortar 6) Beakers Procedure The procedure for Fiehe s test and Aniline chloride is given herewith. Carry out these test following the steps enumerated herewith. Fiehe s test 1) Take 5 g of honey in pestle and mortar. 2) Mix honey solution with 10 ml ether (you will notice that honey or ether will not mix). 3) Now, decant ether extract into porcelain dish. 4) Repeat this extraction twice. Allow the extract to evaporate to dryness at room temperature. 5) Add a large drop of resorcinol solution into the porcelain dish with the residue. 6) The production of cherry red colour indicates a positive reaction. If the Fiehe s test is positive, we go for Aniline chloride test for conformation of adulteration (i.e., presence of commercial sugar). Aniline chloride test 1) Take 5 gm of honey in a porcelain dish. 2) Add 2.5 ml of prepared aniline chloride solution to it and keep stirring.

16 3) In the presence of commercial invert sugar the presence of orange red colour to fuming red within 1 minute indicates a positive test. Honey Observations (Write your observations about both the tests in the table provided herewith). S.No. Test Observation Result 1. Fiehe s test 2. Aniline chloride test Colour Positive/ Negative The given sample of honey was found to be (pure/impure) and.. (unadulterated/adulterated) as Fiehe's and Anniline chloride tests were (negative/positive). Submit the activity for evaluation... Counsellor Signature 35

17 Principles of Food Science ACTIVITY 4 Date:. 36 DETERMINATION OF FRUCTOSE TO GLUCOSE RATIO Aim: To determine the fructose to glucose ratio in honey. Objectives This activity will help you to: check the given samples of honey for any adulteration with commercial sugars, and check the conformance or non-conformance of the samples to the standards for fructose to glucose ratio Principle The major sugars present in honey are fructose, glucose, followed by lower concentration of sucrose and maltose. 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 1.1:1. Other sugars found in small amounts in honey are isomaltose, nigerose, kojibiose, turanose, gentibiose and laminaribose. Although, analysis of honey for physical, chemical and microbiological parameters give a good picture about the quality of honey, but, it is possible that the adulteration with the low cost sugar syrups such as invert syrups or high fructose corn syrup (HFCS) may go undetected. Thus, now-a-days carbon isotope ratio methods are recommended by Association of Official Analytical Chemists (AOAC) to detect the adulteration of expensive honey with cheap HFCS and invert cane sugar. According to this method, the carbon isotope ratio of the honey and that of the protein isolated for that honey should be similar, as nearly all the protein in honey originates from the bee in the form of enzymes that ripen the nectar. The addition of corn syrup to honey will change the carbon isotope ratio of the honey but not of the protein. If the honey has been adulterated with invert sugar or HFCS, the honey will have ratio of the carbon isotope significantly different from that of honey protein. But, here in this exercise we will be carrying out the simple experiment on detection of fructose to glucose ratio titration. Reagents Required Iodine (I 2 ) solution (0.05 N) NaOH N Standard sodium thio sulphate solution = 0.05 N = 12.4 g/l Starch solution (freshly prepared) Materials Required Sample of honey Iodination flask Beaker Burette Procedure Carry out the activity following the steps enumerated herewith. 1) Place accurately 1 gm of honey solution in 250 ml of volumetric flask. 2) Dilute with about 150 ml of water. 3) Mix thoroughly contents of glass and make the volume to 250 ml. 4) Pipette 50 ml of honey solution in a 250 ml stoppered iodination flask. 5) Add 40 ml of Iodine solution.

18 6) Add 25 ml of NaOH solution. Honey 7) Stopper the flask and keep in dark place for 20 minutes. 8) Acidify the solution with 5 ml of H 2 SO 4 and titrate quickly the excess of I 2 against standard Sodium thiosulphate solution. 9) After adding some sodium thiosulphate add few drops of starch solution and see the change of colour from voilet to colourless. 10) Now repeat steps 5 to 9 once again. Start by taking 50 ml water (distilled) instead of honey solution. This is your blank sample. 11) Calculate the fructose glucose ratio with the help of following calculations: Calculations A) Approximate glucose % by mass (W) = (B S) a where, B = volume of sodium (Na) thiosulphate used for blank S = volume of Na thiosulphate used for sample a = mass of honey taken for the test B) Approximate fructose % by mass (X) = Approximate total reducing sugar (% after inversion) w Here total reducing sugar (% after inversion) value can be taken as calculated in Activity 2 earlier. C) True glucose % by mass (Y) = W X D) True fructose % by mass (Z) = Approximatereducingsugar beforeinversion(%) Y Here approximate reducing sugar before inversion value can be taken as calculated in Activity 1. E) Total reducing sugar % by mass = Y + Z F) Fructose to glucose ratio = Truefructose%by mass(z) TrueGlu cose%by mass(y) Results and Observations Record your observations in the format given below: Volume of Na thiosulphate used for blank (B) =.. ml Volume of Na thiosulphate used for sample (S) =. ml Mass of honey taken for analysis (a) =.. g Now calculate the following: A) Approximate glucose % by mass (W) = (B S) a Putting in the values, we get: Approximate glucose % by mass (W) = 37

19 Principles of Food Science B) Total reducing sugars after inversion were found to be (% by mass) = Write down the value of total sugar (inverted) obtained in Activity 2 of this practical from page 33. C) Total reducing sugars before inversion were found to be (% by mass) =.. Write the value of total reducing sugar obtained in Activity 1 of this practical from page 29 D) Approximate fructose % by mass (X) = Approximate total reducing sugar (% after inversion) w Putting in the values we get E) True glucose % by mass (Y) = W X (Putting the values we get): F) True fructose % by mass (Z) = Approximatereducingsugar beforeinversion(%) Y (Putting the values we get): G) Fructose to glucose ratio = (Putting the values we get): Truefructose%by mass(z) TrueGlu cose%by mass(y) 38

20 Inference The sample of honey analyzed gave. fructose to glucose ratio. According to the specifications laid down the fructose to glucose ratio should be. Thus, the sample of honey was found to be conforming/non conforming to the standards. Honey Submit the activity for evaluation. Counsellor Signature 39

21 Principles of Food Science ACTIVITY 5 Date:. DETERMINATION OF ACIDITY Aim: To determine the acidity in the given sample of honey. Objectives This activity will help you to: carry out the acidity test of the given samples of honey, learn about the keeping quality of honey, and check the conformance or non-conformance of the samples to the standards for acidity. Principle The ph of natural honey ranges from 3.4 to 6.1. Acidity of honey is primarily due to presence of acids such as formic acid, gluconic acid, pyruvic acid, malic acid, citric acid and succinic acid. Acidity of a afresh honey is usually very low, 13 to 35 meq/kg. Honey with acidity more than 40 meq/kg is considered as poor in quality. Acidity is determined by titration of a known weight of honey with 0.1M Sodium hydroxide (NaOH). Materials Required 0.1 N NaOH Phenolphthalein indicator Conical flask Pipette CO 2 free water Procedure Now carry out the activity following the steps enumerated herewith: 1) Take 10 g of honey sample in a conical flask. 2) Dissolve in it 75 ml of CO 2 free water (Boiling distilled water and cooling). 3) Mix thoroughly and titrate against standardized NaOH using phenolphthalein as indicator. (Add 1 ml of phenolphthalein during mixing of honey and water). 4) Observe for the change in colour to light pink. 5) Pink colour should persist for at least 10 seconds. 6) Also conduct blank titration with 85 ml of CO 2 free water and phenolphthalein indicator. 7) Calculate the acidity using the formula given herewith: Calculations Acidity is expressed as % formic acid by mass in honey. Equivalent weight of formic acid = 0.23 Acidity = 0.23 Volumeof NaOH Mass of Honey Volume of Volume of Volume of NaOH = NaOH required NaOH required forsample titration for blank titration 40

22 Results and Observations Record your observations as indicated Mass of honey taken for analysis = g Volume of NaOH used =. ml Acidity = 0.23 titre volume Mass Putting in the values we get, Honey Acidity (% by mass) as. Inference Acidity (% by mass) of the given sample was calculated as. According to the specifications the acidity of honey should not exceed by.. % by mass. Thus, the given sample of honey was found to be (conforming/non-conforming) to the standards. Submit the activity for evaluation.. Counsellor Signature 41