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
 Honey
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 30th 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 1.41 1.39 1.37 -
2. Moisture % by mass (maximum) 20 22 25 25
3. Total reducing sugar % by mass 70 65 65 65
(minimum)
4. Sucrose % by mass (maximum) 5.0 5.0 5.0 5.0
5. Fructose – glucose ratio (minimum) 1.0 1.0 1.0 0.9
6. Ash % by mass (maximum) 0.5 0.5 0.5 0.5
7. Acidity expressed as formic acid, % 0.2 0.2 0.2 -
by mass (maximum)
8. Fiehe’s Test -ve -ve -ve -ve
9 Aniline chloride Test -ve -ve -ve -
21
Principles of Food According to PFA Act, 1954 and the rules in 1955, honey has been defined as the
Science 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

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.
22
To assess the quality of honey, total reducing sugars and sucrose content is most Honey
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.

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
Principles of Food Reaction for Non-Reducing Sugars
Science

D) Total Solids and Water

Most genuine honeys contain 13-23 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 infra-
red 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:
24
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
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
Reagents Required Honey

1) Standard invert sugar solution: Weigh accurately 0.985 g of sucrose and dissolve
in 500 ml of water. Add 2 ml of concentrated H2SO4. Boil gently for 30 minutes
and keep aside for 24 hours. Neutralize this with Na2CO3 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 CuSO4 is
calculated by multiplying the titrated value with 0.001 (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.

27
Principles of Food Calculations:
Science
250 ×100 × S
Reducing sugar =
H×M
where,
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 × 0.001
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

Titre value = ………………….

Strength of CuSO4 solution / Fehling factor (S) is:
Titre value × 0.001= ……………………..
Solution of Honey
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
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
250 ×100 × S
Reducing sugar =
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
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
Reagents Required Honey

1) Standard invert sugar solution: Weigh accurately 0.985 g of sucrose and dissolve
in 500 ml of water. Add 2 ml of conc. H2SO4. Boil gently for 30 minutes and
keep aside for 24 hours. Neutralize this with Na2CO3 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 0.001 (mg / ml of the standard
invert sugar solution). This is known as Fehling factor.

31
Principles of Food Note: carry out the titration till you get consecutive titre value. This means you may
Science 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
250 ×100 × S
Total sugars =
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 × 0.001
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 Cu2+ 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

Titre value = ……………………….

Strength of CuSO4 solution / Fehling factor (S) is
Titre value …………… × 0.001= …………………
Solution of Honey (inverted)
Record your observations in the format below according to the procedure you
followed in step 3.
32
 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
250 ×100 × S
Total sugars =
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
Principles of Food
Science
ACTIVITY
3
DETECTION OF ADULTERATION

Date: …………. 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.
34
3) In the presence of commercial invert sugar the presence of orange red colour to Honey
fuming red within 1 minute indicates a positive test.
Observations
(Write your observations about both the tests in the table provided herewith).

S.No. Test Observation

Colour Positive/ Negative

1. Fiehe’s test

2. Aniline chloride test

Result
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
Principles of Food
Science
ACTIVITY
4
DETERMINATION OF FRUCTOSE TO GLUCOSE RATIO

Date: …………. 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 (I2) solution (0.05 N)
NaOH - 0.1 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.
36
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 H2SO4 and titrate quickly the excess of I2
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
(B − S) × 0.004 × 100 × 5
A) Approximate glucose % by mass (W) =
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
0.925
Here total reducing sugar (% after inversion) value can be taken as calculated in
Activity 2 earlier.
C) True glucose % by mass (Y) = W – 0.012 X
D) True fructose % by mass (Z) =
Approximate reducing sugar beforeinversion(%) − Y
0.925
Here approximate reducing sugar before inversion value can be taken as
calculated in Activity 1.
E) Total reducing sugar % by mass = Y + Z
True fructose % by mass (Z)
F) Fructose to glucose ratio =
True Glu cos e % 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:
(B − S) × 0.004 × 100 × 5
A) Approximate glucose % by mass (W) =
a
Putting in the values, we get:

Approximate glucose % by mass (W) = …………………

37
Principles of Food B) Total reducing sugars after inversion were found to be (% by mass) = ……
Science
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
0.925
Putting in the values we get

E) True glucose % by mass (Y) = W – 0.012 X

(Putting the values we get):

F) True fructose % by mass (Z) =

Approximate reducing sugar beforeinversion(%) − Y
0.925
(Putting the values we get):

True fructose % by mass (Z)

G) Fructose to glucose ratio =
True Glu cos e % by mass (Y)
(Putting the values we get):

38
Inference Honey

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.

Submit the activity for evaluation

………………………….
Counsellor Signature

39
Principles of Food
Science
ACTIVITY
5
DETERMINATION OF ACIDITY
Date: …………. 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
CO2 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 CO2 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 CO2 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
0.23 × Volume of NaOH
Acidity =
Mass of Honey
 Volume of   Volume of 
Volume of NaOH =  NaOH required  −  NaOH required 
  
 for sample titration   for blank titration 

40
Results and Observations Honey

Record your observations as indicated

Mass of honey taken for analysis = ……………… g
Volume of NaOH used = ………………. ml
0.23 × titre volume
Acidity =
Mass
Putting in the values we get,

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