Beverages and Confectionary

LAB.
MANUAL 4
MANUAL OF METHODS
OF
ANALYSIS OF FOODS
BEVERAGES (COFFEE, TEA, COCOA, CHICORY)
SUGAR AND SUGAR PRODUCTS &
CONFECTIONERY PRODUCTS
FOOD SAFETY AND STANDARDS AUTHORITY OF INDIA

MINISTRY OF HEALTH AND FAMILY WELFARE
GOVERNMENT OF INDIA
NEW DELHI
2015
ACKNOWLEDGEMENT
Deepest Sense of Gratitude and Indebtedness to all the

Members of the Panel “Method of Sampling and Analysis” and
Experts, who have helped, supported knowledge and insight,
have led the successful completion of Revision of manuals.
Sincere Thanks to the Panel, Chairman for his valuable

guidance, and encouragement and the Secretariat of this
panel who have worked hard throughout
the tenure of revision work.
Deepest Appreciation to the Chairperson, FSSAI and CEO,

FSSAI for the cooperation, support and constant
encouragement, without which the work
would not have seen the light of day.
*******
Beverages, Sugar and Confectionery Product 2015
MANUAL FOR ANALYSIS OF BEVERAGES (COFFEE, TEA,

COCOA, CHICORY), SUGAR AND SUGAR PRODUCTS
AND CONFECTIONERY PRODUCTS
TABLE OF CONTENTS
S.No. TITLE PAGE NO.
PART A- BEVERAGES AND SUGAR AND SUGAR PRODUCTS
1.0 Roasted Coffee 4

2.0 Chicory and Coffee – Chicory Mixture 20
3.0 Soluble (Instant) Coffee Powder 21
4.0 Cocoa 22
5.0 Tea 24
6.0 Honey 29
7.0 Cane Sugar and Refined Sugar 40
8.0 Bura 52
9.0 Gur or Jaggery 53
10.0 Sulphated ash in Dextrose 53
11.0 Starch in Icing Sugar 54
12.0 Total Solids in Liquid Glucose 54
PART B - CONFECTIONERY PRODUCTS
A. SUGAR BOILED CONFECTIONERY AND LOZENGES
A1 Preparation of Sample 57
A2 Determination of Moisture 57
A3 Determination of Sulphated Ash 59
A4 Determination of Sulphated Ash on salt free basis 60
A5 Determination of Ash in dil HCl 61
A6 Test for presence of added synthetic colour 63
A7 Determination of Total Protein 68
A8 Determination of Fat 71
A9 Determination of Reducing Sugar 75
A10 Determination of Sucrose 76
A11 Determination of Sulphur dioxide 77
A12 Determination of Lead, Copper and Zinc 80
A13 Determination of Filth in Candy 83
1

A14 Determination of Starch 86
A15 Determination of Paraffin 88
A16 Determination of Shellac 89
A17 Determination of Alcohol in Syrups 90
A18 Determination of Total Plate Count 91
A19 Determination of Coliform 94
A20 Determination of Yeast and Mould Count 97
A21 Determination of E.coli 102
A22 Determination of Staphylococcus aureus 104
B. CHEWING GUM AND BUBBLE GUM
B1 Preparation of Sample 106

B2 Determination of Moisture 107
B3 Determination of Ash in dil HCl 108
B4 Determination of Sulphated Ash 109
B5 Test for presence of added synthetic colour 110
B6 Determination of Titanium dioxide 112
B7 Determination of Gum- Base Content 113
B8 Determination of Reducing Sugar 115
B9 Determination of Sucrose 116
B10 Determination of Total Protein 116
B11 Determination of Fat 116
B12 Determination of Lead, Copper and Zinc 116
B13 Determination of Filth 116
B14 Determination of Total Plate Count 116
B15 Determination of Coliform 116
B16 Determination of Yeast and Mould Count 116
B17 Determination of E.coli 116
B18 Determination of Staphylococcus aureus 116
C. CHOCOLATE
C1 Preparation of Sample 118

C2 Determination of Moisture 118
C3 Determination of Fat 120
C4 Determination of Non Fat Milk Solids 122
C5 Test for Rancidity 125
C6 Determination of Ash in dil HCl 125
C7 Determination of Milk Fat 126
C8 Determination of Cocoa Solids 130
2

C9 Determination of Reducing Sugar 131
C10 Determination of Sucrose 132
C11 Determination of Chocolate Component of Filled Chocolate 132
C12 Determination of Edible Wholesome Substances 132
C13 Determination of Total Protein 133
C14 Determination of Lead, Copper and Zinc 133
C15 Determination of Filth 133
C16 Determination of Total Plate Count 134
C17 Determination of Coliform 134
C18 Determination of Yeast and Mould Count 134
C19 Determination of E.coli 134
C20 Determination of Staphylococcus aureus 134
D. ICE LOLLIES OR EDIBLE ICES
D1 Determination of Reducing Sugar 134

D2 Determination of Sucrose 134
D3 Determination of Lead, Copper and Zinc 134
D4 Determination of Total Plate Count 135
D5 Determination of Coliform Count 135
D6 Determination of Yeast and Mould Count 135
D7 Determination of E.Coli 135
D8 Determination of Staphylococcus aureus 135
D9 Determination of Salmonella 135
D10 Determination of Listeria monocytogenes 140
3
MANUAL FOR ANALYSIS OF BEVERAGES (COFFEE, TEA,

COCOA, CHICORY), SUGAR AND SUGAR PRODUCTS
AND CONFECTIONERY PRODUCTS
PART A
BEVERAGES AND SUGAR AND SUGAR PRODUCTS
COFFEE - Definitions of different types of Coffee are given under section

2.10.2 of Food Safety and Standards (Food Product Standards and Food
Additives) Regulations, 2011.
1.0 Roasted Coffee
1.1 Preparation of sample:
Grind the sample in a grinder to pass through No. 30 mesh sieve. Mix
well to get a homogenous sample. Store sample in a tightly stoppered bottle,
Withdraw portions for analytical determinations.
(Ref: - A.O.A.C 17th edn, 2000 Official Method 920.91 Roasted Coffee
Preparation of sample)
1. 2 Determination of moisture: (Routine method)
Weigh accurately about 5 gms of sample in a tared aluminium dish.

(About 7.5 cm in dia and 2.5 cm deep). Dry in an air oven at 100 ±20 C for 5 to
6 hours. Cool in a desiccator and weigh. Dry again for 30 minutes, cool in a
dessicator and weigh. Repeat the process of heating and cooling in a
dessicator until the difference in two successive weighings is less than 1 mg.
Record the lowest weight. Carry out the determination in duplicate.
4
1.2.1 Calculation
W1 – W2
Moisture (%) = x 100
(by weight) W1 – W
Where,
W = Weight in gms, of Aluminium dish.

W1 = Weight in gms, of Aluminium dish + sample before drying.
W2 = Weight in gms, of Aluminium dish + dried sample.
(Ref: - I.S.I Handbook of Food Analysis (Part IX) – 1984 page 51 / I.S 3077:
1972 Specification for roasted and ground Coffee)
1.2 A Determination of Moisture - Vaccum Oven method (Reference

method)
1.2 A.1 Apparatus
1. Aluminium dish – 7 cm diameter and about 3 cm height with close

fitting cover.
2. Dessicator
3. Vaccum oven – connect with pump capable of maintaining partial

vaccum in oven A with pressure equivalent to 25 mm Hg and provided
with thermometer passing into the oven in such a way that the bulb is
near the test sample. Connect H2 SO4 gas drying bottle with oven to
admit dry air when releasing vaccum
1.2 A.2 Procedure
Accurately weigh about 5 gm of sample in a dish previously dried at
5
98 –1000 C, cooled in dessicator and weighted with cover soon after attaning
room temperature. Place in oven, lean cover against dish and heat to
constant weight (about 5 1/2 hrs) at 98 – 1000 C at pressure equal to
25 mm Hg. During heating admit slow current of air (about 2 bubbles /
second) through H2SO4 into oven. Carefully admit dry air into oven to bring
to atmospheric pressure. Cover dish, transfer to dessicator and weigh soon
after room temperature is attained. Report percent loss in weight as
moisture.
(Ref: - A.O.A.C 17th edn, 2000 Official Method 968.11 Moisture (Loss on
Drying in Roasted Coffee, Vacuum Oven method 1)
1.3 Determination of total ash:
Weigh accurately about 5 gms of sample in a tared silica / platinum

dish Char the material carefully on a burner and transfer the dish to a muffle
furnace and ash at a temperature of 550 ±10 0 C until the ash is free of
Carbon. Heat the dish again at 550 ± 100 C for 30 minutes Cool in a desiccator
and weigh. Repeat this process of heating for 30 minutes, cooling in a
dessicator and weighing until the difference between two successive
weighing’s is less than 1 mg. Record the lowest weight.
(W2 – W) x 100 x 100

Total ash (% on dry weight) =
(W1 – W) x (100 – M)
Where,
W1 = Weight in gms of Silica dish. + sample

W2 = Weight in gms of Silica dish + ash
W = Weight in gms of empty Silica dish.
M = Moisture % of the sample.
6
Note: – Preserve the dish containing this ash for the determination of acid
insoluble ash
Alternative method of Determination of Total Ash- Use a

programmable muffle furnace that allows a gradual increase in temperature
than charring with a Bunsen burner and inserting the sample into the
furnace at 550 degrees Celsius. The charring technique is often prone to
losses and you can have superheating of samples as they enter the furnace
causing them to ‘explode’ (not in a dramatic way) and lose sample or
contaminate surrounding samples. It’s very quick but not as accurate as
using a programmable muffle. It’s also safer to use a programmable furnace
for the analyst- handling crucibles at 550 Celsius is prone to risk.
Instead of Bunsen burner, hot plate can also be used for charring of samples.
(Ref: - I.S: 3077 – 1972 Specification for Roasted and Ground Coffee
Appendix F / I.S.I Handbook of Food Analysis (Part IX) – 1984 page 52)
1.4 Determination of water soluble ash:

Transfer the ash with the aid of about 25 ml distilled water into a
beaker. Cover with a watch glass and boil for 5 minutes. Filter through an
ashless filter paper (Whatman No. 42 or its equivalent). Collect the filterate
in a 150 ml beaker, wash the filter paper 4 -5 times with hot water until the
filterate no longer turns red litmus blue and collect the washings in the same
beaker. (Reserve the entire filtrate for the determination of alkalinity of
soluble ash) Dry the ashless paper with residue in an oven and transfer to
muffle furnace and ignite at 550ºC for 2 hours. Cool in a desiccator and weigh
(W3) Repeat the process till the difference in two consecutive weighings is
less than 1 mg. Record the lowest weight.
(W2 – W) x 100 x 100
Water in-soluble ash on dry wt basis (%) =
(W1 – W) x (100 – M)
7
Where,
W2 = Weight in gms of Silica dish + water insoluble ash.

W = weight in gm of empty dish
W1 = weight in gm of dish with material
M = Percentage of moisture
1.4.1 Calculation
Water soluble ash percent by wt = A – B
Where,
A = Total ash percent by wt

B = Water insoluble ash percent by wt
Water soluble ash

Water soluble ash of total ash = x 100
(Percent by wt) Total ash
(Ref: - I, S Specification I.S 3077: 1972 Specification for Roasted and Ground
Cofee / I.S.I Handbook of Food Analysis (Part IX) – 1984 page 52)
1.5 Determination of Ash insoluble in dilute HCl:
Boil total ash prepared as in sec. 1.3, with 25 ml of hydrochloric acid

(1: 2.5) for 5 minutes, covering the Silica dish with a watch glass to prevent
spattering. Filter through ashless filter paper (Whatman No. 42 or
equivalent). Wash the entire residue with hot water ( > 85ºC) until the
filtrate does not turn blue litmus paper to red. Dry the ashless paper with the
residue and transfer to muffle furnace and ignite at 550ºC for 2 hours. Repeat
the process of igniting in the muffle furnace, cooling and weighing at ½ hr
8
intervals until the difference in two successive weighings is less than 1 mg.
Cool in a desiccator and weigh (W4).
1.5.1 Calculation
(W2 – W) x 100 x 100

Ash insoluble in dilute HCl (%) =
(on dry wt.) (W1 – W)x (100 – M)
Where,
W2 = weight of dish + acid insoluble ash
W1 = weight of dish + sample
W = weight of dish
M = Percent moisture
(Ref: - I.S Specification I.S: 3077 – 1972 Specification for Roasted and Ground
Coffee / I.S.I. Handbook of Food Analysis (Part IX) 1984) page 52)
1.6 Determination of alkalinity of soluble ash:
To the filtrate reserved during the determination of water soluble ash

(sec.1.4), add 3-4 drops of methyl orange indicator (0.1% in water) and
titrate with 0.1N hydrochloric acid to an orange end point. Note down the
titre value.
Titre value x Normality of HCl
Alkalinity of soluble ash % per =
gm of sample (on dry wt.) Wt. of sample (W1– W) x (100 – M)
Where,
W = weight of empty dish
W1 = weight of dish + sample
9
M = % Moisture of the sample
1.7 Determination of aqueous extract:
Accurately weigh around 2 gms of sample and transfer to a 500 ml

flask. Add 200 ml distilled water and connect the flask with a 50 cm long
water jacketed condenser. Reflux for 1 hour over low flame with occasional
mixing. Cool, and filter through Whatman No 1 filter paper or equivalent,
wash 3 times with 10 – 15 ml water and finally make upto 250 ml in a
volumetric flask. Shake well and pipette 50 ml of aliquot to a tared
aluminium dish.
Evaporate on a steam bath and transfer to 100 ºC air oven and dry for
2 hours. Dry again for 30 minutes, cool in a desiccator and weigh. Repeat this
process of heating for 30 minutes, cooling in dessicator and weighing until
the loss in weight between two successive weighings is less than 1 mg.
Record the lowest weight
(W2 – W1) x 250 x 100 x 100

Aqueous extract (%) = -----------------------------------------
(on dry wt.) W x 50 x (100 – M)
Where,
W= Weight of sample.
W1 = Weight of empty aluminium dish.
W2 = Weight of empty aluminium dish + dried extract.
M = Moisture %.
10
1.8 Determination of Caffeine Content (Bailey Andrew Method)
1.8.1 Procedure
Weigh accurately about 5 gm of sample, transfer to a 250 ml

Erlenmeyer flask, add 3 gm of magnesium oxide and 100 ml of distilled
water. Weigh the flask with contents and boil under a reflux condenser for 45
minutes shaking occasionally. Cool and weigh the flask again and add water
till the original weight is obtained. Mix well and filter through a dry filter
paper directly into a 50 ml graduated flask until exactly 50 ml of the solution
(equivalent to half the quantity of the sample taken for test) is obtained.
Transfer the solution to a 125 ml separator. Wash the graduated flask

with 2 ml of water and add the washings to the separator. Add 4 ml of dilute
Sulphuric acid (1: 9). Extract with five 10 ml portions of chloroform shaking
vigorously for 1 minute for each extraction. Let the emulsion break, then
drain the chloroform into a 125 ml separator. Add 5 ml of Potassium
hydroxide solution (1%). Shake vigorously for 1 minute, let the emulsion
break and drain the chloroform through a cotton plug into a 100 ml Kjeldahl
flask. Extract the Pot hydroxide solution with 5 ml of chloroform and add to
the Kjeldahl flask. To the digestion flask add 1.3 ± 0.5 gm of potassium
sulphate and 40 ± 5 mg mercuric oxide. Rinse down the neck of the flask with
3 ml chloroform.
Place the flask on the digestion rack and concentrate chloroform to

about 20 ml. Distill off chloroform. Add 2 ± 0.1 ml conc sulphuric acid of sp gr
1.84 digest for 1 hour after the acid begins to boil. Cool and add minimum
quantity of water to dissolve the solids. Cool and place a thin film of vaselin at
11
the rim of the flask. Transfer the digest with a few boiling chips to the
distillation apparatus and rinse the flask 5 – 6 times with 1 – 2 ml water.
Place a 125 ml beaker containing a known quantity of standard
sulphuric acid (0.05N). Add 6 ml of conc sodium hydroxide solution ( 1+ 2)
carefully through the side of the still so that it does not mix, and assemble the
distillation apparatus taking care that the dip tube extends well within the
standard sulphuric acid solution contained in the beaker. Mix the contents of
the distillation flask and distill until all ammonia has passed over into the
standard sulphuric acid. Shut off the heater and immediately detach the flask
from the condenser. Rinse the condenser thoroughly with water into the
beaker. Wash the dip tube carefully so that all traces of the condensate are
transferred to the beaker. When all the washings have drained into the
beaker, add 2-3 drops of methyl red indicator and titrate with standard
sodium hydroxide solution (0.1N).
Carry out a blank determination using reagents in the same proportion

without the sample.
1.8.2 Calculation
486.96 (B- A) N
Caffeine on dry basis =
(%) by weight W (100 – M)
Where,
B= volume of standard sodium hydroxide used to neutralize acid in
the blank determination
A= volume of standard sodium hydroxide used to neutralize the
excess acid in the test with the sample
N= Normality of standard sodium hydroxide solution
W= weight in gm of the sample in the aliquot
M= Percentage of moisture in the sample
Note: - For soluble coffee (instant coffee) the quantity of sample taken for
12
test should be 1 gm only
Coffee / I.S.I Handbook of Food Analysis (Part IX) page 53) (Also see A.O.A.C
17th edn, 2000 Official method 960. 25 Caffeine in Roasted Coffee)
1.8A Alternate Chromatographic – Spectrophotometric Method:

Applicable to roasted, soluble (instant) and decaffeinated coffee
1.8A.1 Regents
a) Ammonium hydroxide solution – 1 : 2 (V/V)
b) Sulphuric acid – 4N
c) Diethyl ether – Water Saturated
d) Chloroform – Water Saturated
e) Celite 545
f) Caffeine standard solution – 10, 20, 30 µg /ml in Chloroform.
Accurately weigh 100 mg of caffeine (USP, anhydrous) into 100 ml

volumetric flask, dissolve in CHCl3.and make upto volume. Dilute 10 ml
aliquot to 100 ml with chloroform. Further dilute 10, 20, and 15 ml aliquots
to 100, 100 and 50 ml respectively with chloroform to obtain standard
solutions of 10, 20, and 30 µg / ml
1.8A.2 Apparatus
a) Glass column – 25 x 250 mm size
b) Recording UV – VIS Spectrophotometer – To record 250 – 350 mm
range with matched 1 cm cells.
1.8A.3 Preparation of Sample
a) For roasted Coffee
13
Accurately weigh about 1 gm ground sample and transfer to 100 ml

beaker. Add 5ml NH4OH (1+2) and warm on boiling water-bath for 2
minutes. Cool, transfer to 100 ml volumetric flask and make up to volume
with water. To 5 ml aliquot of the turbid solution add 6 g celite 545 and mix
carefully.
b) For soluble Coffee
Proceed as in (a) taking 0.5 g sample and an aliquot of 3 ml
c) For decaffeinated roasted coffee
Accurately weigh 1 gm of ground sample. Transfer to 100 ml beaker,

add 5 ml NH4OH (1+2) and warm on boiling water bath for 2 minutes. Add 6
gm celite 545 and mix carefully.
d) For decaffeinated soluble coffee
Proceed as in c) taking 0. 5 gm sample
1.8A.4 Preparation of Columns
a) Acid column:
Place fine glass wool and plug into the base of 25 x 250 mm column.
Add 3 ml 4N H2SO4 to 3 g celite and mix well by kneading with spatula.
Transfer into the tube and tamp using gentle pressure and place small glass
wool above the surface.
c) Basic Column:
14
Layer I:
Mix 3 g celite 545 and 2 ml 2N NaOH and place in 25 x 250 mm tube.

Transfer over glass wool plug as in (a) into the column.
Layer II:
Transfer sample plus celite mixture in about 2 g portions to tube

directly over layer I, tamping before adding mixture portion of sample until
homogenous and compact layer is obtained. Dry wash beaker with about 1 g
celite 545, transfer to tube and tamp to uniform mass. Dry wash beaker with
wad of glass wool and transfer to top of basic column.
1.8A.5 Determination:
Mount basic column above acid column. Pass 150 ml water saturated
ether sequentially through basic column to acid column and discard ether.
Then pass 50 ml water saturated ether through acid column and discard
ether Place 50 ml volumetric flask under acid column. Pass 48 ml, water
saturated CHCl3 through acid column washing tip of basic column with first
portions. Dilute contents of volumetric flask (100 ml) to volume with water
saturated chloroform, mix, and read O.D. at 275 against water saturated
chloroform CHCl3 blank, by scanning from 350 to 250 nm. Determine O.D of
standards and use this value to calculate the caffeine percentage.
(Ref: - A.O.A.C 17th edn, 2000 Official Method 979.11 Caffeine in Roasted
Coffee, Chromatographic – Spectrophotometer method)
1.8 B Alternate HPLC method
1.8 B.1 Procedure
15
Dissolve 1.00 gm of sample in 100 ml hot water. Filter 20 ml through a

Millipore filter (0.45µm) under vacuum and apply to a Bond Elut C 18
cartridge or equivalent under vacuum. Elute the caffeine with 5 ml of mobile
phase (0.005 M Sodium acetate: tetrahydrofuran – 95: 5 at pH 5). Collect in a
10 ml flask and make upto volume Inject 20 ul into a Spherisorb ODS, C 18, 5
um packed column 25 cm long x 4 mm internal dia. Elute with the mobile
phase at 1 ml / min, observe absorbance at 280 nm, calibrate with standard
Caffeine solution, 0 - 1 mg Caffeine in 10 ml mobile phase For routine
purposes the HPLC step can be eliminated and the absorbance of eluant from
the cartridge measured at 280 nm in a spectrophotometer.
(Ref:- Pearson’s Composition and Analysis of Foods 9th edn,1991 , page373)
1.9 Microscopic Examination:
Boil about 1 gm of sample with 50 ml of 2% sodium hydroxide for

about 2 - 3 minutes. Dilute and filter and wash the residue with water till the
filtrate is free of alkali. Repeat till the residue gives no colour with water
(treatment with calcium chloride solution and then washing with water may
be done in case the decant still shows some colouring matter. Place a drop of
residue material in glycerine on a clear microscopic slide.
Place a cover slip on the drop of the suspension and see under
microscope. Alternatively boil sample with water so that most of the colour is
extracted. Drain and replace with chloral hydrate. Heat until sufficiently
cleared. Wash out chloral hydrate and stain with phloroglucinol/
hydrochloric acid The microscopic structure as shown in the
photomicrograph given below can be seen:
16
17
18
Note:-
Coffee is characterized by longitudinal and transverse schlerenchymatous
fibres (from pericarp) Chicory has large vessels upto 115 microns across
which have short pits. Roasted cereals such as barley, oats and wheat and
soya may be mixed with coffee and coffee and chicory as coffee substitutes.
Careful microscopic examination will reveal their presence.
(Ref :- I.S Specification I.S 3077 : 1972 Specification for Roasted and Ground
Coffee / I.S.I Hand book of Food Analysis ( Part IX) – 1984 page 49 / F.A.O
Manuals of Food Quality Control 14 /8 pages 318 and 319)
1.9.1 Test for Chicory in Coffee:
Principle:
19
Chicory is a root and contains inulin, hydrolysis of which gives

fructose. Coffee does not contain inulin. Therefore, this method can be used
to test the presence of Chicory in coffee.
Reagents:
(a) Neutral lead acetate – Prepare 10% solution in water.

(b) Conc. HCl.
(c) Seliwanoff reagent – Dissolve 0.05 g of resorcinol in 100 ml of 1:2 HCl.
Procedure:
Clarify 25 ml of 2% aqueous extract of the sample with neutral lead
acetate and filter. To 5 ml of filtrate add 5 ml of Seliwanoff reagent and 1 ml
of conc HCl. Boil for 2 minutes. Appearance of distinct red colour on standing
shows the presence of Chicory in coffee.
Note:-
In addition to microscopic examination and the positive reaction with
seliwanoffs reagent, the presence of chicory may be shown simply by
sprinkling the powder onto water in a measuring cylinder. Coffee floats while
chicory particles start sinking within a few seconds leaving behind a brown
trail of caramel
(Ref: - F.A.O Manuals of Food Quality Control 14 / 8 pages317 and 318)
2.0 CHICORY AND COFFEE - CHICORY MIXTURE
2.1 Preparation of sample – Refer to clause 1.1
2.2 Determination of moisture – Refer to clause 1.2
20
2.3 Determination of total ash on dry basis – Refer to clause 1.3
2.4 Determination of ash insoluble in dil HCl – Refer to clause 1.5
2.5 Determination of aqueous extract – Refer to clause 1.7
2.6 Microscopic Examination – Refer to clause 1.9
2.7 Determination of Caffeine content – Refer to clause 1.8
3.0 SOLUBLE (INSTANT) COFFEE POWDER
3.1 Determination of Moisture – Refer to clause 1.2 A (vacuum oven

method with the following changes)
(1) Keep temperature of oven at 70 ± 1 0 C.

(2) Maintain vacuum at 37. 5 mm Hg
(3) Dry for 16 hours
(4) Admit air at the rate of 1 bubble / second
(5) Calculate moisture (Loss on drying) as under
(M1 – M2)
Moisture (%) = x100
(M1 – M0)
Where
M0 = Weight of empty dish

M1 = weight of dish + sample before drying
M2 = Weight of dish + sample after drying
(Ref: - A.O.A.C 17th edn, 2000, Official Method 979.12 Moisture (Loss on
21
Drying) in Roasted Coffee – applicable to instant coffees – I.S.O – A.O.A.C

method)
3.2 Determination of Total Ash = Refer to clause 1.3
3.3 Determination of Caffeine Content – Refer to clause 1.8.
3.4 Determination of Solubility in boiling water
3.4.1 Procedure
Weigh 2.5 gm of instant coffee powder / coffee- chicory powder in a

500 ml beaker. Then pour 150 ml of freshly boiled water, stir. The product
should dissolve in 30 seconds.
(Ref: - I.S.I Hand book of Food Analysis (Part IX) – 1984 page 58)
4.0 COCOA POWDER
4.1 Preparation of Sample
Mix thoroughly and preserve in tightly stoppered bottle
(Ref: - A.O.A.C 17th edn, Official method 970.20 Cocoa products Preparation
of Laboratory Sample)
4.2 Determination of moisture
Weigh accurately about 2 gm of sample in Platinum / stainless steel

dish. Distribute the material as evenly as possible and place in an air oven
maintained at 100 0 C. Dry to a constant weight. (aluminium dish may be used
22
when ash is not determined on the same sample). Report loss in weight as
moisture.
(Ref: - A.O.A.C 17th edn, 2000, Official method 931.04 Moisture in Cocoa
Products / I, S, I Handbook of Food Analysis (Part IX) – 1984)
4.3 Determination of Cocoa Butter
4.3.1 Apparatus
(1) Soxhlet apparatus – with 250 ml flat bottomed flask
4.3.2 Reagents
(1) Petroleum ether – redistilled below 60 0 C
4.3.3 Procedure
Weigh accurately 10 – 20 gm of the material and transfer it to the fat

extraction thimble. Dry in an oven at 100 0 C for 1 hour. Extract the fat with
Petroleum ether. Continue extraction till 300 ml of petroleum ether have
been circulated. Recover excess solvent and evaporate the last traces on a
steam bath. Dry the fat in an air oven till the difference in two successive
weighings is less than 1 mg.
4.3.4 Calculation
Wt of extracted fat x100 x 100

Cocoa butter (%) =
on moisture free basis Wt of sample (100 – Moisture)
23
(Ref: - I.S.I Hand Book of Food Analysis (Part IX) – 1984 page 24) (Also see
A.O.A.C 17th edn, 2000 Official Method 963.15 Fat in Cocoa Products, Soxhlet
Extraction method)
4.4 Determination of Total ash – Refer to clause 1.3
4.5 Determination of Ash insoluble in dil HCl – Refer to clause 1.5
4.6 Determination of Alkalinity of ash - Refer to clause 1.6
5.0 TEA/INSTANT TEA
5.1 Preparation of sample: Ref. to clause 1.1.
5.2 Determination of moisture: Ref Clause 1.2
5.3 Determination of total ash: Ref Clause. 1.3
(Ref: - I.S 13854: 1994 / I, S, O 1575: 1987 Tea – Determination of Total Ash)
5.4 Determination of Water soluble ash:- Ref Clause. 1.4
(Ref: - I.S 13855: 1993 / I.S.O 1576:1988 Tea – Determination of Water

soluble ash and Water insoluble Ash)
5.5 Determination of Ash insoluble in dilute HCl: Ref Clause. 1.5
(Ref: - I.S 13857: 1993 / I.S.O 1577: 1987 Tea – Determination of Acid
insoluble Ash)
5.6 Determination Aqueous extract: Ref Clause. 1.7
24
5.7 Determination of alkalinity of soluble ash:
Determine as per the procedure given in clause 1.6 (under analysis of coffee).
Express the result as KOH (m/m) on dry basis:
0.0056 x titre value x Normality HCl x 100 x 100

Alkalinity of =
soluble ash % Weight of sample x 0.1 x (100 – moisture %)
(Ref: - I.S 13856: 1993 / I.S.O 1578: 1975 - Tea Determination of Alkalinity of
Water soluble ash)
5.8 Determination of Crude Fibre:
Reagents:
1. 1.25 g Sulphuric acid/100 ml.(1.25 percent w / v)
2. 1.25 g Caustic soda/100 ml free from sodium carbonate.(1.25 % w /
v)
Apparatus:
(i) Condenser – Use condenser that will maintain constant volume

of refluxing solutions.
(ii) Digestion flask – 700-750 ml Erlenmeyer flask is
recommended.
(iii) Filtering cloth –Use filtering cloth of such character that no
solid matter passes through when filtering is rapid. Fine linen
or dress linen with about 18 threads / cm or 45 threads per
inch (i.e the aperture size 0.14 mm and thread thickness 0.42
mm) or its equivalent may be used (Whatman filter Paper No.
25
54 may also be used).
(iv) Muffle Furnace maintained at 525 ± 20 0 C
Procedure:
Weigh accurately about 2 gm of prepared sample and dry in an air

oven maintained at 100 ± 20C for 4 hours. Transfer to the digestion flask. Add
200 ml of boiling 1.25 percent sulphuric acid. Immediately connect to the
condenser and heat (it is essential that the solution boils within one minute
and boiling continues briskly for exactly 30 minutes). Rotate flask frequently
until sample at sides is thoroughly wetted, taking care to keep material from
remaining on the sides of the flask. Immediately filter through linen in fluted
funnel, and wash with boiling water until washings are acid free.
Wash the residue back into the flask with 200 ml of boiling 1.25
percent sodium hydroxide solution using wash bottle marked to deliver 200
ml. Connect flask to reflux condenser and boil briskly exactly for 30 minutes
After 30 minutes remove flask immediately, filter through gooch prepared
with asbestos mat and carefully transfer all the residue into the gooch with
hot water. Wash the residue thoroughly with hot water until the filtrate is
alkali free. Then, wash with about 10 ml alcohol. Dry the gooch crucible at
110ºC to constant weight. Cool and weigh (W1). Transfer the gooch to a
muffle furnace controlled at 525 - 550ºC and ash the material. Cool, weigh
(W2). Loss in weight represents crude fibre.
Calculation:
W1 – W2 100 x 100
Crude fibre % = X x
----------------------
(on dry weight) Wt. of sample (100 – Moisture)
26
(Ref: - I.S.I Hand book of Food Analysis (Part IX) – 1984 page 44)
5.9 Determination of Iron filings and size of the particles:
Spread the entire quantity of the sample in a thin and uniform layer
on a polythene sheet Run a powerful magnet over the sample repeatedly till
no more iron filings cling to the magnet.
Collect the iron fillings in a clean dry and previously weighed
petridish. Note down and express the weight of iron filings and calculate the
content in ppm.
Calculation:
Weight of iron filings in gms x 1000 x 1000

= ppm
Weight of sample
Note: The method specified by Tea Board i.e. Total Iron and Non-
Magnetic Iron by Colorimetry can be also be followed.
5.9.1 Method for the determination of the size of iron particles:
Calibrate an ocular scale against a known stage-micrometre scale.

This is done by placing an ocular scale in the eye piece of a microscope. Focus
the stage micrometre under a desired magnification. Count the number of
ocular scales covering the number of stage micrometre scales and calculate
the factor.
Example: If X number of ocular scales = Y No. of stage micrometer scale then
27
1 ocular scale (factor) = 1 Y/X mm.
Place iron particles under question on a slide and focus at same

magnification. Bring individual particle under the ocular scale. Count the
number of ocular scale covering the two farthest points of the particle.
Multiply this number by the factor in order to get the size of iron particles.
5.9.2 Test for presence of added colouring matter:

Refer to manual on Food Additives
5.9.3 Microscopic Examination:

Follow the same procedure as in clause. 1.9 under analysis of Coffee.
28
SUGAR AND SUGAR PRODUCTS
(HONEY, SUGAR, BURA, JAGGERY, GLUCOSE SYRUP)
6.0 HONEY
6.1 Preparation of sample
If the sample is liquid or strained honey and is free from granulation,

mix thoroughly by stirring or shaking. If granulated place the closed
container in water bath without submerging and heat 30 minutes at 60 0 C,
then if necessary heat at 65 0 C until liquefied. Occasional shaking is essential.
Mix thoroughly and cool rapidly as soon as sample liquefies. Do not heat
honey intended for hydroxyl methyl furfural or diastase determination.
If foreign matter such as wax, sticks, bees, particles of comb etc is

present heat sample to 400C in water bath and strain through cheese cloth in
hot water funnel before sampling.
(Ref: A,O,A,C 17th edn, 2000 Official method 920.180 Honey (Liquid, strained
or comb) Preparation of test sample)
29
6.2 Determination of Moisture:
6.2.1 Refractive Index Method:
Determine Abbe’s refractometer reading of the sample at a constant

temperature near 20ºC and obtain corresponding moisture % from the
following table. If the determination is made at a temperature other than 20 0
C convert the reading to standard temperature of 20 0 C according to the
temperature corrections given below
Relationship between refractive index and water contents of honey:
Water Refractive Water Refractive

Content % Index at 200C Content % Index at 200C
1 2 3 4
13.0 1.5044 19.0 1.4890
13.2 1.5038 19.2 1.4885
13.4 1.5033 19.4 1.4880
13.6 1.5028 19.6 1.4875
13.8 1.5023 19.8 1.4870
14.0 1.5018 20.0 1.4865
14.2 1.5012 20.2 1.4860
14.4 1.5007 20.4 1.4855
14.6 1.5002 20.6 1.4850
14.8 1.4997 20.8 1.4845
15.0 1.4992 21.0 1.4840
15.2 1.4987 21.2 1.4835
15.4 1.4982 21.4 1.4830
15.6 1.4976 21.6 1.4825
15.8 1.4971 21.8 1.4820
16.0 1.4966 22.0 1.4815
16.2 1.4961 22.2 1.4810
16.4 1.4956 22.4 4.4805
16.6 1.4951 22.6 4.4800
16.8 1.4946 22.8 4.4795
17.0 1.4940 23.0 1.4940

17.2 1.4935 23.2 1.4935
17.4 1.4930 23.4 1.4930
17.6 1.4925 23.6 1.4925
30
17.8 1.4920 23.8 1.4920

18.0 1.4915 24.0 1.4915
18.2 1.4910 24.2 1.4910
18.4 1.4905 24.4 1.4905
18.6 1.4900 24.6 1.4900
18.8 1.4895 24.8 1.4895
25.0 1.4940
Temperature corrections to change refractive index
Temperature above 20 0 C - add 0. 00023 per 0 C

Temperture below 20 0 C - substract 0. 00023 per 0 C
(Ref: - F.A.O Manuals of Food Quality Control 14 / 8 page 119 / I.S.I

Hand book of Food Analysis (Part II) – 1984 page 36 / A.O.A.C 17th edn, 2000
Official method 969.38 Moisture in Honey)
6.2.2 Vacuum Oven Drying Method:
Place 20-25 gm pure quartz sand which passes through 500 micron I.S
Sieve and is retained by I.S 180 micron I.S Sieve and a short glass rod in a 6
cm diameter aluminium flat dish. Dry thoroughly and cool in a desiccator and
weigh. Accurately weigh about 5 gm of sample in a beaker and transfer
completely to aluminium flat dish by thorough washing with water. Mix well
and heat on steam-bath for partial drying. Transfer the dish to vacuum oven
and dry the sample at less than 70ºC under 25 mm Hg pressure. After 2 hours
remove the dish to a dessicator allow to cool and weigh. Replace the dish in
the oven for a further period of 1 hour, cool and weigh again Determine the
difference in weight and determine the moisture %.
(Ref: - I.S.I Handbook of Food Analysis (Part II) – 1984 page 35)
31
6.3 Determination of specific gravity
6.3.1 Apparatus
Thermostatically controlled water bath maintained at 27 ± 1 0 C

Specific gravity bottle.
6.3.2 Procedure
Clean and thoroughly dry the sp. gravity bottle and weigh. Fill it upto
the mark with freshly boiled and cooled distilled water maintained at 27 ± 1 0
C and weigh. Remove the water, dry the bottle again and fill it with honey
sample maintained at the same temperature. Weigh the bottle again.
6.3.3 Calculation
C-A
Specific gravity at 27 /27 C =
0
B-A
Where
C = wt of sp. Gravity bottle with honey sample
A = wt of empty sp. Gravity bottle
B = wt of sp. Gravity bottle with water
(Ref: - I.S.I Handbook of Food Analysis (Part II) page 35)
32
6.4 Determination of reducing sugars, total reducing sugars and sucrose

in honey:
Principle:
Invert sugar reduces the copper in Fehling-A solution to a brick red

insoluble cuprous oxide
Reagents:
Fehling A: Dissolve 69.28 g copper sulphate (CuSO4.5H2O) in distilled water.

Dilute to 1000 ml. Filter and store in amber coloured bottle.
Fehling B: Dissolve 346 g Rochelle salt (potassium sodium tartrate) (K Na

C4H4O6. 4H2O) and 100 g NaOH in distilled water. Dilute to 1000 ml. Filter
and store in amber coloured bottle.
Neutral Lead Acetate: Prepare 20% neutral lead acetate solution.

(This reagent is used to clarify sugar solutions)
Potassium Oxalate Solution: Prepare 10% Potassium oxalate(K2C2O4.H2O)

solution. This reagent is used to remove the excess lead used in clarification.
Methylene Blue Indicator: Prepare 1% of methylene blue solution in

distilled water.
6.4.1 Determination of Reducing Sugars:
Weigh accurately 25 gms of sample and transfer to 250 ml volumetric

flask Add10 ml of neutral lead acetate solution and dilute to volume with
water andfilter. Transfer an aliquot of 25 ml of the clarified filtrate to 500 ml
33
volume flask containing about 100 ml water. Add potassium oxalate in small
amounts until there is no further precipitation. Make upto volume. Mix the
solution well and filter through Whatman No. 1 filter paper. Transfer the
filtrate to a 50 ml burette.
Preliminary Titration: Pipet 5 ml each of Fehling A and B into 250 ml

conical flask. Mix and add about 10 ml water and a few boiling chips or glass
beads. Dispense solution. Heat the flask to boiling. Add 3 drops of methylene
blue indicator. Continue the addition of solution dropwise until the blue
colour disappears to a brick-red end point. (The concentration of the sample
solution should be such that the titre value is between 15 and 50 ml). Note
down the titre value.
Final Titration: Pipet 5 ml each of Fehling A and B. Add sample solution

about 2 ml less than titre value of the preliminary titration. Heat the flask to
boiling with in 3 minutes and complete the titration. Perform the titration
duplicate and take the average. Calculate the reducing sugars % as shown
below.
Dilution x Factor of Fehling (in gm) x 100

Reducing Sugars % =
(as Invert Sugar) Weight of sample x titre
6.4.2 Determination of Total Reducing Sugars:
Pipette an aliquot of 50 ml of the clarified, de-leaded filtrate to a 100

ml volumetric flask. Add 5 ml of conc. HCl and allow to stand at room
temperature for 24 hours. Neutralise with conc. NaOH solution followed by
0.1N NaOH. Make up to volume and transfer to 50 ml burette having an offset
tip and perform the titration on Fehlings solution similar to the procedure
described in the determination of reducing sugars.
34
Dilutions x Fehling factor x 100

Total Reducing Sugars % = ------------------------------------------
(as Invert Sugar) Weight of sample x titre
(Ref: - A.O.A.C 17th edn, 2000, Official method 920.183 (b) Sugars (Reducing)
in Honey / I.S.I Hand book of Food Analysis (Part II) – 1984 page 36)
6.4.3 Determination of Factor (for Invert Sugar) of Fehling Solution:
Accurately weigh around 4.75 gms of analar grade sucrose. Transfer

to 500 ml volume flask with 50 ml distilled water. Add 5 ml conc. HCl and
allow to stand for 24 hours
Neutralize with NaOH solution and make up to volume. Mix well and
transfer 50 ml to a 100 ml volumetric flask and makeup to volume. Transfer
to a burette having an offset tip.
Perform the titration of Fehling solution following the similar procedure as

above:
Titre x Weight of sucrose in gm

Fehling Factor =
(for Invert Sugar) 500
35
6.4.4 Determination of Sucrose:
Calculation:
Sucrose %
= (Total reducing sugars / invert sugar % - reducing sugars % ) x 0.95
(Ref: - A.O.A.C 17th edn, 2000, Official method 920.184 Sucrose in Honey /
I.S.I Hand Book of Food Analysis (Part II) – 1984 page 36)
6.5 Determination of Fructose: Glucose ratio
Principle:
Glucose % is determined iodimetrically in a weak alkaline medium

and the value is subtracted from reducing sugars % to arrive at fructose %
and fructose: glucose ratio.
Reagents:
1. 0.1N Iodine: Weigh 13 gm iodine and 20 gm potassium iodide

together and dissolve in water and make up to 1 litre. Store in amber
coloured bottle.
2. 0.2N Sodium bi-carbonate: Dissolve 3.5 gm sodium bicarbonate in 200
ml water.
3. 0.2N Sodium Carbonate: Dissolve 4.25 gm sodium carbonate in 200 ml
water.
4. 25% H2SO4 (v/v).
5. 0.1N Sodium Thiosulphate: Dissolve 25 gm sodium thiosulphate in
boiling distilled water. Cool make up to 1 litre. Filter and store in
amber coloured bottle. Standardize against potassium dichromate.
36
Procedure:
Weigh 2 g of sample to 250 ml volumetric flask and make upto volume

Mixwell and transfer an aliquot of 25 ml to a 250 ml of iodine flask. Pipet 50
ml of 0.1N Iodine and add 50 ml of 0.2N sodium carbonate and 50 ml of 0.2N
sodium bicarbonate solution Allow to stand in dark for 2 hours Acidify with
12 ml of 25% H2SO4 and titrate with standard sodium thiosulphate using
starch as indicator. Carry out blank simultaneously Subtract from titre value
of blank the titre value of sample.
Calculation:
Glucose %
Normality of thiosulphate x dilution x (B – S) x 0.009005 x 100
=
0.1N x weight of sample
Fructose % = Reducing sugars % - glucose %
Fructose %
Fructose: Glucose ratio =
Glucose %
(Ref: - F.A.O Manuals of Food Quality Control 14 / 8 page 125 / Pearson’s

Chemical Analysis of Foods 7th edn, 1976 page 141)
6.6 Test for commercial Invert Sugar:
6.6.1 Fiehe’s Test:
Reagents:
1. 1% Resorcinol: Dissolve 1 gm Resorcinol in 100 ml conc. HCl.

2. Diethyl Ether, AR grade.
37
Procedure:
Dissolve around 2 gms of sample in 50 ml water and extract with

diethyl ether in a separatory funnel. Collect the ether layer in a porcelain
basin and evaporate the ether. Add 4-5 drops of freshly prepared resorcinol
solution.
Appearance of cherry red colour indicates the presence of commercial

invert sugar.
(Note: This test should not be taken as a conclusive test.)
(Ref: - I.S.I. Handbook of Food Analysis (Part II) page 37)
6.6.2 Aniline Chloride Test:
Aniline Chloride Reagent: To 100 ml aniline, add 30 ml 25% HCl.
Procedure:
Introduce about 5 gm of sample into a porcelain basin and add while

stirring 2.5 ml of freshly prepared aniline chloride reagent. In the presence of
commercial invert sugar, the mixture assumes within 1 minute orange red
colour turning dark-red. Yellow to salmon shades are to be disregarded.
(Ref: - I.S.I Handbook of Food Analysis (PartII) page 38)
6.7 Determination of Ash:
Weigh accurately 5 - 10 gm of sample in a previously dried and

weighed silica dish. Char the sample on a burner and transfer the dish to
38
muffle furnace maintained at 550 ºC. Cool in a desicccator and weigh.

Incinerate to a constant weight and calculate the % ash.
(Ref: - I.S.I Handbook of Food Analysis (Part II) page 37)
6.8 Determination of Acidity
6.8.1 Reagents
(1) Standard Sodium Hydroxide solution – 0.05 N

(2) Phenolpthalein indicator – Dissolve 0.5 gm Phenolpthalein in 100 ml
of 50% ethyl alcohol (v/v)
6.8.2 Procedure
Take 10 gm of the sample in a suitable titration flask and dissolve in

75 ml of carbon dioxide free water. Mix thoroughly. Titrate against standard
sodium hydroxide solution using 4-6 drops of phenolpthalein indicator till
pink colour persists for 10 seconds.
Determine blank on water and indicator and correct the volume of

sodium hydroxide solution used.
6.8.3 Calculation
0.23 X V
Acidity as formic acid (%) by weight =
M
Where,
V = corrected volume of 0.05 N sod. Hydroxide used
M = weight in gm of the sample taken for test
(Ref: - I.S.I. Handbook of Food Analysis (Part II) 1984 page 37)
39
7.0 CANE SUGAR AND REFINED SUGAR
7.1 Preparation of Sample
Grind if necessary and mix to a uniform mass. Thoroughly mix raw

sugar (Gur, Jaggery) in minimum time. Break up any lumps on a glass plate or
in a pestle and mortar. Transfer to a dry stoppered container
(Ref: - A.O.A.C 17th edn, 2000 Official method 920. 175 Preparation of
Sample)
Transfer 5 g of the prepared sample in a previously dried, tared

aluminium dish. Cover the dish with the lid and weigh accurately. Remove
the lid and dry the sample at 105 0 ± 1 0C for 3 hours. Cool in a desiccator and
weigh.
Redry 1 hour and repeat process until change in weight between two
successive dryings is less than 2 mg. Report the loss in weight as moisture.
(Ref: - A.O.A.C 17th edn, 2000 Official method 925.45 (b) (except 105 0 C
temperature as per P.F.A) Moisture in Sugars)
Note: - The ICUMSA method (1974) requires a forced draft oven maintained
at a temperature 0f 105 ± 10 C as measured 2.5 ± 0.5 cm above the dishes in
the test. The oven is to be ventilated and the circulation fan fitted with an
interlock switch which opens when the oven door is opened the dishes with
tight fitting lids should be 6-10 cm with a depth of 2-3 cm although the dish
may be made of glass, platinum or Nickel, aluminium is recommended. The
quantity of sample recommended is 20 – 30 gm, the depth of sample in the
40
dish not to exceed 1 cm.
7.3 Determination of Ash:
Follow the same procedure as given for honey under Sec. 6.0.
7.4 Determination of Sucrose:
Weigh exactly around 10 gm of prepared sample and make upto 250

ml volume. Determine the reducing sugars as described in the analysis of
honey in Sec. 6.4.
Take an aliquot of 100 ml in a 500 ml volumetric flask and add 10 ml

of HCl and let stand for 1 ½ days at 25ºC and above. Dilute to 500 ml.
Transfer an aliquot of 100 ml to a 250 ml volumetric flask, neutralize with
NaOH and make up to volume and mix. Take this solution in a burette having
an offset tip. Proceed with the titration against Fehling A and B similarly as
described in the analysis of honey.
Calculation: See under honey analysis.
Sucrose %
= [Reducing sugars % after inversion – Reducing sugars %
before inversion] x (0.95)
7.4.1 Alternate Method (Polarisation method)
7.4.1.1 Principle
An aqueous solution of the sugar sample (26 gm, i.e the normal weight
of sucrose in 100 ml water.) is polarized by means of a saccharimeter which
is calibrated to read 1000 S on the ‘ International Sugar Scale’ under specified
41
conditions.
7.4.1.2 Apparatus
Saccharimeter, calibrated with quartz plates. Basis of calibration of

100 0 points on international sugar scale is polarization of normal solution of
pure sucrose (26.000 gm / 100 ml) at 20 0 C in 200 mm tube using white light
and dichromate filter defined by thecommission. This solution polarized at
200 C must give saccharimeter reading of exactly 100 0 S Temperature of
sugar solution during polarization must be kept constant at 20 0 C.
Following rotations hold for normal quartz plate of international sugar scale:
NormalQuartz Plate =100 0 S = 40.690 ± 0.002 (´= 546.1 nm) at 200C
In general make all polarizations at 20 0 C. For countries where mean
temperature is above 20 0 C. Saccaharimeters may be adjusted at 30 0 C or any
other suitable temperature, provided sugar solution is diluted to final volume
and polarized at this temperature.
7.4.1.3 Procedure
In determining polarization use whole normal weight (26± 0.002 gm)

for 100 ml or multiple for any corresponding volume. Bring solution exactly
to mark at proper temperature and after wiping out the neck of the flask with
filter paper add minimum amount of dry basic lead acetate, shake to dissolve.
Repeating addition till precipitation is complete Pour all clarified sugar
solution on rapid air dry filter. Cover funnel at start of filteration. Reject first
25 ml filterate and use remainder (must be perfectly clear) for polarization.
In no case return whole solution or any part to filter.To remove excess lead
used in clarification add anhydrous Pot or Sod Oxalate to clarified filterate in
small amounts until test for lead in filterate is negative, then refilter Polarise
in 200 mm tube. Other permissible clarifying and decolorizing agents are
42
alumina cream or conc alum solution Temperature correction for

polarization of sugars. Polarisation when made at temperatures other than
20 0 C may be calculated to polarization at 20 0 C by the following formula:
P 20 = P t [1 +0.0003 (t – 20)]
Where, Pt = polarization at temperature read.

(Ref: - A.O.A.C 17th edn, 2000 Official method 925.46 Sucrose in Sugars and
syrups Polarimetric method)
7.4.1B Polarimetric method (Before and After inversion with HCl)
(a) Direct reading - Pipette 50 ml Pb free filterate into 100 ml volumetric

flask, add 2.315 gm NaCl and 25 ml water Dilute to volume with water at 200
C and polarize in 200 mm tube at 20 0 C. Multiply reading by 2 to obtain
direct reading.
(b) Invert Reading - Pipette 50 ml aliquot Pb free filterate into 100 ml

volumetric flask and add 20 ml water. Add little by little while rotating flask
10 ml HCl (sp. gr 1.109). Heat water bath and adjust heater to keep bath at
600 C. Place flask in water bath, agitate continuosly for 3 minutes and leave
flask in bath exactly 7 minutes longer. Place flask at once in water at 20 0 C.
When contents cool to 35 0 C dilute almost to mark,. Leave flask in bath at 20

0 C at least 30 minutes longer and finally dilute to mark.
Mix well and polarize in 200 mm tube provided with lateral branch
and water jacket keeping temperature at 20 0C . Multiply by 2 obtain invert
reading.
43
Calculate Sugar % as follows
100 ( P –I )
S=
132.66 - 0.0794(13-m) - 0.53 ( t – 20 )
Where,
P = direct reading, normal solution

I = Invert reading, normal solution
t = Temperature at which readings are made
m = gm of total solids from original sample in 100 ml inverted solution
(Ref: - A.O.A.C 17th edn, 2000 Official Method 925.48 Sucrose in sugars and
Syrups)
7.5 Determination of Sulphur Dioxide:
Sulphur dioxide is determined by the modified Monier-William’s Method
The apparatus as assembled is shown below:
44
7.5.1 Reagents
(a) Sodium Carbonate Solution- 10 percent (m/v). aqueous
(b) Bromophenol Blue Indicator Solution – Dissolve 0.1 g of

bromophenol blue in 3.0 ml of 0.05 N sodium hydroxide solution and
5ml of ethyl alcohol (90 percent by volume) by gently warming. Make
up the volume of the solution with ethyl alcohol (20 percent v/v) to
250 ml in a volumetric flask.
(c) Hydrogen peroxide solution- Dilute a 30 percent (m/v)

hydrogen peroxide solution with about twice its volume of water and
neutralize the free sulphuric acid that may be present in the hydrogen
peroxide solution with barium hydroxide solution, using
bromophenol blue indicator solution. Allow the precipitate of barium
sulphate to settle, and filter. Determine the concentration of hydrogen
peroxide in the filtrate by titrating with standard potassium
permanganate solution. Dilute the filtrate with cold water so as to
obtain a 3 percent (m/v) solution of hydrogen peroxide.
(d) Concentrated Hydrochloric acid- sp.gr. 1.16
45
(e) Carbon dioxide gas- from a cylinder.
(f) Standard sodium hydroxide solution- approximately 0.1 N,

standardized at the time of the experiment using bromophenol
blueindicator solution.
7.5.2 Procedure
Assemble the apparatus as shown above. Introduce into the flask C, 300 ml of
water and 20 ml of concentrated hydrochloric acid through the dropping
funnel E. Run a steady current of cold water through the condenser F. Boil
the mixture contained in the flask G for a short time to expel the air from the
system in current of carbon dioxide gas previously passed through the wash
bottle A. Weigh accurately about 100 g of the material and mix with the
minimum quantity of water so as to make the diluted material easily flow
down to the dropping funnel. Introduce the diluted material into the flask C
through the dropping funnel E. Wash the dropping funnel with a small
quantity of water and run the washing into the flask C. Again boil the mixture
contained in the flask C in a slow current of carbon dioxide gas (passed
previously through the wash bottle A) for one hour. Just before the end of the
distillation, stop the flow of water in the condenser. (This causes the
condenser to become hot and drives over residual traces of sulphur dioxide
retained in the condenser.) When the delivery tube H, just above the
Erlenmeyer flask j, becomes hot to touch, remove the stopper J immediately.
Wash the delivery tube H and the contents of the Peligot tube L with
water into Erlenmeyer flask. Cool the contents of the Erlenmeyer flask to
room temperature, add a few drops of bromophenol blue indicator and
titrate with standard sodium hydroxide solution.(Bromophenol blue is
unaffected by carbon dioxide and gives a distinct change of color in cold
46
hydrogen peroxide solution). Carry out a blank determination using 20 ml of

conc hydrochloric acid diluted with 300 ml of water.
7.5.3 Calculation
0 .032000 (V-v) x 1000 x 1000 x N

Sulphur dioxide, mg/kg =
W
Where,
V = volume in ml of standard sodium hydroxide solution
required for the test with the material.
v = volume in ml of standard sodium hydroxide solution
required for the blank determination;
N = normality of standard sodium hydroxide solution; and
W = weight in g of the material taken for the test
(Ref: - I.S.I Handbook of Food Analysis (Part II) -1984 page 8)
Note: Rosaniline method can also be used as alternative method for

determination of sulphur dioxide.
47
7.6.3 Procedure
7.6.3.1 Platinizing the electrodes of the conductivity cell
Wash the electrodes of conductivity cell first with warm chromic acid
solution and then several times with distilled water. Support the electrodes
in an inclined position in the chloroplatinic acid solution and connect by way
of a commutator to a 4 volt lead accumulator and rheostat. Adjust the current
so that the evolution of gas is slow. Reverse the current every 30 seconds.
Thus continue to pass the current for 15 minutes. Disconnect the
conductivity cell wash it with distilled water thoroughly and fill with dil
solution of sulphuric acid. Elecrtolyze the solution of sulphuric acid for ½
hour to remove occluded gases, reversing the current every 30 seconds.
Wash the cell wall with conductivity water.
Note: The cleanliness of the cell is confirmed by determining the conductivity

of the conductivity water, washing out the cell and making a second
determination of the conductivity water. Two successive determinations
shall give concordant measurement of the conductivity if the cell is clean.
7.6.3.2 Determination of the cell constant
Wash the conductivity cell with conductivity water. Then rinse with
the standard Potassium chloride solution. Transfer sufficient quantity of Pot
chloride solution so that the electrodes are well within the solution, taking
care that no air bubbles are enclosed between the electrodes. Place the
conductivity cell in a thermostat. Maintain the temperature of the thermostat
at 35 ± 10C. Ensure that all the connections made are with fairly thick copper
wire and tight. When Pot chloride solution has attained the temperature of
the bath, measure the observed conductivity of the solution. Report twice the
measurement by replacing a fresh Pot Chloride solution.
48
Calculate the cell constant as follows

C
K =
O1
Where,
K = cell constant
C = specific conductivity of Pot chloride solution at 350 C, that is 3.3.1X 10 3
Mhos / cm
O 1 = Observed conductivity of Pot Chloride solution
7.6.3.3 Determination of specific conductivity
Dissolve 10 gm of the material (accurately weighed) in 200 ml of

conductivity water. Wash the conductivity cell thoroughly with distilled
water and then with conductivity water and later rinse with test solution
twice. Determine the observed conductivity at 350 C. Repeat with a fresh
sample of test solution and take the average value.
Determine the conductivity of conductivity water at 35 0C in the same
manner.
Calculate the specific conductivity x 106 of 5 % (w/ v) aqueous solution at 35

0 C as follows:
S = [O 2 – (0.9 X O 3) ] x K x 10 6
Where,
S = specific conductivity of test solution X 10 6

O2 = observed conductivity of test solution
O3 = observed conductivity of conductivity water
49
K = cell constant
(Ref: - I.S.I. Handbook of food analysis (Part II) – 1984, page 7)

(Ref: - IS 15729: Sugar and sugar products)
7.7 Determination of Calcium oxide
7.7.1 Apparatus
(1) Calibrated Brix spindle

(2) Brix Cylinder
(3) Conical flasks - 250 ml capacity
(4) Beakers – 100 and 200 ml capacity
(5) Funnels
(6) Pipettes- calibrated at 10 ml
7.7.2 Reagents
(1) EDTA solution – Weigh accurately 6.6473 gm EDTA into a beaker ,

dissolve in distilled water and make upto 1000 ml to obtain exactly M
/ 56 solution
(2) Ammonia Liquor
(3) Lead Subacetate
(4) Potassium Ferrocyanide powder
(5) Potassium iodide

Eriochrome Black – T – weigh 0.1 eriochrome black T in a 100 ml
volumetric flask and dissolve the same in rectified spirit or absolute
50
alcohol. Make upto volume and use as indicator.
7.7.3 Procedure
Make a 15 0 Brix solution of the sample. Transfer about 150 ml of the
solution to a conical flask. Clarify the solution with Lead subacetate. Transfer
about 60 ml of the clarified solution to a dry conical flask or flask previously
rinsed with the clarified solution. Add Potassium Ferrocyanide powder little
by little till no further precipitate forms. Shake thoroughly and filter. Test the
filterate with Pot. Iodide. Collect the lead free filterate in a conical flask
Pipette out 10 ml of lead free filterate in a clean conical flask previously
rinsed with distilled water and dried. Add 5 – 6 drops of liquor ammonia and
4-5 drops of indicator when a pink colour appears. Titrate against EDTA
solution shaking the flask after each addition of EDTA solution. The end point
is indicated by a sharp change of colour from red to blue. Note down the
volume of the titrant.
7.7.4 Calculation
Calcium oxide mg / 100 gm = V X 100 mg per litre of diluted solution
(Ref :- I.S.I. Handbook of Food Analysis (Part II) – 1984 page 9)
7.8 Determination of Reducing Sugars :-
The following methods can be used for reference:

 Luff school method, GS 1-5 for reducing sugars,
 Modified offner method, GS 2-6 for white ad refined sugars
IS 15729 also has adopted the modified Offner Method
51
8.0 BURA:
8.1 Determination of Ash insoluble in dilute HCl:
Weigh 5 gm of sample in a silica dish. Char the sample and ash in a

furnace at 550ºC.Add 25 ml of 1:2.5 Hydrochloric acid to the ash. Cover the
dish with a watch glass and boil for 5 minutes. Cool, filter through an ashless
filter paper (Whatman No. 42 or 41) and wash the residue well with hot
water until acid-free. Return the filter paper to the silica dish and incinerate
at 525ºC. Cool, weigh and calculate as % acid insoluble ash.
(Ref: - I.S.I. Handbook of Food Analysis (Part IX) 1984) page 52)
8.2 Determination of total sugars expressed as sucrose:
Follow the procedure given under cane sugar for sucrose. Invert the
solution with HCl Conduct the titration and calculate as given under.
Total sugars % expressed as sucrose = Total reducing sugars % x 0.95
52
9.0 GUR OR JAGGERY:
Determine the moisture using a vacuum oven at 70ºC.
9.2 Determination of extraneous matter insoluble in water:
Take 10 gm of sample, add 200 ml hot distilled H2O and bring to

boiling. Allow to cool to room temperature. Filter through a tared gooch
crucible having a bed of asbestos or sintered glass filter Wash the residue
with hot water till the filtrate is sugar-free (perform Molisch test). Dry the
gooch crucible or sintered glass filter at 135 ± 20 C and weigh. Express as %
insoluble matter.
(Ref: - I.S.I Handbook of Food Analysis (Part II) – 1984 page10)
9.3 Determination of total Ash:
Follow the procedure as given under analysis of Honey. (Sec. 3.5)
9.4 Determination of ash-insoluble in dilute HCl:
Follow the procedure as given under Analysis of Bura. (Sec 5.1).
10.0 Determination of Sulphated Ash in Dextrose:
Weigh accurately about 10 gm 5 g sample into a silica dish, add 0.5 ml

of conc sulphuric acid or 5 ml of 10% (by weight) H2SO4. Heat on hot plate or
burner to carbonize the sample (perform in a hood with exhaust facility).
Then ash in the furnace at 550 ºC. Cool, add again 2 ml of 10% H2SO4,
53
evaporate on steam bath and dry on hot plate and again ash at 550 ºC to
constant weight. Express as % sulphated ash.
(Ref: - I.S.I. Handbook of Food Analysis (Part II) – 1984 page 18)
11.0 Determination of Starch in Icing Sugar:
If the iodine test for starch is positive, proceed as follows. Weigh

suitable quantity of sample and dissolve with 100 ml of hot water. Cool, add
equal volume of alcohol, stir and let stand for 2 hours. Filter the solution
through Whatman No. 1 filter paper. Wash the precipitate with 50% alcohol
until the washings does not answer the Molisch test for sugars.
Transfer the precipitate with 200 ml hot water into a flask. Add 20 ml
HCl and connect the reflux condenser and heat in boiling water bath for 2.5
hours. Cool, neutralise with NaOH and dilute to 500 ml. Determine % of
glucose by Lane Eynon’s method.
Calculation:
Starch % = Glucose % x 0.90
(Ref: - Modified A.O.A.C method, 17th edn, 925.50, Starch in Confectionery,
last para)
12.0 Determination of Total solids in Liquid Glucose
12.1 Apparatus
(1) Moisture dish – approx 75 mm dia and 25 mm high

(2) Dessicator
(3) Vaccum oven
(4) Nickel scoop
54
(5) Glass stirring rod

(6) Diatomaceous earth – Neutral to litmus when moistened with
water. If a commercial grade is used, wash it by percolation with
water slightly acidulated with hydrochloric acid until the effluent
is acidic to litmus. Then rewash with water (not acidulated) until
the effluent is neutral to litmus. Finally dry in an oven at about
1050 C,
12.2 Procedure
Place about 10 gm of diatomaceous earth and the glass rod in the

weighing dish and dry them in an air oven at about 105 0 C. Cool in a
dessicator and weigh. Repeat drying and cooling in dessicator and weighing
till constant weight is achieved. Weigh accurately about 5 gm of the material
in the nickel scoop, mix it with 5 ml of water and run on the diatomaceous
earth using glass stirring rod. Wash the scoop three times with 2 ml water
and transfer the washings to diatomaceous earth working the contents of the
dish into a thick paste with the stirring rod. Leave the stirring rod in the dish.
Place the moisture dish in the vacuum oven and dry contents of dish at 100
0C. Cool the dish in a dessicator and weigh.. Repeat drying in the vacuum
oven, cooling and weighing till constant weight is obtained.
12.3 Calculation
Total solids % by wt = 100 - [100(M + A – B)]

M
Where
M = weight in gm of the material taken for test
A = weight in gm of moisture dish with glass rod and

diatomaceous earth.
55
B = weight in gm of moisture dish with glass rod,

diatomaceous earth and dehydrated material.
(Ref: - I.S 873: 1974 Specification for Liquid Glucose / I.S.I. Handbook of Food
Analysis (Part III) – 1984 Page64)
56
PART B
CONFECTIONERY PRODUCTS
A. SUGAR BOILED CONFECTIONERY & LOZENGES
Sugar Boiled Confectionery - As per FSSA, Sugar boiled confectionery is a

processed composite food article made from sugar with or without doctoring
agents such as cream of tartar by process of boiling whether panned or not. It
may be center filling or otherwise which may be in the form of liquid, semi-
solid or solids with or without coating of sugar or chocolate or both.
Lozenges - As per FSSA, it is a confection made mainly out of pulverised

sugar, or icing sugar with binding materials such as edible gums, edible
gelatine, liquid glucose or dextrin and generally made from cold mixing
which does not require primary boiling or cooking of the ingredients.
A.1 Preparation of sample
If composition of entire product is desired, grind and mix thoroughly.

If product is composed of layers or of distinctly different portions and it is
desired to examine these individually, separate with knife or other
mechanical means as completely as possible, and grind and mix each test
portion thoroughly.
th
[Ref: - A.O.A.C 18 edn, 2005 Official Method 925.49 Preparation of Test
sample]
A.2 Determination of Moisture - Vacuum Oven method
Apparatus
(1) Aluminum dish – 75mm diameter and about 25mm height with
close fitting cover
57
(2) Dessicator
(3) Vacuum oven
Procedure
1. Accurately weigh about 5 g of sample in a dish previously dried and

weighed. Distribute the material as evenly as practicable over the bottom
of the dish by gentle sidewise movements.
2. Place dish in vacuum oven, remove cover of dish and dry the material for
two hours at 65 ± 1º C at a pressure not exceeding 50mm of Hg. During
heating admit slow current of air into oven.
3. Cover dish, transfer to dessicator and weigh soon after room temperature
is attained.
4. Redry for one hour and repeat the process till the difference between the
two successive weighing is less than 2 mg. Report percent loss in weight
as moisture %.
Calculations
(W3 - W2)
Moisture content, % by mass = X 100
W1
Where,
W1 = Weight of prepared sample taken for test in g
W2 = Weight of empty moisture dish in g
W3 = Weight of (dish + dried sample) in g
[Ref: - IS: 6287-1985 (Reaffirmed 2010) Methods of Sampling and Analysis

for Sugar Confectionery]
58
A.3 Determination of Sulphated Ash
Reagents
(1) Sulphuric Acid - 10 percent (m/m)
Procedure
1. Accurately weigh about 5 g of the prepared sample into a 9-cm diameter

platinum basin.
2. Add 5 ml of sulphuric acid to the material in the dish. Gently heat the dish
on a hot plate until the material is well carbonized and then increase the
heat until the evolution of sulphuric acid fumes ceases.
o
3. Ash the carbonized matter in a muffle furnace at 550 ± 25 C.
4. Cool the ash and moisten it with 2-3 ml of sulphuric acid. Heat strongly on
a hot plate until sulphuric acid fumes ceases to be evolved and finally ash
o
in the muffle furnace at 550 ± 25 C for two hours.
5. Cool in a desiccator and weigh.
o
6. Heat again in a muffle furnace for 30 minutes at 550 ± 25 C. Cool in a
desiccator and weigh.
7. Repeat the process of heating in the muffle furnace for 30 minutes,
cooling and weighing till the difference between two successive weighing
is less than 1 mg. Record the lowest mass.
Calculation
M1 x 100
Sulphated ash, % by mass = X 100
M2
Where,
M1 = mass in g of the sulphated ash
M2 = mass in g of the prepared sample taken for the test.
59

A.4 Determination of Sulphated ash on salt free basis - delete
Procedure:
1. Determine Sodium Chloride content in the sample separately (as

mentioned in A.4.1)
2. Convert the quantity of Sodium Chloride obtained to Sodium Sulphate
(2NaCl = Na2 SO4) i.e. 117: 142
3. Ratio of Na 2 SO4 / Na Cl = 142 / 117 = 1.21
4. Multiply the Sod. Chloride content obtained with 1.21.
5. Deduct the value thus obtained from the sulphated ash to obtain
sulphated ash on salt free basis.
A.4.1 Titration method
Apparatus
(1) Pipette — Graduated, 1 ml capacity

(2) Conical flask — Glass-stoppered, 250 ml capacity
Reagents
(1) Standard AgNO3 Solution — 0.1 N

(2) Potassium chromate indicator solution – 5% solution in water
Procedure
1. Weigh appropriate sample quantity in a conical flask.
2. Add 100 ml of boiling water.
60
3. Let it shake for 30 minutes to 1 hour, ensuring complete dissolution of

salt present in sample.
4. Filter the contents through a filter paper.
5. Titrate the filtrate thus obtained against standard 0.1 N AgNO3 using
Potassium chromate as indicator, till brown colored end point persists for
30 seconds.
6. If the colour of the sample solution interferes in titration, an alternative
potentiometric method can be used.
Calculation
58.44  N  (A-B)
Sodium chloride, % by mass = ------------------------
Where,
N = normality of AgNO3 solution,
A = volume, in ml, of AgNO3 solution in the blank titration
B = volume, in ml, of AgNO3 solution in the sample titration, and
W = weight, in g, of the sample.
th
[Ref: - A.O.A.C 18 edn, 2005 Official Method 960.29 Salt in Butter & A.O.A.C
th
18 edn, 2005 Official Method 971.27 Sodium chloride in canned vegetables]
A.5 Determination of Ash Insoluble in dilute HCl
Reagents
(1) Dilute hydrochloric acid - Approx 5 N (445ml in 1litre)
61
Procedure
1. Weigh accurately about 5 g of the prepared sample in a tared, clean and

dry platinum basin of 100ml capacity.
2. Carbonize the material in the dish with the flame of a burner.
3. Complete the ignition by keeping in a muffle furnace at 550 + 25ºC until
gray ash results.
4. Cool in a desiccator.
5. To the ash, add 25ml of the dilute hydrochloric acid, cover with a watch
glass and heat on a small flame of a burner to near boiling.
6. Allow it to cool and filter the contents of dish through Whatman filter
paper No. 42 or its equivalent. Wash the filter paper with hot water until
the washings are free from chlorides (To check this, add few drops of 2M
Nitric acid and 0.1 M Silver nitrate solution to the filtrate obtained. No
precipitate or milky turbidity should occur in the solution, if it is chloride-
free.)
7. Return the filter paper and the residue to the dish. Keep it in an air oven
maintained at 105 +2ºC for about three hours. Ignite in the muffle furnace
at 550 +25ºC for one hour.
8. Cool the dish in a desiccator and weigh.
9. Heat again for 30 minutes in the muffle furnace, cool and weigh.
10. Repeat this process of heating for 30 minutes, cooling and weighing till
the difference between two successive weighing is less than one
milligram. Note the lowest mass.
Calculation
Acid insoluble ash, percent by mass = 100 x M1

M2
Where,
M1 = mass in g of the acid insoluble ash
62
M2 = mass in g of the prepared sample taken for the test

A.6 Test for the presence of added synthetic colour
Paper Chromatographic Separation of Synthetic Food Colours
The general scheme for identifying synthetic food colours present in

foods normally involve preliminary treatment of the food, extraction of the
colour from the prepared solution of the food, separation of colours in case of
mixtures and identification of the separated colours.
Apparatus
(1) Pipette
(2) Beaker
(3) Flask
Reagents
(1) White knitting wool:- Extract pure white wool in a soxhlet extractor with
petroleum ether for 2-3 hrs to remove fat. Boil in very dilute solution of
sodium hydroxide and then in water to free it from alkali.
(2) Paper: Whatman No. 1 chromatographic paper.
(3) Solvents
 1 ml (0.88 sp. gr) ammonia + 99 ml water

 2.5% aqueous sodium chloride
 2% sodium chloride in 50% ethanol
63
 Acetic acid solution in water (1:3)

 Iso-butanol-ethanol-water (1: 2 : 1, v/v)
 n-butanol-water-glacial acetic acid (20 : 12 : 5, v/v)
 Iso-butanol-ethanol-water (3 : 2 : 2, v/v). to 99 ml of this add 1ml of
 (0.88 sp gr.) ammonia
 80 gm phenol in 20 gm water.
Procedure
1. Preliminary treatment of food: Assuming that an acidic colour is present,

the preliminary treatment involves removing interfering substances and
obtaining the dye in acid solution prior to boiling with wool.
 Non-alcoholic beverages e.g. soft drinks: As most foods in this group are
acidic they can be usually treated directly with wool, otherwise, slightly
acidify the food with acetic acid.
 Alcoholic liquids (e.g. Wine): Boil to remove alcohol and acidify if
necessary as in (a).
 Starch based foods (e.g. cakes, custard powder etc): Grind 10 g of sample
thoroughly with 50 ml of 2 % ammonia in 70% alcohol, and allow it to
stand for an hour and centrifuge. Pour the separated liquid into a dish
and evaporate on water bath. Take up the residue in 30 ml dilute acetic
acid.
 Candied fruits: Treat as in (c).
 Products with high fat content (e.g. Sausages, meat, fish paste): De-fat the
sample with light petroleum and extract the colour with hot water
(acidify etc. as usual). Note that oil soluble colours tend to give coloured
solutions in organic solvents.
If the extraction is difficult treat with warm 50-90% acetone or alcohol
(which precipitates starch) containing 2% ammonia. The organic solvent
should be removed before acidifying as in (c).
64
2. Extraction of the colour from the food: Introduce about 20 cm length of

woollen thread into a beaker containing about 35 ml of the prepared
acidified solution of the sample and boil for a few minutes till the woollen
thread is dyed. Take out the woollen thread and wash it with tap water.
Transfer the washed woolen thread to a small beaker containing dilute
ammonia and heat again. If the colour is stripped by the alkali, the
presence of an acid coal-tar dye is indicated. Remove the woollen thread.
Make the liquid slightly acidic and boil with a fresh piece of woollen
thread. Continue boiling until the colour is taken by the woollen thread.
Extract the dye from the woolen thread again with a small volume of
dilute ammonia, filter through a small plug of cotton and concentrate the
filtrate over a hot water bath. This double stripping technique usually
gives a pure colour extract. Natural colours may also dye the wool during
the first treatment, but the colour is not usually removed by ammonia.
Basic dyes can be extracted by making the food alkaline. with ammonia,
boiling with wool and then stripping with dilute acetic-acid. At present,
all the permitted water soluble coal-tar dyes are acidic, hence an
indication of the presence of a basic dye suggests that an unpermitted
colour is present
3. Identification of the separated food colours by paper chromatography:

Draw a pencil-line parallel to the bottom edge of the paper Whatman
No.1) at about 2 cm distance. Spot the concentrated solution of the
unknown dye on the line together with a series of spots (about 2 cm
apart) of aqueous solutions of standard permitted dyes of similar colour
and dry. Run the chromatogram, by ascending technique, using a selected
solvent. Solvent No.5 is often helpful for general purposes. Identify the
colour in the sample by matching its spot with the spot of the standard
colour and confirm by co spotting
65
4. Determination of Synthetic food colours in food products:
(1) For samples containing single colour
(a) Preparation of standard curve: Stock solution: Weigh 0.1 g m of each

reference colour and dissolve in 0.1N HCl in separate 100 ml
volumetric flasks and make up the volume with 0.1N HCl in each case.
Working standard: Pipette 0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 ml of stock
solution of each of the reference colours into series of clean and dry
100 ml volumetric flasks and dilute to volume with 0.1N HCl.
Determine the optical densities of each of the reference colours at the
respective wave length of maximum absorption (refer table) Obtain
the standard curve for each colour by plotting optical density against
concentration.
(b) Determination in sample by column chromatography: Transfer a

known weight of the sample (approximately 5 - 10 gm) into a glass
stoppered separatory funnel. Extract the colour with 70% acetone.
Shake acetone extract with petroleum ether (40-60ºC) in order to
remove carotenoids and other natural pigments, if any. Continue
extraction with petroleum ether until petroleum ether extract is
colourless. Pass the acetone extract containing only coal-tar food
colours through a column (2.1 · 45 cm) containing aluminium oxide
acidified with 1% HCl. Elute the adsorbed colour with 1% ammonia.
Evaporate the eluate to dryness on a hot waterbath, dissolve the
residue with 0.1 N HCl, transfer quantitatively to a 100 ml volumetric
flask and make up the volume with 0/1N HCl. Determine the optical
density of the dye solution at the wavelength of maximum absorption.
Calculate the dye concentration from the standard curve.
66
TABLE SHOWING ABSORPTION MAXIMA
OF PERMITTED FOOR COLOURS
Name of the Colour Absorption maxima (nm)
Carmosine 516
Ponceau 4 R 507
Erythrosine 527
Green FCF 624
Indigo Carmine 609
Brilliant Blue FCF 630
Tartrazine 427
Sunset yellow FCF 482
(2) For samples containing mixture of colours
(a) Paper Chromatography:
Extract the colours present in the samples and isolate as described

under column chromatography. Make up the purified dye solution to a
known volume with water (5 ml).
Spot an aliquot (approximately 0.5 to 1.0 ml) of the purified dye on

Whatman No.1 filter paper as a band and develop the chromatogram
using butanol : acetic acid: water (20:5:12) solvent system. After
drying, cut out the coloured spots on the chromatogram and elute
with 0.1 N HCl. Prepare a blank by cutting an equivalent strip from
plain portions of the chromatogram and elute with 0.1 N HCI. Make up
67
the eluate to a known volume (100 ml) with 0.1 N HCI and determine
the dye content as described under column chromatography.
(b) Thin Layer Chromatography:
Extract the colours present in the sample as described under column

chromatography. Concentrate the eluate and make up to known
volume with water (5 ml).
Preparation of TLC Plate:
To 50 g of silica gel without binder add 50 ml of starch solution (0.6

gm of soluble starch dispersed in 100 ml or glass distilled water heated to
boiling to gelatinise starch) and 50 ml of 1.25% solution of disodium salt of
ethylene diamine tetra acetic acid , Mix the slurry well. Spread slurry using
applicator on glass plates (20 · 20 cm) to thickness of 0.5 mm. Allow the plate
to air dry and then dry at 120ºC for two hours. Spot an aliquot of the purified
dye on TLC plate and develop the chromatogram using isoamyl : glacial acetic
acid : water (40 : 20 : 20) solvent system. Remove the plate and dry. Scrape
out the colour spots on the plate and transfer to test tubes. Elute the colour
using 0.1N HCl. Prepare a blank by scraping from the plain portions of the
plate. Make up the eluates to a known volume (100 ml) with 0.1N HCl.
Determine the dye content as described under column chromatography.
(Ref: - Manual Methods of Analysis for Adulterants and Contaminants in

Food, I.C.M.R 1990 Page 56)
A.7 Determination of Total Protein (in Milk Toffee)
Apparatus
(1) A recommended distillation assembly is shown below - The assembly

consists of a round bottom flask A of 1000 ml capacity fitted with a rubber
68
stopper through which passes one end of the connecting bulb tube B. the
other end of the bulb B is connected to the condenser C which is attached, by
means of a rubber tube, to a dip tube D which dips into a known quantity of
standard sulphuric acid contained in a beaker E of 250 ml capacity.
(2) Kjeldahl flask – 500 ml capacity
Distillation assembly for Protein Estimation
Reagents
(1) Anhydrous Sodium sulphate
(2) Copper Sulphate
(3) Concentrated Sulphuric Acid- sp gr 1.84
(4) Sodium Hydroxide Solution- Dissolve about 225 g of sodium

hydroxide in 500 ml of water
(5) Standard Sulphuric Acid - 0.1 N
(6) Methyl red indicator solution- Dissolve one g of methyl red in 200 ml
of Rectified spirit (95 percent v/v)
69
(7) Standard sodium hydroxide solution -0.1N
*Boric acid can also be used instead of sulphuric acid.
Procedure
1. Transfer carefully about one to two grams of the sample accurately

weighed, to the Kjeldhal flask, taking precaution to see that particles of
the material do not stick to the neck of the flask.
2. Add about 10 g of anhydrous sodium sulphate, 0.2 to 0.3 g of copper

sulphate and 20 ml of concentrated sulphuric acid.
3. Place the flask in an inclined position. Heat below the boiling point of the
acid until frothing ceases. Increase heat until the acid boils vigorously and
digests for 30 minutes after the mixture becomes clear and pale green in
colour. Cool the flask.
4. Transfer quantitatively to the round-bottomed flask with water the total

quantity of water used being about 200 ml. Add a few pieces of pumice
stones to avoid bumping. Add about 50 ml of Sodium hydroxide solution
(which is sufficient to make the solution alkaline) carefully through the
side of the flask so that it does not mix with the acid solution but forms a
separate layer below the acid layer.
5. Assemble the apparatus as shown above taking care that the dip tube
extends below the surface of the standard sulphuric acid solution
contained in the beaker.
6. Mix the contents of the flask by shaking and distill until all the ammonia
has passed over into the standard sulphuric acid. Missing from the
document.
70
7. Shut off the burner and immediately detach the flask from the condenser.
Rinse the condenser thoroughly with water into the beaker. Wash the dip
tube carefully so that all traces of the condensate are transferred to the
beaker.
8. When all the washings have been drained into the beaker, add two or
three drops of methyl red indicator solution and titrate with the standard
sodium hydroxide solution. Carry out a blank determination using all
reagents in the same quantities but without the sample to be tested.
Calculation
8.75 x (B-A) x N
Total Protein (N x 6.25), % by mass =
M
Where,
B = volume in ml of the standard sodium hydroxide solution used

to neutralize the acid in the blank determination
A = volume in ml of the standard sodium hydroxide solution used
to neutralize the excess of the acid in the test with the material
N = Normality of the standard sodium hydroxide solution
M = mass in g of the material taken for the test

*DUMAS method can be used as an alternate method for estimation of

nitrogen content (Ref. EN ISO 16634-2008)
A.8 Determination of Fat
(in Milk Toffee and Butter Toffee)
71
A.8.1 Simple Extraction (in Butter Toffee)
Apparatus
(1) Mojonnier fat extraction tube or any other similar apparatus

(2) Flasks
Reagents
(1) Diethyl ether
(3) Petroleum ether
Procedure
1. Dissolve 10 gm sample in 10 ml warm water, and introduce into

Mojonnier fat extraction tube or similar apparatus.
2. Add 25 ml peroxide free ethyl ether.
3. Cork the tube and shake vigorously for 1 minute.
4. Add 25 ml of Petroleum ether and shake again for 30 seconds.
5. Let stand for 30 minutes or until separation is complete.
6. Draw off the ether layer containing fat in a previously dried and weighed
flask.
7. Repeat the extraction twice.
72
8. Pool the ether extract, recover excess solvent and dry the fat for 1 hour at
0
100 C. Cool and weigh.
9. Fat must be dried by keeping the flasks for 30 minutes and weighed, till
constant mass is achieved.
Calculation
M1 x 100 x 100
Fat, % on dry basis=
M2 x (100 – M)
Where,
M1 = Weight in g of the fat
M2 = Weight in g of sample taken
M = Moisture % in the sample

A.8.2 Extraction of Fat by Rose Gottleib Method (in Milk Toffee)
Apparatus
(1) Mojonnier fat extraction tube or similar apparatus.
Reagents
(1) Concentrated Ammonia - sp.gr. 0.88

(2) Ethyl Alcohol 95 to 96 percent (v/v)
(3) Diethyl ether - sp.gr. 0.720 (peroxide free)
73
º
(4) Petroleum ether – boiling range 40 to 60 C, recently distilled
Procedure
1. Introduce 4 g sample into a Mojonnier extraction tube or similar

apparatus. Dilute to 10 ml with water.
2. Add 1.2 ml Ammonia solution and mix thoroughly. Add 10 ml alcohol and
mix.
3. Then add 25 ml ether and shake vigorously for about 30 seconds and
finally add 25 ml petroleum ether and shake again for about 30 seconds.
4. Let stand for 20 minutes or until separation of liquids is complete.
5. Draw off as much as possible of ether fat solution (usually 0.5 to 0.8 ml is
left) into a weighed flask through a small rapid filter.
6. Again extract liquid remaining in tube, this time with 15 ml each of ether
and petroleum ether; shake vigorously for about 30 seconds with each
solvent and let settle. Proceed as above, washing mouth of tube and filter
with a few milliliters of mixture of equal parts of two solvents.
7. For accuracy, repeat extraction. If previously solvent-fat solution has
been drawn off closely, third extraction usually yields approximately up
to 1 mg fat or about 0.02 percent with 4 gm sample.
8. Slowly evaporate solvent on steam bath and then dry fat in an oven
0
maintained at 100 C to constant mass.
9. Test purity of fat by dissolving in a little petroleum ether. If residue
remains, wash out fat completely with petroleum ether, dry the residue,
weigh and calculate the mass of the fat.
Calculation
M1
Fat, % by mass = X 100
M2
Where
74
M1 = Weight in g of the fat
M2 = Weight in g of sample taken

A.9 Determination of Reducing Sugars:
Reagents:
(1) Fehling A: Dissolve 69.28-g copper sulphate (CuSO4.5H2O) in distilled

water. Dilute to 1000 ml. Filter and store in amber coloured bottle.
(2) Fehling B: Dissolve 346 g Rochelle salt (potassium sodium tartrate) (K Na

(3) Carrez 1 – Add 21.9 g Zinc acetate and 3 ml acetic acid in a 100 ml
volumetric flask. Make up the volume with water.
(4) Carrez 2 – 10.6 % aqueous solution of Potassium ferrocyanide.
(5) Methylene Blue Indicator: Prepare 1% of methylene blue solution in

distilled water.
Procedure
1. Weigh accurately about 5 g sample, transfer to a 200 ml volumetric flask

dissolve in warm water, dilute to about 150 ml.
75
2. In case solution is not clear, add 5 ml of Carrez 1 solution followed by 5

ml of Carrez 2 solution.
3. Make up to 200 ml. Filter through a dry filter paper.
4. Titrate the solution obtained as such to determine % Reducing sugars.
Preliminary Titration:
Pipet 5 ml each of Fehling A and B into 250 ml conical flask. Mix and
add about 10 ml water and a few boiling chips or glass beads. Dispense
solution. Heat the flask to boiling. Add 3 drops of methylene blue indicator.
Continue the addition of solution dropwise until the blue colour disappears
to a brick-red end point. (The concentration of the sample solution should be
such that the titre value is between 15 and 50 ml). Note down the titre value.
Final Titration:
Pipet 5 ml each of Fehling A and B. Add sample solution about 2 ml

less than titre value of the preliminary titration. Heat the flask to boiling with
in 3 minutes and complete the titration. Perform the titration duplicate and
take the average. Calculate the reducing sugar % as shown below.
Dilution x Factor of Fehling (in gm)

Reducing Sugars % = X 100
Weight of sample x Titre value
A.10 Determination of Sucrose
Take an aliquot of the filterate obtained in Reducing sugar method

and invert it with Hydrochloric acid in a water bath at 60º C by keeping for
10 minutes. Cool immediately and neutralize with sodium hydroxide and
76
finally with sodium carbonate. Make upto volume and determine reducing
sugar by Lane and Eynon method.
Dilution x Factor of Fehling (in gm)

Reducing Sugars % = X100
(as Invert Sugar) Weight of sample x Titre value
Determination of Factor (for Invert Sugar) of Fehling Solution:
Accurately weigh around 4.75 g of analar grade sucrose. Transfer to

500 ml volume flask with 50 ml distilled water. Add 5 ml conc. HCl and allow
to stand for 24 hours Neutralize with NaOH solution and make up to volume.
Mix well and transfer 50 ml to a 100 ml volumetric flask and makeup to
volume. Transfer to a burette having an offset tip.

above:
Titre x Weight of sucrose in gm

Fehling Factor =
(as Invert Sugar) 500
Calculation:
Sucrose %
= (Total reducing sugars / invert sugar % - reducing sugars %) x 0.95

A.11 Determination of Sulphur Dioxide
77
Sulphur dioxide is determined by the modified Monier-William’s Method –

The apparatus as assembled is shown below:
Assembly for determination of Sulphur dioxide
Reagents
(1) Sodium Carbonate Solution - 10 percent (m/v) aqueous
(2) Bromophenol Blue Indicator Solution – Dissolve 0.1 g of bromophenol

blue in 3.0 ml of 0.05 N sodium hydroxide solution and 5 ml of ethyl
alcohol (90 percent by volume) by gently warming. Make up the
volume of the solution with ethyl alcohol (20 percent v/v) to 250 ml
in a volumetric flask.
(3) Hydrogen peroxide solution - Dilute a 30 percent (m/v) hydrogen

peroxide solution with about twice its volume of water and neutralize
the free sulphuric acid that may be present in the hydrogen peroxide
solution with barium hydroxide solution, using bromophenol blue
indicator solution. Allow the precipitate of barium sulphate to settle,
and filter. Determine the concentration of hydrogen peroxide in the
filtrate by titrating with standard potassium permanganate solution.
78
Dilute the filtrate with cold water so as to obtain a 3 percent (m/v)

solution of hydrogen peroxide.
(4) Concentrated Hydrochloric acid - sp.gr. 1.16
(5) Carbon dioxide gas - from a cylinder
(6) Standard sodium hydroxide solution - approximately 0.1 N,

standardized at the time of the experiment using bromophenol blue
indicator solution.
Procedure
1. Assemble the apparatus as shown above.

2. Introduce into the flask C, 300 ml of water and 20 ml of concentrated
hydrochloric acid through the dropping funnel E.
3. Run a steady current of cold water through the condenser F.
4. Boil the mixture contained in the flask G for a short time to expel the air
from the system in current of carbon dioxide gas previously passed
through the wash bottle A. Check the pressure of carrier gas entering the
receiving flask by monitoring the gas bubbling rate.
5. Weigh accurately about 100 g of the material and mix with the minimum
quantity of water so as to make the diluted material easily flow down to
the dropping funnel. Introduce the diluted material into the flask C
through the dropping funnel E. Wash the dropping funnel with a small
quantity of water and run the washing into the flask C.
6. Again boil the mixture contained in the flask C in a slow current of carbon
dioxide gas (passed previously through the wash bottle A) for one hour.
Just before the end of the distillation, stop the flow of water in the
condenser. (This causes the condenser to become hot and drives over
residual traces of sulphur dioxide retained in the condenser.)
79
7. When the delivery tube H, just above the Erlenmeyer flask J, becomes hot
to touch, remove the stopper J immediately. Wash the delivery tube H and
the contents of the Peligot tube L with water into Erlenmeyer flask.
8. Cool the contents of the Erlenmeyer flask to room temperature, add a few
drops of bromophenol blue indicator and titrate with standard sodium
hydroxide solution. Bromophenol blue is unaffected by carbon dioxide
and gives a distinct change of color in cold hydrogen peroxide solution).
Carry out a blank determination using 20 ml of conc. hydrochloric acid
diluted with 300 ml of water. Blank must be carried out, treating the
same way as sample, with same boiling time and gas bubbling rate.
Calculation
Sulphur dioxide, mg/kg = 0.032000 (V-v) x 1000 x 1000 x N
W
Where
V = volume in ml of standard sodium hydroxide solution required for the test

with the material
v = volume in ml of standard sodium hydroxide solution required for the
blank determination;
N = normality of standard sodium hydroxide solution; and
W = weight in g of the material taken for the test
[Ref: - I.S.I Handbook of Food Analysis (Part II) -1984 page 8]
A.12 Lead, Copper and Zinc
Equipment:
80
(1) Atomic absorption spectrophotometer (Flame)

(2) Crucibles
(3) Hot plate
(4) Calibrated weighing balance
(5) Muffle Furnace
Reagents:
(1) Nitric acid

(2) Water
(3) Hydrochloric acid — (1:1)
(4) Standard stock solution (Certified Reference Materials) – 1000 mg/L of
Pb, Cu & Zn (traceable to NIST)
Procedure:
1. Sample preparation: Homogenize product, if necessary using non-

contaminating equipment.
2. Weigh about 10-20 g of the sample to nearest 0.01g in a crucible, and dry
on a hot plate at 100˚C.
3. Place the dish in muffle furnace at initial temperature not higher than
100˚C. Increase temperature at a maximum rate of 50˚C per hour to
450˚C. Let dish stand for at least 8 hours or overnight.
4. Take crucible out of furnace and let cool.
5. Wet ash with 1-3 ml of water and evaporate on hot plate.
6. Put crucible back in furnace at no more than 200˚C and raise temperature
(50-100˚C per hour) to 450˚C. Proceed for 1-2h or longer.
7. Repeat procedure until product is completely ash i.e. ash should be
white/ grey or slightly covered.
8. Add 5 ml (1:1) HCl to crucible ensuring that all ash comes into contact
with acid. Evaporate acid on hot plate.
81
9. Dissolve residue in 10-30 ml 0.1M HNO3. Cover with watch glass and let
stand for 1-2 hours. Make up to a particular volume.
10. Treat blank in the same way as prepared sample.
11. Standard preparation: Add 10 ml of standard stock solution into 100 ml
volumetric flask and make up the volume using deionized water.
(Pb/Cu/Zn 100 mg/L solution)
12. Preparation of standard working solution: To a series of 100 ml one-mark
volumetric flasks, pipette 0.2, 0.5, 1.0, 2.0 and 5.0 ml of the standard
working solution and 1ml of concentrated nitric acid and then dilute to
mark with deionized water. These solutions then contain lead
concentration 0.2, 0.5, 1.0, 2.0 and 5.0 µg of Pb/Cu/Zn per ml of the
solution.
13. Aspirate both standard & sample solutions into the AAS.
Calculation:
Determine the Pb/Cu/Zn concentration in the sample solution by

extrapolating the value from the standard curve plotted using conc. of
different standard solution vs. absorbance reading.
(S - B) × Dilution
Pb / Cu / Zn, mg/kg =
Weight of sample
Where,
S = Concentration of Pb/Cu/Zn in sample
B = Concentration of Pb/Cu/Zn in blank
[Ref: - A.O.A.C 18th edn, 2005 Official Method 999.11]
82
A.13 Filth in candy
Apparatus
(1) Microscope
(2) Magnetic stirrer – hot plate
(3) Sieve
(4) Wire basket – 8 cm diameter and 3 cm height, made from No. 8 screen
and with wire handles.
Reagents
(1) HCl (1+70)

(2) Turgitol anionic 7 – sodium heptadecyl sulphate (SIGMA chemical co.)
(3) Mineral oil – Paraffin oil, white, light, 125/ 135 Saybolt universal viscosity
(38), sp. gravity 0.840-0.860 (24)
(4) Isopropanol
(5) Chloroform
(6) Floatation liquid – mineral oil and heptane (85 +15)
(7) Dichloromethane
Procedure
(1) In hard candy, gum drops, gum, starch or pectin based candies
1. Dissolve in boiling HCl.

2. Filter through rapid paper on hirsch funnel.
3. And examine microscopically.
(2) In hard boiled candy difficult to filter and In All water insoluble candy
except those containing confectioners corn flakes, wheat bran, or other
83
cereal fillers, and those whose major constituents, excluding chocolate

coating, consists primarily of ground nutmeats (eg. Peanut butter, almond
paste etc.)
1. Weigh 225g test portion into 1.5 to 2 litre beaker.
2. Add 1 litre 5% solution of turgitol and heat in steam bath for 10
minutes. Stir 5 to 10 minutes on magnetic stirrer – hot plate.
3. Sieve portion wise on number 230 sieve. If residue on sieve is small,
transfer directly to ruled filter paper; otherwise, transfer
quantitatively to 2 litre trap flask, using 40% isopropanol.
4. Bring volume to 1 litre with 40% isopropanol and add 50 ml HCl.
5. Gently stir on magnetic stirrer – hot plate, while heating to full boil.
6. Immediately transfer flask to cool stirring unit and add 40 ml light
mineral oil.
7. Stir magnetically for 2 minutes, let it stand for 1 minute; then slowly
fill flask with 40% isopropanol by running liquid down stoppered rod
while top of stopper is maintained just above liquid. After filling flask,
gently stir settled plant material for 5 to 10 seconds with stoppered
rod.
8. Let stand undisturbed for 2 minutes and immediately trap off.
9. Add 25 ml light mineral oil, stir by hand gently for 30 seconds, and let
stand for 10 minutes. Repeat trapping. Wash flask neck thoroughly
with isopropanol and transfer washings to beaker containing
trappings.
10. Filter onto ruled paper and examine microscopically.
(3) In water insoluble candies containing confectioners corn flakes, wheat

bran, or other cereal fillers, and those whose major constituents,
excluding the chocolate coating, consists primarily of finely ground
nutmeats (eg. Peanut butter, almond paste etc.)
84
1. Proceed as mentioned in (2); through sieving on number 230

sieve.
2. Wash residue on sieve with isopropanol.
3. Form filter paper around 600 ml beaker, moistening with water to
make paper pliable. Insert paper into 91 mm buchner, wash with
isopropanol, and aspirate to near dryness.
4. Quantitatively transfer residue on sieve to filter paper cup with
isopropanol and add enough isopropanol to cover residue.
5. After 1 minute apply vacuum until dripping ceases.
6. Place paper cup containing sieved residue in 1 litre beaker, add
200-ml chloroform, and boil 5 minutes on steam bath.
7. After few minutes of cooling, lift paper, drain, and transfer to
200ml fresh chloroform. Repeat for 5 minutes, boil and drain.
8. Return paper cup to buchner and apply vacuum until dripping
ceases. Cover residue with isopropanol for 5 minutes, reapply
vacuum, and continue to aspirate for 5 minutes after visible
dripping ceases.
9. Transfer quantitatively to 2 litre trap flask, using 40%
isopropanol. Bring volume to 1 litre with 40% isopropanol and
add 50 ml HCl.
10. Gently stir on magnetic stirrer – hot plate, while heating to full
boil.
11. Immediately transfer flask to cool stirring unit and add 40 ml
floatation liquid.
12. Stir magnetically for 2 minutes, let it stand for 1 minute; then
slowly fill flask with 40% isopropanol by running liquid down
stoppered rod while top of stopper is maintained just above liquid.
After filling flask, gently stir settled plant material for 5 to 10
seconds with stoppered rod.
13. Let stand undisturbed for 2 minutes and immediately trap off.
85
14. Add 25 ml floatation liquid, stir by hand gently for 30 seconds, and
let stand for 10 minutes. Repeat trapping. Wash flask neck
thoroughly with isopropanol and transfer washings to beaker
containing trappings.
15. Filter onto ruled paper and examine microscopically.
(4) In chocolate candy coating
1. Heat 400ml dichloromethane in 800ml beaker to 30-35 ˚C and keep at

this temperature.
2. Place test portion of candy in wire basket.
3. Move basket up and down through dichloromethane until chocolate
coating dissolves. Rinse each candy center with fine stream of
dichlormethane from wash bottle and save center. Repeat with
balance of test sample.
4. Stir dichloromethane-chocolate suspension and pour through No. 140
sieve.
5. Transfer residue from sieve to filter paper and examine
microscpically. Examine candy centers by appropriate method as in
(1), (2) and (3).
[Ref: - A.O.A.C 18th edn, 2005 Official Method 971.34 Filth in candy
(Floatation method)]
A.14 Starch in Confectionery
Reagents
(1) Malt extract – Prepare infusion of freshly ground malt just before use. For
every 80 ml malt extract required digest 5 g ground malt with 100 ml
water at room temperature 2 hours or 20 minutes if mixture can be
86
stirred by electric mixer. Filter to obtain clear extract, refiltering first

portion of filterate if necessary. Mix infusion well.
Procedure
1. Measure 25 ml of solution of uniform mixture (representing 5 g test

portion) into 30 ml beaker, or add to beaker 5 g finely ground test portion
(previously extracted with ether, if test sample contains much fat); add
enough water to make 100 ml; heat to 60˚C (avoiding, if possible
gelatinizing starch); and let stand for 1 hour, stirring frequently to secure
complete solution of sugar.
2. Transfer to wide-mouth bottle, rinse beaker with little warm water and
cool.
3. Add equal volume alcohol and mix and let stand for less than 1 hour.
4. Centrifuge until precipitate is closely packed on bottom of bottle and
decant supernate through hardened filter.
5. Wash precipitate with successive 50 ml portions of alcohol, 50% by
volume, by centrifuging and decanting through filter until washings are
sugar-free by following test:
Add to test-tube few drops of washing, 3-4 drops 20% alcoholic α-

naphthol solution, and 2 ml water. Shake well, tip tube, let 2 to 5 ml H2SO4
flow down sides of tube, and then hold tube upright. If sugar is present,
interphase of two liquids is coloured faint to deep violet; on shaking,
whole solution becomes blue-violet.
6. Transfer residue from bottle and hardened filter to beaker with 50 ml

water.
7. Immerse beaker in boiling water, and stir constantly 15 minutes or until,
all starch is gelatinized.
87
8. Cool to 55˚C, add 20 ml malt extract, and hold at this temperature for 1
hour, or until residue treated with I2 solution shows no blue tinge upon
microscopic examination. Cool, dilute to 250 ml and filter.
9. Place 200 ml of filterate in flask, add 20 ml HCl (sp. Gr. 1.125), connect
with reflux condenser, and heat in boiling water-bath 2.5 hours.
10. Cool, nearly neutralize with 10% NaOH solution, finish neutralization
with Na2CO3 solution, and dilute to 500 ml.
11. Mix solution thoroughly, pour through dry filter, and determine glucose
in aliquots by Munson – Walker method. Conduct Blank determination on
same volume of malt extract as used with test portion and correct
weighed glucose accordingly.
Weighed glucose obtained x 0.925 = Weighed Starch
A.15 Paraffin in Confectionery
Procedure
1. To solvent extract in flask (as obtained in the above method for Ether
Extract), add 10 ml alcohol and 2 ml NaOH solution (1+1); connect flask
with reflux condenser and heat 1 hour on water-bath or until
saponification is complete.
2. Remove condenser and keep flask on bath until alcohol evaporates and
residue is dry.
3. Dissolve residue as completely as possible in approx. 40 ml water and
heat on bath, shaking frequently.
4. Wash into separator, cool, and extract with 4 successive portions of
petroleum ether, collecting extracts in weighed flask or capsule.
5. Evaporate petroleum ether and dry to constant weight at 100˚C. Any
phytosterol or cholesterol present in fat could be extracted with the
88
paraffin, but the amount is so insignificant that it may generally be

disregarded.
A.16 Shellac in Confectionery
Procedure
1. Place 50 g test portion in 400 ml beaker.

2. Add 50 ml mixture of Benzene and absolute alcohol (1+1), and cover with
watch-glass. Heat to boiling point on steam bath, and simmer few
minutes, stirring occasionally.
3. Decant liquid into tared, round 100 ml glass dish with flat bottom approx.
7 cm diameter.
4. Extract once more with Benzene-alcohol mixture, and finally rinse with
two 25 ml portions of absolute alcohol, simmering and stirring each time.
With moist sugar candy, avoid over heating to prevent pieces from
sticking together. Add each extract to glass dish previously placed on
steam bath.
5. Evaporate until alcohol is just removed, rotating dish as it goes to dryness
in order to spread extract uniformly on the bottom surface. Avoid baking
shellac on dish. If fat appears to be present, wash with three 15 ml
portion of petroleum ether, stirring and warming. Decant through rapid
filter.
6. Add mixture of 25 ml iso-amyl alcohol (B.P. – 129-132°C) and 25 ml
Benzene to filter, and filter back to dish.
7. Heat on steam bath with stirring, cool somewhat, and transfer solution
with suspended matter to 125 ml separator. Rinse dish with 25 ml hot
(approx. 60°C) water, and add to separator; shake well and filter and
wash water if necessary.
8. Repeat washings with water twice (or until washings are colourless),
rinsing dish well around sides with first portion of liquid.
89
9. Finally, filter solution of shellac into tared dish, rinsing separator and
filters with little absolute alcohol.
10. Evaporate to dryness on steam bath, rotating dish to give uniform film.
11. If much fat was extracted in original benzene extraction, wash final
shellac residue with 25 ml petroleum ether, warming and stirring. Decant,
dry on steam bath and in 100˚C oven and weigh.
12. After weighing, check for complete removal of sugars by thoroughly
rinsing dish and surface of shellac with hot water, warming on steam
bath, decanting, rinsing down with alcohol and evaporating with care to
give uniform film on dish.
13. Dry and reweigh.
A.17 Alcohol in syrups
Procedure
1. Collect n beaker syrup from enough pieces of confectionery to yield 30 –

50 g, strain into weighed beaker and weigh.
2. Place syrup in 250-300 ml distillation flask, dilute with half its volume of
water, attach flask to vertical condenser and distill almost 50 ml, or as
much of liquid as possible without causing charring. Foaming may be
prevented by adding little tannin or piece of paraffin approx. size of pea.
3. Cool distillate, dilute to volume with water, and mix well.
4. Determine specific gravity of distillate.
Calculate % alcohol by weight or volume in candy filling by using Table - %
by volume at 15.56˚C of ethyl alcohol corresponding to apparent specific
gravity at various temperatures & % by weight corresponding to various
percentages by volume at 15.56˚C in mixtures of ethyl alcohol and water.
*For microbial analysis refer ‘Microbiology Manual’
90
A.18 Determination of Total Plate Count (CFU/gm)
Materials and equipment

The following media and reagents (1-4) are commercially available and
are to be prepared and sterilized according to the manufacturer's
instructions.
(1) Plate count agar (PC)/Nutrient Agar (NA)
(2) Peptone water diluent (0.1%)(PW)
(3) Sodium 2, 3, 5 triphenyltetrazolium chloride, TTC (0.1%) (optional)
(4) 1N HCl and 1N NaOH
(5) pH meter or paper capable of distinguishing to 0.3 to 0.5 pH units within
a range of 5.0 to 8.0
(6) Stomacher, blender or equivalent for sample
preparation/homogenization.
(7) Incubator capable of maintaining the growth temperature required for
the specific type of aerobic bacteria being enumerated (i.e. for
psychrophilic bacteria: 15 – 20°C, for mesophilic bacteria: 30 – 35°C, and
for thermophilic bacteria: 55°C) and 45°C waterbath
(8) Colony counting device (optional)
Procedure
The test shall be carried out in accordance with the following instructions:
(1) Handling of Sample Units

1. During storage and transport, the following shall apply: with the
exception of shelf stable products, keep the sample units refrigerated
(0-5°C). Sample units of frozen products shall be kept frozen.
2. Thaw frozen samples in a refrigerator or under time and temperature
conditions which prevent microbial growth or death.
91
3. Analyze sample units as soon as possible after receipt in the

laboratory.
(2) Preparation of Media

1. Prepare plate count/Nutrient agar and dispense in appropriate
quantities. Sterilize.
2. Clean surface of working area with a suitable disinfectant.
3. Clearly mark the duplicate Petri plates.
(3) Preparation of Dilutions

1. Prepare sterile 0.1% peptone water diluent.
2. To ensure a truly representative analytical unit, agitate liquid or free
flowing materials until the contents are homogeneous. If the sample
unit is a solid, obtain the analytical unit by taking a portion from
several locations within the sample unit.
3. Prepare a 1:10 dilution of the food by aseptically homogenizing 25 g
or mL (the analytical unit) into 225 mL of the required diluent. If a
sample size other than 25 g or mL is used, maintain the 1:10 sample to
dilution ratio, such as 11 g or mL into 99 mL.
Note:
1. Check the pH of the food suspension. If the pH is outside the range of 5.5-
7.6, adjust the pH to 7.0 with sterile NaOH or HCl.
2. Prepare succeeding decimal dilutions as required, using a separate sterile
pipette for making each transfer.
3. Shake all dilutions immediately prior to making transfers to ensure
uniform distribution of the microorganisms present.
(4) Plating
92
1. Agitate each dilution tube to re-suspend material that may have

settled out during preparation.
2. Pipette 1 mL or 0.1 mL of the required dilutions to appropriately
marked duplicate Petri plates.
3. In the case of products that tend to adhere to the bottom of the plates,
add the inoculum to 1.0 mL of sterile diluent previously placed in the
Petri plate.
4. Pour 12-15 mL of tempered agar into each plate, and mix by rotating
and tilting.
5. Allow to solidify. Plates should be poured not more than 15 min after
preparation of dilutions.
(5) Incubation
Incubate plates in the inverted position for 48 h ± 4 h. Incubation
temperature is dependent on the growth temperature requirements of
the target organisms (for psychrophilic bacteria: 15 – 20°C, for
mesophilic bacteria: 30 – 35°C, and for thermophilic bacteria: 55°C). The
plates used to enumerate psychrophilic and thermophilic bacteria may be
incubated up to 5 days.
(6) Counting Colonies

1. Count colonies promptly after the incubation period.
2. If possible, select plates with 15-300 colonies (including pinpoint
colonies). If counts do not fall within this range select plates that fall
nearest to the 15-300 range.
For counting of colonies in cfu/gm or cfu/ ml the formula is as follows:
c
N= ____________________
(n1+0.1n2)d
93
Where,
n= the number of counts
n1= the number of plates counted in the first dilution
n2= the number of plates counted in the second dilution
d= the dilution from which the first counts were obtained (for example, 10-
1)
Round the results obtained with above formula to two significant figures.
(7) Differentiation of Colonies from Interfering Particles

Food particles such as meat, milk powder, etc., often interfere with the
enumeration of the plates. This can be eliminated by making one extra
plate of each dilution containing interfering particles and holding it under
refrigeration as a control for comparison during counting.
Alternatively, after incubation flood plates with 2 mL of 0.1% 2,3,5,

triphenyltetrazolium chloride. Gently rock plates from side to side to
cover the entire area with solution. Pour off excessive solution and allow
the plates to remain at room temperature for 3 hrs. in an inverted
position. The bacteria reduce the indicator to a formazan which colours
the colonies red and aids in distinguishing the food particles. Colonies
cannot be picked for isolation after this method has been used.
(8) Reporting:
Total Plate Count: cfu/g
A.19 Determination of Coliform Count (CFU/gm)

The following media and reagents are commercially available and are to be
prepared and sterilized according to the manufacturer's instructions.
94
(1) Violet Red Bile Agar.

(2) Peptone water diluent (0.1%) (PW)/ N-Saline.
a range of 5.0 to 8.0.
the specific type of aerobic bacteria being enumerated i.e. at 35°C.
(6) Colony counting device (optional).
Procedure

exception of shelfstable products, keep the sample units refrigerated
3. Analyze sample units as soon as possible after receipt in the
laboratory.
(2). Preparation of Media

1. Prepare VRB agar and dispense in appropriate quantities. Sterilize.
(3). Preparation of Dilutions

95

Note:


4. Plating
1. Agitate each dilution tube to resuspend material that may have settled
out during preparation.
2. Pipette 1 mL or 0.1 mL of the required dilutions to appropriately
marked duplicate Petri plates.
3. In the case of products that tend to adhere to the bottom of the plates,
add the inoculum to 1.0 mL of sterile diluent previously placed in the
Petri plate.
4. Pour 12-15 mL of tempered agar into each plate, and mix by rotating
and tilting.
5. Allow to solidify. Plates should be poured not more than 15 min after
preparation of dilutions.
96
6. Incubation
1. Incubate plates in the inverted position for 24 h ± 4 h at 35±2°C.
2. Avoid crowding or excessive stacking of plates to permit rapid
equilibration of plates with incubator temperature.
3. Counting Colonies
2. If possible, select plates with 30-300 colonies (including
pinpoint colonies). If counts do not fall within this range select
plates that fall nearest to the 30-300 range.
c
N =
(n1+0.1n2) d
Where;
d= the dilution from which the first counts were obtained (for example, 10-
1)
4. Reporting: Total Coliform Count : cfu/g
A.20 Enumeration of Yeasts and Moulds (CFU/gm)
Materials and special equipment

The following media and reagents are commercially available and are
to be prepared and sterilized according to the manufacturer's instructions.
97
These agars are suitable for yeast and mould count in food products:
(1) Chloramphenicol Yeast extract Glucose Agar (CYGA)

(2) Potato dextrose agar with chloramphenicol (PDA-C)
(3) 20% sucrose (diluent additive for osmophiles)
(4) Malt extract agar containing 50% (w/w) sucrose
Others:
(1) Peptone water (0.1%) (PW)

(2) 1N HCl and 1N NaOH
(3) Gram stain solutions
(4) Stomacher, blender or equivalent
(6) Light microscope
(8) Incubator (darkened) capable of maintaining 22 to 25°C,
Procedure
Each sample unit shall be analyzed individually. The test shall be carried
out in accordance with the following instructions:
1. Handling of Sample Units

exception of shelfstable products, keep the sample units refrigerated
3. Analyze the sample units as soon as possible after receipt at the
laboratory.
98
2. Preparation of Medium
Prepare the appropriate media for the analysis being carried out.
Note:
2. Mark clearly the duplicate petri plates identifying sample, sample unit,
dilution and date of inoculation.
3. Preparation of Dilutions
1. Prepare 0.1% peptone water as diluent. An appropriate solute, such as
20% sucrose, should be added to the diluent when enumerating
osmophiles in foods such as syrups and fruit juice concentrates. In
addition, a 2% solution of sodium citrate, pre-warmed to 45°C, can be
used as diluent for high-fat foods such as cheese, butter.
2. Prepare a 1:10 dilution of the food by aseptically blending 25 g or mL
(the analytical unit) into 225 mL of the required diluent, as indicated
in Table I. If a sample size other than 25 g or mL is used, maintain the
1:10 sample to dilution ratio, such as 11 g or mL into 99 mL.
Note:
1. Stomach, blend or shake the food sample.

2. Blend or stomach for the minimum time required to produce a
homogeneous suspension.
3. Verify the pH of the suspension. If the pH is not between 5.5 and 7.5,
adjust the pH to 7.0 with a sterile solution of 1N NaOH or 1N HCl.
4. If the 1:10 dilution is prepared in a dilution bottle, it should be mixed by
shaking the tube using vortex mixer.
99
6. Because mould propagules may settle out within a few minutes, it is

important to shake all dilutions immediately prior to making transfers to
ensure uniform distribution of the microorganisms present.
4. Plating
1. Agitate each dilution bottle to resuspend material that may have
settled out during preparation.
2. Moulds should be enumerated by a surface spread-plate technique
rather than with pour plates. This technique provides maximal
exposure of the cells to atmospheric oxygen and avoids heat stress
from molten agar. Agar spread plates should be dried overnight
before being inoculated. Spread 0.1 mL onto duplicate plates.
5. Incubation
Incubate plates undisturbed in an upright position at 22 to 25°C for 3-5
days. Incubate plates in the dark. Normally, count colonies on plates after
5 days. Examine on the third day and if mould colonies are numerous,
count them and then count again on the fifth day, if possible.
Note:
Handle the plates as little as possible when counting on third day so spores
will not be dislodged, which may result in secondary growth.
6. Counting Colonies and Examining Growth

Count colonies, distinguishing, if required, yeast colonies from mould
colonies, according to their colonial morphology. Microscopic
examination with crystal violet stained smears may be necessary to
distinguish yeast colonies from some bacterial colonies that may look like
yeast.
100
If possible, select plates with 15-150 colonies. Determine the identity of

pin-point colonies microscopically. If counts do not fall within this range,
select plates that fall nearest to the 15-150 range. If the mycoflora
consists primarily of moulds, the lower population range is selected; if
primarily yeast colonies, the upper limit is counted.

2. If possible, select plates with 15-150 colonies (including pinpoint
colonies). If counts do not fall within this range select plates that fall
nearest to the 15-150 range.

c
N =
(n1+0.1n2)d
Where;
d= the dilution from which the first counts were obtained(for example, 10-
1)
Alternatively,
Wet mounts and gram stains of several diverse types of cells per sample
should be examined to confirm that bacteria are not present. Yeast cells and
asexual mould spores are generally gram-positive, whereas mould mycelia
are gram-negative.
7. Recording Results
Calculate the average count (arithmetic mean) of the duplicate plates
101
Avoid creating erroneous ideas of precision and accuracy when

computing counts
8. Reporting: Yeast and Mould Count : cfu/g
9. Precautions
1. Some yeasts and moulds can be infectious or can cause allergic
responses, therefore, it is important to be fairly cautious when
working with fungi. Ideally, plates should be held in incubators, not in
an open room. Plate lids should generally only be removed for
procedures such as the preparation of a slide for microscopic
examination.
2. Flamed needles should be cooled before making transfers to avoid
dispersal of conidia and other cells. Cultures should never be smelled.
A.21 Detection of E. Coli per gm

The following media and reagents are commercially available and are to
be prepared and sterilized according to the manufacturer's instructions.
(1) Tergitol – 7 Agar (T-7)
(2) McConkey Agar (MC)
(3) Eosien Methylene Blue (EMB)Agar
(4) Nutrient Broth
(5) Peptone water diluent (0.1%)(PW)/ N-Saline
a range of 5.0 to 8.0.
102
Procedure

1. Prepare McConkey, EMB, T-7 agar and dispense in appropriate
(2) Preparation of Dilutions

Note:
(3) Pre-Enrichment
103
Take 10 ml of diluted sample in 90 ml NB and incubate for 24 hrs. at

35±2°C
(4) Selective Enrichment

1. Transfer a loopful of the inoculum from pre-enrichment culture in to
T-7 agar and incubate for 24 hrs at 35±2°C.
2. Pick golden yellow colonies from the T-7 agar and streak on to MC and
EMB agar and incubate the plates for 24 hrs at 35±2°C.
3. Observe for characteristic colonies if Pink red colonies and green
metallic sheen colonies on MC and EMB agar respectively.
(5) Biochemical Identification Tests

The biochemical tests conducted and interpreted as the reaction of the
biochemical reagents as below;
1. Gram stain
2. Indole test
3. Voges Proskauer test
4. Citrate Utilization
5. Growth on Macconkey broth at 44 ±0.50C
6. Carbohydrate Utilization for acid and gas
7. H2S Production test
8. Motility test
9. Urease test
(6) Reporting: E. coli : cfu/g; Present or Absent / g
A.22 Detection of S.aureus per g

104
(1) Baird Parker Agar

(2) Nutrient Agar
Procedure
1. Prepare Baird Parker (BP) agar and dispense in appropriate

(2) Pre-Enrichment

or mL (the analytical unit) into 225 mL of the required Diluent. If a
2. Take 10 ml of above diluted sample in 90 ml 0.1% Peptone and
incubate for 24 hrs. at 37±2°C
105
1. Transfer a loopful of the inoculum from pre-enrichment culture in to

BP agar and incubate for 30 hrs at 37±2°C.
2. Observe for characteristics shiny black colonies with grey margin on
the BP agar.
(4) Biochemical Identification Tests

1. Gram Staining
2. Coagulase Test
(5) Reporting:
Staphylococcus aureus Present or absent / gm
B. CHEWING GUM AND BUBBLE GUM
As per FSSA, Chewing and bubble gum shall be prepared from

chewing gum base or bubble gum base, natural or synthetic, non-toxic; cane
sugar and liquid glucose (corn syrup).
B.1 Preparation of sample:
Cut into small bits/ pieces around 50-75 g and mix well. Store in an airtight
container.

for Sugar Confectionery / A.O.A.C 17th edn, 2000 Official Method 925.49
Preparation of Test sample]
106
B.2 Determination of Moisture - Vacuum Oven method
Apparatus
(1) Aluminum dish – 75mm diameter and about 25mm height with close
fitting cover.
(2) Dessicator
(3) Vacuum oven
Procedure

weighed. Distribute the material as evenly as practicable over the bottom
of the dish by gentle sidewise movements.
6-8 hours at 70ºC under 25 mm Hg pressure. During heating admit slow
current of air into oven.
is attained.
as moisture %.
Calculations
(W3 - W2)
Moisture content, % by mass = x 100
(W3 - W2)
Where
107

B.3 Ash insoluble in dilute HCl
Reagents
(1) Dilute hydrochloric acid - Approx 5 N (445ml in 1litre)
Procedure

dry platinum basin of 100 ml capacity.
2. Carbonize the material in the dish with the flame of a burner. Complete
the ignition by keeping in a muffle furnace at 550 + 25˚C until gray ash
results. Cool in a desiccator.
the washings are free from chlorides. (To check this, add few drops of 2M
free.)
maintained at 105 +2˚C for about three hours. Ignite in the muffle furnace
at 550 +25˚C for one hour. Cool the dish in a desiccator and weigh.
6. Heat again for 30 minutes in the muffle furnace, cool and weigh.
108
Calculation
M2
Where
B.4 Determination of Sulphated Ash
Reagents
(1) Sulphuric Acid - 10 percent (m/m)
Procedure
1. Accurately weigh about 5 g of the prepared sample into a 9-cm diameter
platinum basin.
2. Add 5 ml of sulphuric acid to the material in the dish.
3. Gently heat the dish on a hot plate until the material is well carbonized
and then increase the heat until the evolution of sulphuric acid fumes
ceases.
4. Ash the carbonized matter in a muffle furnace at 550 ± 25˚C.
5. Cool the ash and moisten it with 2-3 ml of sulphuric acid. Heat strongly on
a hot plate until sulphuric acid fumes ceases to be evolved and finally ash
in the muffle furnace at 550 ± 25˚C for two hours.
6. Cool in a desiccator and weigh.
109
7. Heat again in a muffle furnace for 30 minutes at 550 ± 25˚C. Cool in a

desiccator and weigh.
8. Repeat the process of heating in the muffle furnace for 30 minutes,
cooling and weighing till the difference between two successive weighing
is less than 1 mg. Record the lowest mass.
Calculation
M1
Sulphated ash, % by mass = x 100
M2
Where,
M1 = mass in g of the sulphated ash
M2 = mass in g of the prepared sample taken for the test.

B.5 Test for presence of added synthetic colour
As per Clause A.6
B.6 Determination of Titanium Di-oxide in Chewing Gum and Bubble

Gum
Reagents
(1) Titanium di-oxide standard solution: (0.1 mg/ml) - Weigh accurately 50

mg titanium di-oxide in a 250 ml beaker. Add 15 g anhydrous sodium
sulphate and 50 ml conc. Sulphuric acid. Add 1 or 2 glass beads, cover
with watch glass and heat on a hot plate to boil and dissolve. Cool and add
110
100 ml distilled water accurately with stirring. (If the solution is cloudy
warm on a steam bath to clarify) Cool and transfer to 500-ml volumetric
flask containing 200-ml water. Make up to volume.
(2) Sulphuric Acid – 1:9 volume
(3) Hydrogen peroxide – 30% grade
(4) Sodium Sulphate – Analar grade
Procedure:
1. Accurately weigh 2-3 grams of prepared sample in a 100-ml silica dish.

2. Char the material on a burner and ash in muffle furnace at 800ºC for 3-4
hours.
3. Cool, add 2-g anhydrous sodium sulphate and 10 ml conc. H2SO4, cover
with watch glass and bring to boiling on a hot plate and dissolve.
4. Cool thoroughly and rinse the watch glass carefully with 30-ml water.
Transfer to 100-ml volumetric flask. (If solution is cloudy heat on steam
bath to clarify) cool and dilute to volume with water.
5. Transfer 3-ml aliquot of the sample solution to 5 ml volumetric flask or
graduated cylinder. Dilute to volume with 10% H2SO4. Add 0.2 ml 30%
H2SO4. Mix well.
6. Measure the absorbance at 408 nm against a prepared blank. Determine
the concentration of TiO2 in sample using a standard curve.
Preparation of Standard Curve:
Transfer 0, 1, 2, 3 and 4 ml of TiO2 standard solution (0.1 mg/ml) to 5-

ml volumetric flask or graduated cylinder. Dilute to volume with 1+ 9 H2SO4.
Mix well. Measure the absorbance of the colour at 408 nm in a
spectrophotometer and prepare a standard curve.
Calculation:
111
Dilution x mg TiO2 x Dilution x Absorbance of sample x

100
TiO2 (in g %) =
Wt. of sample x Dilution x Absorbance of standard x
1000
th
[Ref: -. A.O.A.C 17 edn, 2000 Official Method 973.38 Titanium Dioxide in
Cheese]
B.7 Determination of Gum-Base Content
Reagents and Apparatus:
(1) Chloroform, analar grade

(2) Soxhlet extraction apparatus
(3) Whatman thimble
Procedure:
1. Weigh 4-5 g of sample (W) in a Whatman thimble and extract the gum in
a continuous extraction apparatus (Soxhlet extractor) with chloroform
for 8 hours.
2. Distill off or evaporate the chloroform extract on a steam bath and
transfer the flask to an oven maintained at 100ºC and dry it for 4-5 hours.
3. Cool in a desiccator and weigh (W2).
4. Chloroform extract must be dried by keeping the flasks for 30 minutes
and weighed, till constant mass is achieved.
Calculation:
W2 – W1
Gum base Content % =
100 x W
112
Where
W2 = Weight of flask and extracted gum, in g

W1 = Weight of empty flask, in g
W = Weight of sample taken for test, in g
[Ref: - IS: 6747-1981 (Reaffirmed 2010) Specification for Chewing Gum and
Bubble Gum]
B.8 Determination of Reducing sugars and Sucrose
Reagents
(1) Fehling A: Dissolve 69.28 g copper sulphate (CuSO4.5H2O) in distilled

(3) Neutral Lead Acetate: Prepare 20% neutral lead acetate solution. (This
reagent is used to clarify sugar solutions)
(4) Potassium Oxalate Solution: Prepare 10% Potassium oxalate
(K2C2O4.H2O) solution. This reagent is used to remove the excess lead
used in clarification.
distilled water.
Determination of Reducing Sugars:
1. Weigh accurately 25 g of sample and transfer to 250 ml volumetric flask.

2. Add 10 ml of neutral lead acetate solution and dilute to volume with
water and filter. Transfer an aliquot of 25 ml of the clarified filtrate to 500
ml volume flask containing about 100 ml water.
113
3. Add potassium oxalate in small amounts until there is no further

precipitation. Make upto volume.
4. Mix the solution well and filter through Whatman No. 1 filter paper.
Transfer the filtrate to a 50 ml burette.
Preliminary Titration:
Pipet 5 ml each of Fehling A and B into 250 ml conical flask. Mix and add
about 10 ml water and a few boiling chips or glass beads. Dispense solution.
Heat the flask to boiling. Add 3 drops of methylene blue indicator. Continue
the addition of solution dropwise until the blue colour disappears to a brick-
red end point. (The concentration of the sample solution should be such that
the titre value is between 15 and 50 ml). Note down the titre value.
Final Titration:
Pipet 5 ml each of Fehling A and B. Add sample solution about 2 ml

less than titre value of the preliminary titration. Heat the flask to boiling with
in 3 minutes and complete the titration. Perform the titration duplicate and
take the average. Calculate the reducing sugar % as shown below:
Dilution x Factor of Fehling (in g) x 100

Reducing Sugars % =
Determination of Total Reducing Sugars:
Pipette an aliquot of 50 ml of the clarified, de-leaded filtrate to a 100

ml volumetric flask. Add 5 ml of conc. HCl and allow standing at room
temperature for 24 hours. Neutralize with conc. NaOH solution followed by
0.1N NaOH. Make up to volume and transfer to 50 ml burette having an offset
tip and perform the titration on Fehling solution similar to the procedure
described in the determination of reducing sugars.
114
Dilution x Factor of Fehling (in g) x 100

Reducing Sugars % =
[Ref: - A.O.A.C 17th edn, 2000, Official method 920.183 (b) Sugars (Reducing)
in Honey / I.S.I Hand book of Food Analysis (Part II) – 1984 page 36]
Determination of Factor (for Invert Sugar) of Fehling Solution:
Accurately weigh around 4.75 g of analar grade sucrose. Transfer to

500 ml volume flask with 50 ml distilled water. Add 5 ml conc. HCl and allow
to stand for 24 hours Neutralize with NaOH solution and make up to volume.
Mix well and transfer 50 ml to a 100 ml volumetric flask and makeup to
volume. Transfer to a burette having an offset tip.

above:
Factor Titre x Weight of sucrose (in g)

Reducing Sugars % =
Fehling Factor = Titre x Weight of sucrose in gm

B.9 Determination of Sucrose
Calculation
Sucrose %
= (Total reducing sugars / invert sugar % - reducing sugars %) x 0.95
[Ref: - A.O.A.C 17th edn, 2000, Official method 920.184 Sucrose in Honey /
I.S.I Hand Book of Food Analysis (Part II) – 1984 page 36]
115
B.10 Determination of Total protein
As per Clause A.7
B.11 Determination of fat
As per Clause A.8
B.12 Determination of Pb, Cu & Zn
As per Clause A.12
B.13 Determination of filth
As per Clause A.14
B.14 Determination of TPC
Refer Microbiology Manual
B.15 Determination of Coliform
B.16 Determination of Yeast & Mould
B.17 Determination of E.Coli
B.18 Determination of S. aureus
116
C. CHOCOLATE
As per FSSA, chocolate is a homogenous product obtained by an

adequate process of manufacture from a mixture of one or more the
ingredients, namely cocoa beans, cocoa nib, cocoa mass, cocoa press cake and
cocoa dust, including fat reduced cocoa powder with or without addition of
sugars, cocoa butter, milk solids including milk fat.
Types of chocolate
Milk chocolate: Milk chocolate is the homogenous product obtained by an

adequate process of manufacture from one or more of cocoa nib, cocoa mass,
cocoa press cake, cocoa powder with sugar and milk solids including milk fat
& cocoa butter.
Milk covering chocolates: Milk covering chocolate is same as milk chocolate

and is suitable for covering purposes.
Plain chocolate: Plain chocolate is the homogenous product obtained by an

adequate process of manufacture by one or more cocoa nib, cocoa mass,
cocoa press cake, cocoa powder including milk fat & cocoa butter.
Plain covering chocolates: Plain covering chocolate is same as plain

chocolate and is suitable for covering purposes.
Filled Chocolate: Filled chocolate is a product having an external coating of

chocolate with a center clearly distinct through its composition from the
external coating. Filled chocolate does not include flour, confectionery, pastry
and biscuit products.
Composite chocolate: Composite chocolate is a product containing at least

60% m/m of chocolate and edible wholesome substances such as fruits, nuts
etc.
117
Blended chocolate: Blended chocolate is a blend of milk and plain

chocolates in varying proportions.
C.1 Preparation of Sample
 Melt the product in a beaker at a temperature of 45-50˚C. Pour the melted

sample on a marble slab and mix thoroughly with a spatula till the
product is solidified and transfer to a stoppered glass bottle. Store in a
cool place.
 Chill the material until hard and then grate or shear to a fine granular
condition Mix thoroughly and transfer to a stoppered glass bottle. Store in
a cool place.
 Alternatively melt in a suitable container by placing container in water
bath at about 50 ˚C. Stir frequently until test portion melts and reaches
temperature of 45 – 50 ˚C, remove from bath, stir thoroughly and while
still hot remove test portion for analysis using glass or metallic tube
provided with close fitting plunger to expel test portion from tube or
disposable plastic syringe.
th
[Ref: - I.S 1163: 1971 Specification for Chocolate/ A.O.A.C 17 edn, 2000,
Official Method 970.20 Cocoa Products – Preparation of sample]
C.2 Determination of Moisture - Vacuum Oven method
Apparatus
(1) Aluminum dish – 75mm diameter and about 25mm height with close
fitting cover.
(2) Dessicator
(3) Vacuum oven
118
Procedure

weighed.
2. Distribute the material as evenly as practicable over the bottom of the
dish by gentle sidewise movements.
o
six hours at 80 ± 1 C at a pressure not exceeding 5 mm of Hg. During
heating admit slow current of air into oven.
is attained.
as moisture %.
Calculations:
(W3 - W2)
Moisture content, % by mass = x 100
W1
Where:

119
C.3 Determination of Fat
Apparatus
(1) Buchner funnel – of 9 cm size.
(2) Soxhelet Apparatus - with 250 ml flat bottom extraction flask
Reagents
(1) 8 M Hydrochloric acid - sp.gr. 1.16
(2) Filter aid - a suitable brand
(3) Petroleum ether - redistilled below 60˚C
(3) Sodium sulphate - anhydrous
Procedure
1. Weigh accurately about 10 to 20 g of the prepared sample into a 400-ml

beaker and add 30 ml of water and 25 ml of hydrochloric acid.
2. Heat for 30 minutes on a steam bath, with frequent stirring.
3. Add 5 g of filter aid and 50 ml of ice-cold water and chill for 30 minutes in
ice-cold water.
4. Fit a heavy piece of linen into the buchner funnel and moisten with water.
5. Apply gently suction and pour over it a suspension of 3 g of filter aid in 30
ml of water. Filter the hydrolyzed mixture by gentle suction, rinsing the
beaker three times with ice-cold water, taking care to leave a layer of
liquid on the filter.
6. Finally wash three times with ice-cold water and suck dry.
7. Transfer the filter-cake from the funnel to the original beaker, using a
small piece of filter paper to transfer any material adhering to the funnel.
8. Wash the funnel with petroleum ether into the beaker and evaporate the
ether on a steam bath.
120
9. Break up the cake with a glass rod and allow it to remain on the steam
bath until the contents are so dry as to enable pulverizing easily. Place in
an oven at 100 + 2˚C for one hour.
10. Add 15 g of powdered anhydrous sodium sulphate and mix well. Transfer
the mixture to the fat extraction thimble of the soxhlet apparatus. Wash
the beaker with 50 ml of petroleum ether and transfer the washings to
the thimble. Extract the fat with petroleum ether so that at least 300 ml
has been circulated.
11. Transfer the extract to a tared dish and evaporate the petroleum ether on
a steam bath.
12. Dry the fat till the difference in weight between successive weighing is
not more than 1 mg.
Calculation
Total Fat % by mass = 10000 x w

(on moisture free basis) W x (100-M)
Where,
w = weight in g of fat
W = weight in g of prepared sample taken for the test
M = moisture, percent by weight, in the prepared sample
Note: - In case of plain covering chocolate, fat can be extracted directly in a

soxhlet apparatus
[Ref: - I.S 1163: 1971 Specification for Chocolate / I.S.I Handbook Of Food
Analysis (Part IX) 1984 page 20]
121
th
Also see A.O.A.C 17 edn, 2000 Official Method 963.15 Fat in Cocoa Products
– Soxhlet Extraction Method
C.4 Determination of Non Fat Milk Solids
Reagents
(1) Petroleum ether
(2) Sodium Oxalate solution – Approximately one percent (w/v)
(3) Glacial acetic acid
(4) Tannic acid solution- approximately 10 percent (w/v)
(5) Concentrated sulphuric acid- sp. Gr. 1.84
(6) Catalyst mixture- 1.0 g of selenium and 5.0 g of mercuric oxide intimately
mixed together.
(7) Alkali solution- prepared by dissolving 300 g of sodium hydroxide and 10

g of sodium thiosulphate in 500 ml of water.
(8) Standard Sulphuric Acid-Approximately 0.1 N
(9) Methyl Red Indicator solution- Dissolve one gram of methyl red in 200 ml
of rectified spirit (95 percent by volume).
(10) Standard Sodium Hydroxide Solution- approximately 0.1 N
Procedure
1. Weigh accurately about 10 g of the prepared sample and extract the fat by
shaking and centrifuging with two consecutive portions each of 100 ml of
petroleum ether. Remove the last traces of ether from the extracted
residue in an air oven.
122
2. Shake the de-fatted residue with 100 ml of water for 4 minutes and then
add 100 ml of sodium oxalate solution. Stopper and shake vigorously for
3 minutes.
3. Allow this mixture to stand for 10 minutes, shake again for 2 minutes and
then centrifuge for 15 minutes.
4. Pipette 100 ml of the clear supernatant liquid into 250-ml beaker and add
one milliliters of glacial acetic acid, stir gently, stand for a few minutes
and then add 4 ml of freshly prepared tannic acid solution and stir.
5. Allow the precipitate to settle and filter through a Whatman filter paper
No. 42 overlaid with paper pulp, in a 7cm Buchner funnel, wash twice
with the sodium oxalate solution containing one percent (w/v) of the
glacial acetic acid and two percent (w/v) of tannic acid solution.
6. Digest the precipitate in a Kjeldahl flask with 20 ml of sulphuric acid, 15 g
of sodium sulphate and 1 g of the catalyst, for 30 minutes after the
mixture has become clear.
7. Cool the contents of the flask. Transfer quantitatively to a round-bottom
flask, with water, the total quantity used being about 200 ml. Add with
shaking a few pieces of pumice stone to prevent bumping.
8. Add 50 ml of alkali solution carefully over the side of the flask so that it
does not mix at once with the acid solution but forms a layer below the
acid.
9. Assemble the apparatus, taking care that the tip of the condenser extends
below the surface of the sulphuric acid contained in the beaker.
10. Mix the contents of the flask by shaking and distill until all ammonia has
distilled over into the standard sulphuric acid.
11. Detach the flask from the condenser and shut off the burner. Rinse the
condenser thoroughly with water into the beaker. Wash the tip carefully
so that all traces of condensate are transferred to the beaker.
12. When all the washings have drained into the beaker, add two or three
drops of the methyl red indicator solution and titrate with standard
123
sodium hydroxide solution Carry out a blank using all reagents in the
same quantities but without the sample to be tested.
Calculation
Non- fat milk solids % by mass = 3126.2 x (B-A) x N

W
(on moisture free basis)
Where,
B = volume in ml of standard sodium hydroxide solution used to neutralize

the acid in the blank determination
A = volume in ml of standard sodium hydroxide solution used to neutralize
the excess of acid in the test with the sample
N = normality of standard sodium hydroxide solution
W = weight in g of the sample taken for the test
[Ref: - I.S 1163: 1971 Specification for Chocolate / I.S.I Handbook of Food
Analysis ( Part IX) 1984 Page 23]
th
Also See A.O.A.C 17 edn, 2000 Official Method 939.02 Protein ( Milk ) in
Milk Chocolate - Kjeldahl Method
Note: - Milk solids can also be determined from the orotic acid content which
remains unchanged by heating during manufacture
th
[Ref: - Pearsons Composition and Analysis of Foods 9 edn page384 – see
method at clause 15.5 of Methods of Analysis for Cereal and Cereal Products]
124
C.5 Test for Rancidity
Reagent
Phloroglucin dihydrate: 0.1% in diethyl ether
Procedure
1. Take 10g of prepared sample.

2. Add 10ml of 0.1% Phloroglucin dihydrate solution.
3. Appearance of pink colour indicates presence of rancidity.
[Ref: - IS: 7679-1978]
C.6 Determination of Ash Insoluble in dilute HCl
Reagents
(1) Dilute hydrochloric acid - Approx 5 N (445 ml in 1 litre).
Procedure

dry platinum basin of 100ml capacity.
2. Carbonize the material in the dish with the flame of a burner.
o
3. Complete the ignition by keeping in a muffle furnace at 550 ± 10 C until
gray ash results.
4. Cool in a desiccator.
the washings are free from chlorides. (To check this, add few drops of 2M
free.)
125
maintained at 105 +2˚C for about three hours. Ignite in the muffle furnace
at 550 ± 10˚C for one hour.
8. Cool the dish in a desiccator and weigh. Heat again for 30 minutes in the
muffle furnace, cool and weigh.
Calculation
M2
Where,
[Ref: - IS: 3077-1972 Specification for roasted and ground coffee/ IS: 1163-
1992 (Reaffirmed 2009) Specification for chocolates/ I.S.I. Handbook of Food
Analysis (Part IX) 1984 page 52]
C.7 Determination of Milk Fat (in Milk Chocolate, White Chocolate)
Procedure
1. Extract sufficient quantity of fat (generally 7 g) from 25-30 g of sample

using Soxhlet extraction method.
2. Weigh 5 g of fat and determine R.M Value.
Extrapolate the milk fat content from the observed R.M value taking the
standard value of 28 for pure milk fat.
126
Determination of RM Value of extracted fat
Requirements
Apparatus
(1) Flat-Bottom Boiling Flask— The flask (A)) shall be made of resistance
glass.
(2) Still-Head — The still-head (B) shall be made of glass tubing of wall
thickness 1.25 ± 0.25 mm. A rubber stopper, fitted below the bulb of the
longer arm of the still-head, and used for connecting it to the flask, shall have
its lower surface 10 mm above the center of the side-hole of the still-head.
(3) Condenser — The condenser (C ) shall be made of glass.
(4) Receiver — The receiver (D) shall be a flask, with two graduation marks
on the neck.
(5) Asbestos Board — An asbestos board (E), 120 mm diameter, 6 mm in

thickness, with a circular hole about 65 mm in diameter, shall be used to
support the flask over the burner.
(6) Bunsen Burner
(7) Reichert- Meissl Distillation Apparatus
Reagents
(1) Glycerine
(2) Conc. NaOH Solution: 50 %(w/w). Dissolve NaOH in an equal weight of

water and store the solution in a bottle protected from carbon dioxide. Use
the clear portion free from deposit.
(3) Pumice Stone Grains —1.4 to 2.0 mm in diameter.
127
(4) Dilute H2SO4 Solution -1 N.
(5) Standard NaOH solution-0.1 N.
(6) Phenolphthalein Indicator — Dissolve 0.1 g of phenolphthalein in 100 ml

of 60 % rectified spirit.
(7) Ethyl Alcohol — 90%, v/v neutral to phenolphthalein.
Procedure
1. Weigh accurately 5.00 ± 0.01 g of the filtered oil or fat into the boiling
flask.
2. Add 20 g of glycerol and 2 ml of conc. NaOH solution from a burette to
which access of carbon dioxide is prevented and whose orifice is wetted
before running in the liquid, the first few drops from the burette being
rejected.
3. Heat the flask and its contents with continuous shaking on a gauze over
the naked flame until the fat, including any drops adhering to the upper
parts of the flask, has been saponified and the liquid becomes perfectly
clear. Avoid overheating during this saponification.
4. Cover the flask with a watch glass, and allow the flask to cool a little. Add
90 ml of boiling distilled water, which has been vigorously boiled for
about 15 min. After thorough mixing, the solution should remain clear. If
the solution is not clear (indicating incomplete saponification) or is
darker than light yellow (indicating overheating), repeat the
saponification with a fresh sample of the oil or fat. If the sample is old, the
solution may sometimes be dark and not clear.
5. Add 0.6 to 0.7 g of pumice stone grains and 50 ml of dilute sulphuric acid,
and immediately connect the flask with the distilling apparatus. Place the
flask on the asbestos board.
128
6. After the fatty acids have melted and separated into a clear liquid layer on
gentle warming, heat the flask without altering the flame so that 110 ml
of liquid distils over in the course of 19-21 min.
7. The distillation is considered to begin when the first drop forms in the
still head.
8. Keep the water flowing in the condenser at a sufficient speed to maintain
the temperature of the outgoing water from the condenser between 15°C
and 20°C.
9. Collect the distillate in a graduated flask.
10. As soon as 110 ml have distilled over, stop heating the boiling flask and
replace the graduated flask by a measuring cylinder of about 25 ml
capacity to catch washings.
11. Close the graduated flask with the stopper, and, without making the
contents, place it in a water-bath at 15°C for 10 min, making sure that the
100-ml graduation mark is below the level of the water. Swirl round the
contents of the flask from time to time.
12. Dry the outside of the flask and then mix the distillate by closing the flask
and inverting it four or five times, but do not shake.
13. Filter through a dry Whatman No. 4 filter paper. Reject the first 2-3 ml of
the filtrate and collect the rest in a dry flask.
14. Pipette 100 ml of the filtrate in a titration flask, add 0.1 ml of
phenolphthalein indicator solution and titrate with standard 0.1N NaOH
solution until the liquid becomes slightly pink.
15. Run a blank test without the fat but using the same quantities of reagents
and following the same procedure.
Calculations:
Milk Fat, % by mass = (RV – 0.2) x F
(on dry Basis) 26
129
Where,
RV = Reichert value obtained for extracted fat
F = Total Fat % in the sample

0.2 = Reichert value of cocoa butter
26 = Reichert value of milk fat
[Ref: - IS: 1163-1992 (Reaffirmed 2009) Specification for chocolates and IS:
548- 1964 Sampling, Physical and Chemical Tests for Oils & Fats]
C.8 Determination of Cocoa Solids
Procedure
1. Treat 50 g milk chocolate with three 100 ml portions of ether in

centrifuge bottle, centrifuging and decanting after each addition.
2. Dry residue in bottle and crush to powder with flat end glass rod. Shake
with 100 ml 1% Sodium oxalate Na2C2O4 (w/v) and let stand 30 minutes.
3. Centrifuge and decant, wash in bottle with three 100 ml portions of water
at room temperature shaking well each time until no cocoa material
adheres to the bottle. Centrifuge 10 – 15 minutes after each washing and
decant.
4. Wash residue in the same fashion with two 100 ml portions of alcohol
and one 100 ml portion of ether. With the aid of small portions of ether,
transfer residue resulting from ether, alcohol and aqueous extract to
tared aluminium dish provided with tight fit cover. Use small amount of
acetone and policeman to transfer any material that sticks to bottom.
5. Evaporate liquid carefully on steam bath and dry residue in oven at 100
˚C.
130
6. Cool dish in dessicator and weigh till constant mass is achieved.
To obtain moisture free and fat free cocoa mass multiply the weight of
residue with factor 1.43.
To obtain weight of chocolate liquor multiply the weight of residue with

factor 2.2 (This factor is based on fat content of 54% in chocolate liquor). The
correction factor is calculate by using said formula.
th
[Ref: - A.O.A.C 18 edn, 2005 Official Method 931.05 Cocoa solids of
chocolate liquor]
C.9 Determination of Reducing Sugars
Reagents:
(1) Fehling A: Dissolve 69.28-g copper sulphate (CuSO4.5H2O) in distilled

(3) Neutral Lead Acetate: Prepare 20% neutral lead acetate solution. (This
reagent is used to clarify sugar solutions)
(4) Potassium Oxalate Solution: Prepare 10% Potassium oxalate
(K2C2O4.H2O) solution. This reagent is used to remove the excess lead
used in clarification.
distilled water.
Procedure & Calculations:
As per Clause A.9
131
C.10 Determination of Sucrose

As per Clause A.10
C.11 Determination of Chocolate Component of Filled Chocolate
Procedure
1. Weigh to the nearest 0.1g, 500g of the filled chocolate.

2. Scrape the chocolate coating and separate the filling. Filling should not be
included in the analysis.
3. Weigh the filling to the nearest 0.1g.
Calculation
(M1 - M2) x 100

Chocolate component, % by mass =
M1
Where,
M1 = mass in g, of the filled chocolate taken for test
M2 = mass in g, of the filling
[Ref: - .IS: 1163-1992 (Reaffirmed 2009) Specification for Chocolates]
C.12 Determination of Edible Wholesome Substances
Procedure
1. Weigh to the nearest 1.0g, 500g of the product containing fruits, nuts etc.
132
2. Break the sample into small into small pieces and place them in 1 litre
glass/ metal container.
3. Cover the sample with meted cocoa butter and place container in a warm
oven until the added ingredients can be separated upon stirring.
4. Sieve contents through a 20 mesh sieve and allow the liquid to drain
completely.
5. Next soak the sieve containing ingredients in trichloroethylene and stir
gently for a minute or two.
6. Remove cleaned nuts, fruits etc, onto a tray and let the solvent evaporate.
Weigh to the nearest 0.1g.
Calculation
Wholesome ingredients,% by mass = Mass of residue x 100

Sample wt.
[Ref: - .IS: 1163-1992 (Reaffirmed 2009) Specification for Chocolates]
C.13 Determination of Total protein
As per Clause A.7
C.14 Determination of Pb, Cu & Zn
As per Clause A.12 or else ICPMS can be used as an alternate method
C.15 Determination of filth
As per Clause A.14
133
C.16 Determination of TPC
C.17 Determination of Coliform
C.18 Determination of Yeast & Mould
C.19 Determination of E.Coli
C.20 Determination of S. aureus
D. ICE LOLLIES or EDIBLE ICES
D.1 Determination of Reducing sugars
As per Clause A.9
D.2 Determination of Sucrose
As per Clause A.10
D.3 Determination of Pb, Cu & Zn
As per Clause A.12
134
D.4 Determination of TPC
D.5 Determination of Coliform
D.6 Determination of Yeast & Mould
D.7 Determination of E.Coli
D.8 Determination of S. aureus
D.9 Isolation of Salmonella per 25 gm

The procedure consists of five distinct stages. The initial handling of

the food and the non-selective enrichment stage (preenrichment) vary
according to the type of food examined.
i) Non-Selective Enrichment (Preenrichment).

The test sample is initially inoculated into a non-inhibitory liquid medium to
favour the repair and growth of stressed or sublethally-injured salmonellae
arising from exposure to heat, freezing, desiccation, preservatives, high
osmotic pressure or wide temperature fluctuations
135
ii) Selective Enrichment

Replicate portions of each preenrichment culture are inoculated into two
enrichment media to favor the proliferation of salmonellae through a
selective repression or inhibition of the growth of competing microorganims.
iii) Selective Plating

Enrichment cultures are streaked onto selective differential agars for the
isolation of salmonellae Purification Presumptive Salmonella isolates are
purified on MacConkey agar plates or BSA plates.
iv) Biochemical Screening

Isolates are screened using determinant biochemical reactions.
v) Serological Identification
Polyvalent and/or somatic grouping antisera are used to support the
tentative identification of isolates as members of Salmonella spp.
Materials and Equipment

(1) Nutrient Broth (NB).
(2) Brilliant Green Water.
(3) Buffered Peptone Water (BPW).
(4) Brilliant Green Broth (TBG).
(5) Selenite Cystine Broth (SC).
(6) Bismuth Sulfite Agar (BS).
(7) Brilliant Green Sulfa Agar (BGS).
(8) MacConkey Agar,
(9) Rappaport Vassiliadis medium (RV)
(10)Nutrient Agar.
(11)Triple Sugar Iron Agar (TSI).
(12)Lysine Iron Agar (LIA).
(13)Urea Agar (Christensen's).
(14)Commercial biochemical test kits.
(15)Polyvalent and single grouping somatic (O) and flagellar (H) antisera.
136
(16)N- Saline.
(17)Blender, stomacher or other homogenizing device.
(18)Incubator or water bath capable of maintaining 35±2°C and 44°C.
Procedure
Analyze samples as soon as possible. If necessary, store samples under time
and temperature conditions that will prevent the growth or death of native
microflora.
(2) Non-selective Enrichment (Pre-enrichment)

Sample Analysis
The required analytical unit is dispersed into a suitable non-selective

enrichment broth Nutrient broth (NB) and buffered peptone water (BPW)
are equally reliable and can be used interchangeably as general purpose
preenrichment. If the pH of the preenrichment mixture lies outside the range
of 6.0 - 7.0, adjust with 1N NaOH or 1N HCl.
Note:
If the sample unit consists of a container with little food material, thoroughly
rinse the interior of the container with a suitable preenrichment broth
medium and incubate the rinse in a sterile flask. This eventuality is more
frequently encountered in situations involving consumer complaints or food
poisoning investigations. A positive Salmonella and a negative medium
control should be set up in parallel with the test samples. Incubate the
preenrichment mixture and the positive and negative controls at 35±2°C for
18 - 24 h.
Note:
137
The negative medium control should not show any evidence of growth after
incubation whereas the absence of growth in the positive control would
invalidate test results.

With a sterile pipette, transfer 1.0 mL of the preenrichment culture into each
of 9 mL of selenite cystine (SC) and Rappaport Vassiliadis broths.
Incubate SC and RV broths for 24±2 h at 35±2°C and 42±2°C, respectively.
(4) Selective Plating

Streak replicate loopsful of each selective enrichment culture onto BSA and
BG agar to obtain well isolated colonies. The enrichment cultures may be
streaked onto additional plating media for the isolation of Salmonella.
Incubate plates at 35±2°C for 24±2 h. If colonies suggestive of Salmonella
have not developed on BSA and BGA plates, incubate for an additional 24±2
h. Examine incubated plates for colonies suggestive of Salmonella. Typical
Salmonella usually occur as pink to fuchsia colonies surrounded by red
medium on BG agar, and as black colonies on BS agar with or without a
metallic sheen, and showing a gradual H2S- dependent blackening of the
surrounding medium with increasing incubation time.
(5) Biochemical Screening

With a sterile needle, inoculate suspect colonies into the biochemical media
or in commercial diagnostic kits that would yield equivalent results. Incubate
the biochemical media for 18-24 h at 35±2°C.
Note:
Erroneous biochemical results may be obtained if tubes are not loosely

capped during incubation.
138
Commercially available diagnostic kits may be used to obtain detailed

biochemical profiles of bacterial isolates. If none of the isolates from a
particular analytical unit are suggestive of Salmonella, the analytical unit is
considered to be free of salmonellae. If the presence of Salmonella is
suspected, proceed with serological testing. If serological testing is not to be
performed within 72 h, inoculate suspect isolates into nutrient agar slants
and incubate at 35±0.5oC for 24±2 h. Store the agar slants at refrigerator (4
to 10oC) temperature. Nutrient agar slants that have been stored for more
than 72 h should not be used for serological testing. Prepare fresh agar slants
for this purpose.
(6) Serological Identification Testing with somatic polyvalent antiserum

1. Mark the following sections on an agglutination plate: C+ (positive
control), C-(negative control) and T (test culture).
2. Add one drop of physiological saline to each of the areas marked T
and C+, and two drops to the area marked C-.
3. Remove sufficient culture material from a triple sugar iron, lysine iron
or nutrient agar slant to prepare a heavy suspension in the test area
(T) and in the negative control (C-) area. The inoculum should be
withdrawn from the slope portion of agar slants.
4. For the positive control, prepare a heavy suspension of a known
Salmonella
5. Culture in the area marked C+.
6. Prepare somatic polyvalent antisera as directed by the manufacturer;
add one drop to each of the areas marked T and C+.
7. Mix the culture-saline-antiserum suspensions in T and C+ and the
saline-culture mixture in C- with a sterile needle or loop. Tilt the slide
preparation back and forth for 1 min.
139
8. Hold the slide against a dark background and observe for

agglutination. Salmonella cultures usually agglutinate within 1 min.
9. False positive reactions from microorganisms that are closely related
to Salmonella may occur. Such misleading reactions can be resolved
through further testing with somatic grouping and flagellar antisera.
10. The serological test for a given culture is invalidated if the negative
control shows agglutination (autoagglutination).
(7) Reporting: Salmonella - Present or Absent / 25g
D.10 Detection of Listeria monocytogenes per gm

(1) Half Fraser Broth
(2) Fraser Broth
(3) Oxford Agar
(4) Palcam Agar
(5) Nutrient Agar
140
Procedure

1. Prepare Half Fraser, Fraser broth, Oxford and Palcam agar and dispense
in appropriate quantities. Sterilize.
4. Pre-Enrichment
or mL (the analytical unit) into 225 mL of the required Diluent.
2. Take 10 ml of above diluted sample in 90 ml Enrichment broth and
incubate for 24 hrs. at 30±2°C
3. Take 0.1 ml of above in 10 ml of Fraser broth and incubate for 48 hrs.
at 37±2°C
5. Selective Enrichment
1. Transfer a loopful of the inoculum from enrichment culture in to
Oxford and Palcam agar and incubate for 48 hrs at 37±2°C.
2. Observe for characteristics colonies on the Palcam and Oxford agar.
6. Biochemical Identification Tests
1. Gram Staining
2. Catalase Test
3. Carbohydrate Fermentation Test
4. Nitrate Reduction Test
5. Haemolysis Test
6. Oxidase Test
7. Indole Test
8. Urease Test
141
9. H2S Test
10. VP Test
11. MR Test
Reporting: Listeria monocytogenes Present or absent / gm
…………..
142
*The methods mentioned in the manual needs to be verified/ validated before they are put
in use by the laboratory.
Food Safety and Standards Authority of India
(Ministry of Health and Family Welfare)
FDA Bhawan, Kotla Road,
New Delhi-110002
www.fssai.gov.in

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LAB.

FOOD SAFETY AND STANDARDS AUTHORITY OF INDIA

Deepest Sense of Gratitude and Indebtedness to all the

Sincere Thanks to the Panel, Chairman for his valuable

Deepest Appreciation to the Chairperson, FSSAI and CEO,

MANUAL FOR ANALYSIS OF BEVERAGES (COFFEE, TEA,

S.No. TITLE PAGE NO.

PART A- BEVERAGES AND SUGAR AND SUGAR PRODUCTS

1.0 Roasted Coffee 4

PART B - CONFECTIONERY PRODUCTS

A. SUGAR BOILED CONFECTIONERY AND LOZENGES

S.No. TITLE PAGE NO.

B. CHEWING GUM AND BUBBLE GUM

B1 Preparation of Sample 106

C1 Preparation of Sample 118

S.No. TITLE PAGE NO.

D. ICE LOLLIES OR EDIBLE ICES

D1 Determination of Reducing Sugar 134

MANUAL FOR ANALYSIS OF BEVERAGES (COFFEE, TEA,

COFFEE - Definitions of different types of Coffee are given under section

1.0 Roasted Coffee

1.1 Preparation of sample:

1. 2 Determination of moisture: (Routine method)

Weigh accurately about 5 gms of sample in a tared aluminium dish.

W = Weight in gms, of Aluminium dish.

1.2 A Determination of Moisture - Vaccum Oven method (Reference

1.2 A.1 Apparatus

1. Aluminium dish – 7 cm diameter and about 3 cm height with close

3. Vaccum oven – connect with pump capable of maintaining partial

1.2 A.2 Procedure

Accurately weigh about 5 gm of sample in a dish previously dried at

1.3 Determination of total ash:

Weigh accurately about 5 gms of sample in a tared silica / platinum

(W2 – W) x 100 x 100

W1 = Weight in gms of Silica dish. + sample

Alternative method of Determination of Total Ash- Use a

1.4 Determination of water soluble ash:

W2 = Weight in gms of Silica dish + water insoluble ash.

Water soluble ash percent by wt = A – B

A = Total ash percent by wt

Water soluble ash

1.5 Determination of Ash insoluble in dilute HCl:

Boil total ash prepared as in sec. 1.3, with 25 ml of hydrochloric acid

(W2 – W) x 100 x 100

1.6 Determination of alkalinity of soluble ash:

To the filtrate reserved during the determination of water soluble ash

M = % Moisture of the sample

1.7 Determination of aqueous extract:

Accurately weigh around 2 gms of sample and transfer to a 500 ml

Record the lowest weight

(W2 – W1) x 250 x 100 x 100

1.8 Determination of Caffeine Content (Bailey Andrew Method)

Weigh accurately about 5 gm of sample, transfer to a 250 ml

Transfer the solution to a 125 ml separator. Wash the graduated flask

Place the flask on the digestion rack and concentrate chloroform to

Carry out a blank determination using reagents in the same proportion

test should be 1 gm only

1.8A Alternate Chromatographic – Spectrophotometric Method:

Accurately weigh 100 mg of caffeine (USP, anhydrous) into 100 ml

1.8A.3 Preparation of Sample