Important methods for analysis of

fruits and vegetable and their

products
 TSS
• Total soluble solids
• Total soluble solids (TSS) are determined by means of brix
hydrometers which measures the specific gravity or by using hand
refractometer which measures refractive index
• Brix is a measure of soluble solids only in case of pure sucrose
solutions
• Generally, the fruit juices contain more sugar than any other soluble
constituent and hence, brix provides a useful guide of soluble solids
or sugar contents
• Brix hydrometer is used to determine the percentage of brix in
syrup while Abbe refractometer gives refractive index as well as
degree brix
• The Refractive index of the test solution is measured at 20°C using a
refractometer and the soluble solid content is determined by the
use of tables correlating refractive index with soluble solids
• Degrees Brix (symbol °Bx) is the sugar content of
an aqueous solution
• One degree Brix is 1 gram of sucrose in 100 grams
of solution and represents the strength of the
solution as percentage by mass
• If the solution contains dissolved solids other
than pure sucrose, then the °Bx only
approximates the dissolved solid content
• The °Bx is traditionally used in the wine, sugar,
carbonated beverage, fruit juice, maple syrup and
honey industries.
• Apparatus/machinery/equipments required:
• Hand refractometer
• Abbe-refractometer
• Distilled water
• Procedure for estimation
1. Calibrate the refractometer with a drop of distilled water, adjust
the scale to 0 %
2. Wipe the prism with a cotton swab.
3. i) Cut a piece of fruit and squeeze a drop of juice on the prism of
the refractometer.
ii) Place a drop of juice/squash/syrup on the prism.

• iii) Place small quantity of jam/jelly in muslin cloth and squeeze and
place on the prism.

• 4. Read the value directly through eye piece of refractometer

towards the light at room temperature.
5. Correct the readings to standard temperature of 20°C if readings
are made at temperature other than 20oC
 Temperature correction for the standard model of sugar refractometer calibrated at 20oC

Temperature Scale reading for soluble solids contents, %

0C
5 10 15 20 25 30 40 50 60 70
Corrections to be subtracted
15 0.29 0.31 0.33 0.34 0.34 0.35 0.37 0.38 0.39 0.40

16 0.24 0.25 0.26 0.27 0.28 0.28 0.30 0.30 0.31 0.32

17 0.18 0.19 0.20 0.21 0.21 0.21 0.22 0.23 0.23 0.24

18 0.13 0.13 0.14 0.14 0.14 0.15 0.15 0.15 0.16 0.16

19 0.06 0.06 0.07 0.07 0.07 0.07 0.08 0.08 0.08 0.08

Corrections to be added
21 0.07 0.07 0.07 0.07 0.08 0.08 0.08 0.08 0.08 0.08

22 0.13 0.14 0.14 0.15 0.15 0.15 0.15 0.16 0.16 0.16

23 0.20 0.21 0.22 0.22 0.23 0.23 0.23 0.24 0.24 0.24

24 0.27 0.28 0.29 0.30 0.30 0.31 0.31 0.31 0.32 0.32

25 0.35 0.36 0.37 0.38 0.38 0.39 0.40 0.40 0.40 0.40
Fruit products TSS(oB)
Most fruit juices 10-15
Perlette grape juice 11-18
Lime juice 6-14
Lemon Juice 5-9
Sauces Not less than15
Ketchup Not less than25
Squashes Not less than 40
Jam Not less than 68
Jelly and Marmalade Not less than 65
Syrups/Sharbats Not less than 65
Preserve and Candies Not less than 68
 Acid Value
• Most of the fruit, vegetables and their products contain
acid or mixture of acids
• The acids may occur naturally in the fruit and vegetables or
may be added during manufacture of different products or
by lactic acid or acetic acid fermentation
• Generally citric acid is added in most fruit products while in
pickles, sauces and ketchup acetic acid is used
• The acids are mainly responsible for the tartness or sour
taste, thus estimation of acidity is used as the measure of
tartness
• They also helps in preservation by lowering the pH of the
finished products.
• Acidity in the sample is measured by titrating a given sample
against a standard alkali solution of known concentration using
phenolphthalein as an indicator to a light pink colour
• However, for highly coloured products like tomato, mixed fruit jam,
accurate determination of end point may be difficult by using
indicator, thus for such samples, acidity is measured by titrating the
sample against a standard alkali to a pH 8.1 using pH meter or using
electrometric titrimeter or the sample is further diluted to almost
colourless
• The acidity is expressed in terms of predominant acid present in the
product using standard expression
• The list of common predominant acids and their equivalent weight
present in different fruit and fruit products is in next slide
 Predominant acids in some fruits and processed products

Acid Fruit/vegetable/products Equivalent weight

Acetic acid Sauce, ketchup, pickle in vinegar 60.05
Citric acid Citrus fruit, most fruit products, 64.04
mango,guava
Lactic acid Curd and sauerkraut 90.08
Oleic acid Olive 282.46
Malic acid Apple, pear, apricot, peach, plum 67.05
Tartaric acid Grapes, tamarind 75.04
• Apparatus, reagents and glassware required

• Sodium hydroxide - 0.1 N

• Phenolphthalein solution -1.0 %
• Volumetric flask - 100 ml capacity
• Conical flask - 250 ml capacity
• Burette - 10-100ml capacity
• Magnetic stirrer
• pH Meter
• Preparation of reagents

• N/10 Sodium hydroxide: Dissolve 40.005g NaOH pellets in water

and dilute to one litre in a volumetric flask. Standardize by titrating
against standard H2SO4 solution (N/10). For standardization of
H2SO4 against Sodium carbonate (Na2CO3), heat analytical grade
Na2CO3 at 1050 C for 8 hours.
• N/10 Na2CO3 Solution: Pipette 25ml of 0.1 N H2SO4 solution ¬into
250ml conical flask. Add about 100ml water and few drops of
bromophenol blue. Add standard 0.1 N Na2CO3 solution from
burette to a blue end point or pH 4.1 using pH meter.
• 1% Phenolphthalein solution: Dissolve 1g phenolphthalein in
100ml ethanol.
• Sample preparation: Crush the sample (fruit,
vegetable, jam, pickle etc) in a blender or
pestle & mortar and mix thoroughly to obtain
pulp. Weigh the material, add some water and
boil for 1hr replacing the water lost in
evaporation. Cool and transfer to a volumetric
flask, make up to a known volume. Filter if
necessary. For juice, squash, cordial etc dilute
(if necessary) without boiling or crushing.
• Procedure for determination of acidity

• Dilute an aliquot of sample to known volume and place in

titration flask.
• Add few drops of 1% phenolphthalein as an indicator
• Titrate with N/10 NaOH to faint pink colour using burette or
pipette.
• Note the titre value and calculate % titratable acidity as
predominant acid.

• Note: For highly coloured products, dilute small volume of the

sample (5 ml) with large volume of distilled water (300-400ml). The
colour becomes so pale that the indicator colour change during
titration can be observed easily.
• Procedure for determining acidity of coloured products
using pH meter:

• Pipette 10ml of sample in 250ml beaker and add

90ml distilled water.
• Agitate diluted sample using magnetic stirrer.
• Immerse electrode of pH meter into the solution and
titrate with N/10 NaOH from a burette to a pH value
8.1. At this pH, phenolphthalein turns from colourless
to pink.
• Note the titre value and calculate titratable acidity as
percent of predominant acid.
• Calculation

% Acidity = Titre × normality of alkali × volume made × equivalent weight of acid × 100
Wt of sample × volume of aliquot ×1000
 Vitamin C
(Ascorbic acid)
• Fruit, vegetables and their products are
important sources of ascorbic acid.
• The ascorbic acid is present in sufficient quantity
in aonla, guava, grapefruit, citrus fruits like
lemon, pineapple, strawberry fruits etc.
• The products manufactured from these fruits are
also considered as rich in ascorbic acid and the
contents available in the commodities can be
detected by using 2, 6 dichlorophenol -
indophenol visual titration method
2, 6 dichlorophenol - indophenol visual
titration method
• The method is based on reduction of 2, 6
dichlorophenol – indophenols dye
• The dye, which is blue in alkaline solution and red in
acidic solution, is reduced by ascorbic acid to a
colourless form
• The reduction is quantitative and specific for ascorbic
acid in solutions in the pH range of 1.0 - 3.5
• In estimation of ascorbic acid, the prepared sample is
titrated against standard 2, 6 dichlorophenol –
indophenols dye to a pink end point
• The titre is then used to calculate the ascorbic acid in
the sample.
• Apparatus, reagents and glassware required

• 3% metaphosphoric acid (HPO3): Dissolve 3g sticks of HPO3 in distilled water

(100 ml). prepare 1 litre solution of 3% HPO3 as it also required in sample
preparation, 0.1% oxalic acid can also be used in place of metaphosphoric acid.
• Ascorbic acid solution: Weigh 100 mg L-ascorbic acid and dissolve in 3% HPO3
and make volume up to 100 ml with HPO3. Dilute 10 ml to 100 ml with 3% HPO3
(1ml=0.1mg ascorbic acid)
• Sodium bicarbonate
• Dye solution: Dissolve 50 mg of the Sodium salt of 2, 6 dichlorophenol
indophenol dye in 150 ml hot glass distilled water containing 42 mg of Sodium
bicarbonate. Cool and dilute with distilled water to 200 ml. Store in refrigerator
and standardize.
• Beakers - 100, 250ml
• Volumetric flasks - 100, 250ml
• Measuring cylinder - 250ml
• Pipette - 10ml
• Standardization of dye: To 5 ml of Standard
ascorbic acid (1ml=0.1mg) and 5 ml HPO3.
Titrate this solution with the dye solution to a
pink colour which should persist for 15
seconds. Determine the dye factor i.e.
ascorbic acid per ml of the dye.
• Sample preparation:

• Fresh Fruits/vegetables: Crush/grind fruit or vegetable parts

(known weight) along with 3% HPO3, make to volume (100 ml) with
HPO3. Filter or centrifuge.
• Fruit Juices: To 10-20 ml juice add 3% HPO3 and make volume to
100 ml. Filter or centrifuge.
• Solid and semi solid food: Take 10 g sample, blend with 3% HPO3
and make up to 100 ml with HPO3. Filter or centrifuge.

• Procedure for titration: Take (2-10ml) the HPO3 extract of the

sample and titrate with the standard dye to a pink end point
persisting for at least 15 seconds. The titre of the sample should be
such that titre should not exceed 3-5 ml.
• Samples containing sulphur dioxide: Sulphur
dioxide when present in fruit products like
squash, jam, drinks etc reduces the indophenol
dye and this interferes in the ascorbic acid
estimation. To eliminate the SO2 interference use
formaldehyde condensation method as under:

• To 10ml filtrate in a test tube, add 1ml of 40%

formaldehyde and 0.1ml of HCl, keep for 10
minutes and titrate as earlier. Record titre and
calculate ascorbic acid.
• Calculation
Dye factor = 0.5
titre

mg of ascorbic acid = Titre X Dye Factor X Volume made up X 100

per 100 gm Aliquot of extract X Wt. Of sample taken
 Sugar
(Reducing, non reducing & total)
• Glucose and fructose in the fruits represent
reducing sugars while sucrose or cane sugar
added to the fruit products represents the non-
reducing sugar
• They are estimated by using Lane and Eynon
method which measures sugar as reducing sugar
and total sugar as invert sugar
• Non-reducing sugar is determined by subtracting
the total reducing sugar from reducing sugar and
multiplying the remainder with 0.95 factor.
• Invert sugar reduces the copper in Fehling’s solution to red,
insoluble cuprous oxide. The sugar content in a food sample is
estimated by determining the volume of the unknown sugar
solution required to completely reduce a known volume of Fehling’s
solution. Glucose and other sugars are capable of reducing oxidizing
agents and are called reducing sugars and this property is used for
the estimation of sugars. The cupric ion in Fehling’s solution is
reduced to cuprous state which precipitates as red cuprous oxide
(Cu2O). Only reducing sugars reduce the copper solution.

• CuSO4 + 2NaOH → Cu (OH)2 + Na2SO4

• Cu (OH)2 → CuO (Cupric oxide)
• 2CuO + CHO → Cu2O (Cuprous oxide) + COOH
Apparatus, reagents and glassware required
Beakers - 250ml
Volumetric flasks - 250ml
Measuring cylinder - 250ml
Pipette - 10ml
Burette - 50ml
Hot plate
Fehling’s solution-A: Dissolve 69.28g copper sulphate (CuSO4.5 H2O) in water, dilute to
1 litre and filter.(Standard Fehling’s solution A or 1)
Fehling’s solution-B: Dissolve 346g of Rochelle salt (Potassium Sodium Tartrate, KNa
C4H4O6.4H2O) and 100g NaOH in water, make volume to 1 litre.(Standard Fehling’s
solution B or 2)
Methylene blue indicator (1%): Dissolve 1g in 100ml water.
45% neutral lead acetate solution: Dissolve 225g of neutral lead acetate in water and
make up to 500ml. It is used as clarifying agent.
22% Potassium oxalate solution: Dissolve 110g Potassium oxalate (K2C2O4. H2O) in
water and make volume to 500ml. This is used for neutralizing excess of lead acetate.
Standard invert sugar: Weigh 10g of sucrose into 250ml increase in 1 litre flask, add
100ml water and 5ml concentrated HCl for hydrolysis. Allow to stand for 3 days at 20-
25OC or 7 days at 15oC for inversion to take place and then make up to volume.
• Neutralization of standard invert sugar: Pipette 25ml of standard invert solution in
to a 100ml volumetric flask; add 50ml water and few drops of Phenolphthalein
indicator. Neutralize with 20% NaOH until solution turns pink. Acidify with 1N HCl
adding it drop wise until pink color disappears. Make up to 100ml with water
(1ml=2.5mg invert sugar).
Standardization of Fehling’s solution: Mix 5ml Fehling A + 5ml Fehling B solution
in 250ml conical flask. Add 25-50ml water and heat the flask. Add standard invert
sugar solution from the burette dropwise till the solution turns brick red. Add few
drops of Methylene blue indicator and add drop-wise invert solution, when the
blue color disappears, note the titre value of invert solution, repeat the titration
and calculate factor for Fehling’s solution as under:-
• Procedure for estimation of reducing sugars
Take 10-20g juice/squash/drink in 250 ml volumetric flask, add 100 ml water neutralize with 1 N NaOH. Add 2ml
45% lead acetate. Shake well and keep for 10 minutes. Add few drops of potassium oxalate solution to remove
excess of lead acetate. Make volume to 250ml with water and filter.
OR
Take 10-25 g of sample (fresh fruit or fruit product) and grind in a pestle and mortar, blend in blender, add 100 ml
water. Neutralize solution with 1 N NaOH. Boil gently for 1 hour with stirring. Replace water lost during
evaporation, cool and transfer to 500 ml volumetric flask. Make volume 500 ml and filter through whatman filter
paper.
Pipette 100 ml aliquot from filtrate in 250ml flask, add 2 ml 45% lead acetate, let it stand for 10 minutes then add
few drops of 22% potassium oxalate solution and made volume to 250 ml with water and filter.
• Add 2 ml 45% neutral lead acetate solution.
• Pipette 10ml of mixed Fehling’s solution (5 ml each of Fehling A and B) and few ml of water into 250ml conical
flask.
• Heat the flask containing mixed Fehling’s solution on hot plate and add the sample (clarified sugar) solution drop
wise from the burette/pipette till faintest blue colour remains.
• Add 2-3 drops of methylene blue indicator and complete the titration till the color changes to brick red.
• At the end point, note the readings and calculate the reducing sugars.
• Procedure for estimation of Total Sugar
Follow the method for preparation of deleaded samples from fruits or fruit
products as explained under reducing sugars (Practical-8).
• Take 50 ml of the clarified deleaded solution from step 1 into a 250 ml conical flask
• Add 5 g of citric acid and 50 ml of water boil gently for 10 minutes to complete the
inversion of sucrose and allow to cool.
• Transfer the inverted solution to a 250 ml volumetric flask and neutralize with 1 N
NaOH using phenolphthalein as indicator and make up the volume to 250 ml with
water. Place this solution into the burette and use for filtration.
• Pipette 10ml of mixed Fehling’s solution (5 ml of Fehling’s A and B) and few ml of
water into 250 ml conical flask.
• Heat the flask containing Fehling’s solution on hot plate and add sample (clarified
sugar) solution drop wise from the burette till faintest blue color remains.
• Add 2-3 drops of methylene blue indicator and complete the titration till the
colour changes to brick red precipitates.
• Note the titre value and calculate the total as well as non –reducing sugars from
the following equations.
 Analysis of starch
• Starch is the most common digestible polysaccharide found in
foods, and is therefore a major source of energy in our diets
• In its natural form starch exists as water-insoluble granules (3 - 60
mm), but in many processed foods the starch is no longer in this
form because of the processing treatments involved (e.g., heating)
• It consists of a mixture of two glucose homopolysaccharides:
amylose (500-2000 glucose units) which is linear, and amylopectin
(>1,000,000 glucose units) which is extensively branched
• These two kinds of starch have different physiochemical properties
and so it is often important to determine the concentration of each
individual component of the starch, as well as the overall starch
concentration
 Analysis of starch
• Sample preparation
• The starch content of most foods cannot be
determined directly because the starch is contained
within a structurally and chemically complex food
matrix
• In particular, starch is often present in a semi-
crystalline form (granular or retrograded starch) that is
inaccessible to the chemical reagents used to
determine its concentration
• It is therefore necessary to isolate starch from the
other components present in the food matrix prior to
carrying out a starch analysis
 Analysis of starch
• In natural foods, such as legumes, cereals or tubers, the starch granules are usually
separated from the other major components by drying, grinding, steeping in water,
filtration and centrifugation
• The starch granules are water-insoluble and have a relatively high density (1500
kg/m3) so that they will tend to move to the bottom of a container during
centrifugation, where they can be separated from the other water-soluble and less
dense materials
• Processed food samples are normally dried, ground and then dispersed in hot 80%
ethanol solutions
• The monosaccharides and oligosaccharides are soluble in the ethanol solution,
while the starch is insoluble
• Hence, the starch can be separated from the sugars by filtering or centrifuging the
solution
• If any semi-crystalline starch is present, the sample can be dispersed in water and
heated to a temperature where the starch gelatinizes (> 65 °C). Addition of
perchloric acid or calcium chloride to the water prior to heating facilitates the
solubilization of starches that are difficult to extract
 Analysis of starch
• Analysis methods. Once the starch has been extracted there are a
number of ways to determine its concentration:
– Specific enzymes are added to the starch solution to breakdown the
starch to glucose. The glucose concentration is then analyzed using
methods described previously (e.g., chromatography or enzymatic
methods). The starch concentration is calculated from the glucose
concentration.
– Iodine can be added to the starch solution to form an insoluble starch-
iodine complex that can be determined gravimetrically by collecting,
drying and weighing the precipitate formed or titrimetrically by
determining the amount of iodine required to precipitate the starch.
– If there are no other components present in the solution that would
interfere with the analysis, then the starch concentration could be
determined using physical methods, e.g., density, refractive index or
polarimetry.
 Analysis of starch
• The amylose and amylopectin concentrations in a sample can be
determined using the same methods as described for starch once
the amylose has been separated from the amylopectin
• This can be achieved by adding chemicals that form an insoluble
complex with one of the components, but not with the other, e.g.
some alcohols precipitate amylose but not amylopectin
• Some of the methods mentioned will not determine the
concentration of resistant starch present in the sample
• If the concentration of resistant starch is required then an
additional step can be added to the procedure where
dimethylsulfoxide (DMSO) is added to dissolve the resistant starch
prior to carrying out the analysis.
 Pectin
• Pectin is a structural acidic heteropolysaccharide
• Its main component is galacturonic acid, a sugar
acid derived from galactose
• It is produced commercially as a white to light
brown powder, mainly extracted from citrus
fruits, and is used in food as a gelling agent,
particularly in jams and jellies
 Determination of pectin
• Colloid titration
• Estimation as calcium pectate
• Ethanol yield method
• Titration of de-esterified pectin after acid
precipitation
• FTIR spectra analyzed were obtained using the
corrected peak areas at 1639.4
• High performance size-exclusion chromatography
(HPSEC)
 Chlorophyll
• Mixture of pigments found in chloroplats of plant
cells
• First isolated and named by Joseph Bienaimé
Caventou and Pierre Joseph Pelletier in 1817
• The presence of magnesium in chlorophyll was
discovered in 1906, and was that element's first
detection in living tissue
• Give green color to leaves
• Responsible for photosynthesis
• Two types a & b
Chlorophyll a Chlorophyll b
 Measurement of chlorophyll content

• Measurement of the absorption of light is complicated by the solvent used

to extract the chlorophyll from plant material, which affects the values
obtained
• In diethyl ether, chlorophyll a has approximate absorbance maxima of
430 nm and 662 nm, while chlorophyll b has approximate maxima of
453 nm and 642 nm
• The absorption peaks of chlorophyll a are at 465 nm and 665 nm.
Chlorophyll a fluoresces at 673 nm (maximum) and 726 nm. The peak
molar absorption coefficient of chlorophyll a exceeds 105 M−1 cm−1, which
is among the highest for small-molecule organic compounds
• In 90% acetone-water, the peak absorption wavelengths of chlorophyll a
are 430 nm and 664 nm; peaks for chlorophyll b are 460 nm and 647 nm;
peaks for chlorophyll c1 are 442 nm and 630 nm; peaks for chlorophyll c2
are 444 nm and 630 nm; peaks for chlorophyll d are 401 nm, 455 nm and
696 nm
• By measuring the absorption of light in the red and far red regions, it is
possible to estimate the concentration of chlorophyll within a leaf
 Measurement of chlorophyll content

• Ratio fluorescence emission can be used to measure

chlorophyll content. By exciting chlorophyll a fluorescence
at a lower wavelength, the ratio of chlorophyll fluorescence
emission at 705±10 nm and 735±10 nm can provide a linear
relationship of chlorophyll content when compared with
chemical testing
• The ratio F735/F700 provided a correlation value of r2 0.96
compared with chemical testing in the range from
41 mg m−2 up to 675 mg m−2
• Gitelson also developed a formula for direct readout of
chlorophyll content in mg m−2. The formula provided a
reliable method of measuring chlorophyll content from
41 mg m−2 up to 675 mg m−2 with a correlation r2 value of
0.95
 Determination of chlorophyll content
• Prepare extracting solution as follows
– Dissolve 0.35 g NH4OH in 100 mL distilled water to form 0.1 N
NH4OH solution
– Mix 0.1 N NH4OH solution with the reagent-grade acetone in a
ratio of 1:9 (v:v) (NH4OH:acetone)
• Grind the sample with extracting solution
• Repeat extraction process
• Centrifuge
• Take absorbance at 645nm and 663 nm on
spectrophotometer
• Absorbance should be between 0.2 to 0.8
• If dilution is required, dilute using 80% aqueous acetone

Perform all steps under diminished light and cold conditions

 Calculations
• Chlorophyll a (mg/mL)= 12.7 A663-2.69 A645
• Chlorophyll b (mg/mL)= 22.9 A645-4.68 A663
– where:A645=absorbance at a wavelength of 645
nm;
– A663=absorbance at a wavelength of 663 nm
• Total Chlorophyll (mg/mL) = Chlorophyll a +
Chlorophyll b

This will give concentration of chlorophyll in solution whose absorbance is

measured. Using sample weight, total volume of extract and dilution factor, calculate
amount of chlorophyll in sample
 β-Carotene
• β-Carotene is an organic, strongly colored red-
orange pigment abundant in fungi,plants, and
fruits.
• It is a member of the carotenes, which are
terpenoids (isoprenoids)
• Among the carotenes, β-carotene is
distinguished by having beta-rings at both
ends of the molecule
 β-Carotene
• β-Carotene is the most common form of
carotene in plants.
• When used as a food coloring, it has the E
number E160a
 β-Carotene
• Plant carotenoids are the primary dietary source of provitamin A
worldwide, with β-carotene as the best-known provitamin A
carotenoid
• Carotenoid absorption is restricted to the duodenum of the small
intestine
• One molecule of β-carotene can be cleaved by the intestinal
enzyme β,β-carotene 15,15'-monooxygenase into two molecules of
vitamin A
• Absorption efficiency is estimated to be between 9 and 22%
• The absorption and conversion of carotenoids may depend on the
form of β-carotene (e.g., cooked vs. raw vegetables, or in a
supplement), the intake of fats and oils at the same time, and the
current stores of vitamin A and β-carotene in the body
 β-Carotene
• Retinol activity equivalents (RAEs)

– 1 µg RE = 1 µg retinol
– 1 µg RAE = 2 µg all-trans-β-carotene from supplements
– 1 µg RAE = 12 µg of all-trans-β-carotene from food
– 1 µg RAE = 24 µg α-carotene or β-cryptoxanthin from food
• International Units

– 1 µg RAE = 3.33 IU retinol

– 1 IU retinol = 0.3 μg RAE
– 1 IU β-carotene from supplements = 0.3 μg RAE
– 1 IU β-carotene from food = 0.05 μg RAE
– 1 IU α-carotene or β-cryptoxanthin from food = 0.025 μg RAE1
 Determination of β-Carotene
• Pigment extraction for β-carotene analysis This was carried out according to the method of the
Association of Official Analytical Chemists (AOAC, 1980). In to a conical flask containing
50ml of 95% ethanol,10g of the macerated sample was placed and maintained at a temperature
of 70-80oC in a water bath for 20minutes with periodic shaking. The supernatant was
decanted, allowed to cool and its volume was measured by means of a measuring cylinder
and recorded as initial volume. The ethanol concentration of the mixture was brought to
85% by adding 15ml of distilled water and it was further cooled in a container of ice water for
about 5minutes. The mixture was transferred in to a separating funnel and 25ml of petroleum
ether (pet-ether) was added and the cooled ethanol was poured over it. The funnel was swirled
gently to obtain a homogenous mixture and it was later allowed to stand until two separate
layers were obtained. The bottom layer was run off into a beaker while the top layer was
collected in to a 250ml conical flask. The bottom layer was transferred in to the funnel and re-
extracted with 10ml pet-ether for 5-6 times until the extract became fairly yellow. The entire
pet-ether was collected in to 250ml conical flask and transferred in to separating funnel for re-
extraction with 50ml of 80% ethanol. The final extract was measured and poured in to sample
bottles for further analysis. Measurement of absorbance The absorbance of the extracts was
measured using a spectrophotometer (model 22UV/VIS) at a wavelength of 436nm. A cuvette
containing pet-ether (blank) was used to calibrate the spectrophotometer to zero point. Samples of
each extract were placed in cuvettes and readings were taken when the figure in the
display window became steady. The operation was repeated 5-6 times for each sample and
average readings were recorded. The concentration of β-carotene was calculated using Bear-
Lamberts Law, which states that the absorbance (A) is proportional to the
concentration(C) of the pigment, as represented by the equation: A ∞L (if concentration(C) is
constant). A=ECL; C=A/EL
 Anthocyanins
• Anthocyanins are water-soluble vacuolar pigments that, depending on their pH,
may appear red, purple, blue or black
• Food plants rich in anthocyanins include the blueberry, raspberry, black rice, and
black soybean, among many others that are red, blue, purple, or black
• Some of the colors of autumn leaves are derived from anthocyanins
• Anthocyanins belong to a parent class of molecules called flavonoids
• They occur in all tissues of higher plants, including leaves, stems, roots, flowers,
and fruits
• Anthocyanins are derived from anthocyanidins by adding sugars
• They are odorless and moderately astringent.
• Although approved to color foods and beverages in the European Union,
anthocyanins are not approved for use as a food additive because they have not
been verified as safe when used as food or supplement ingredients
• There is no conclusive evidence that anthocyanins have any effect on human
biology or diseases
 Anthocyanins
• Anthocyanins are water-soluble vacuolar pigments that, depending on their pH,
may appear red, purple, blue or black
• Food plants rich in anthocyanins include the blueberry, raspberry, black rice, and
black soybean, among many others that are red, blue, purple, or black
• Some of the colors of autumn leaves are derived from anthocyanins
• Anthocyanins belong to a parent class of molecules called flavonoids
• They occur in all tissues of higher plants, including leaves, stems, roots, flowers,
and fruits
• Anthocyanins are derived from anthocyanidins by adding sugars
• They are odorless and moderately astringent.
• Although approved to color foods and beverages in the European Union,
anthocyanins are not approved for use as a food additive because they have not
been verified as safe when used as food or supplement ingredients
• Anthocyanins are approved for use as food colorants in the European Union,
Australia, and New Zealand, having colorant code E163
• There is no conclusive evidence that anthocyanins have any effect on human
biology or diseases
 Structure of Anthocyanins
• The anthocyanins, anthocyanidins with sugar
group(s), are mostly 3-glucosides of the
anthocyanidins
• The anthocyanins are subdivided into the
sugar-free anthocyanidin aglycones and the
anthocyanin glycosides
• As of 2003, more than 400 anthocyanins had
been reported
 Structure of Anthocyanidin

Selected anthocyanidins and their substitutions

Anthocyanid
R3′ R4′ R5′ R3 R5 R6 R7
in
Aurantinidin −H −OH −H −OH −OH −OH −OH
Cyanidin −OH −OH −H −OH −OH −H −OH
Delphinidin −OH −OH −OH −OH −OH −H −OH
Europinidin −OCH3 −OH −OH −OH −OCH3 −H −OH
Pelargonidin −H −OH −H −OH −OH −H −OH
Malvidin −OCH3 −OH −OCH3 −OH −OH −H −OH
Peonidin −OCH3 −OH −H −OH −OH −H −OH
Petunidin −OH −OH −OCH3 −OH −OH −H −OH
Rosinidin −OCH3 −OH −H −OH −OH −H −OCH3
• Structure, pH, temperature, light, oxygen, metal ions, intramolecular association,
and intermolecular association with other compounds (copigments, sugars,
proteins, degradation products, etc.) generally are known to affect the color and
stability of anthocyanins
• Anthocyanins may be used as pH indicators because their color changes with pH;
they are red or pink in acidic solutions (pH < 7), purple in neutral solutions (pH ≈
7), greenish-yellow in alkaline solutions (pH > 7), and colorless in very alkaline
solutions, where the pigment is completely reduced

Red cabbage extract at low pH (left) to high pH (right)

 Determination of Total Anthocyanin Content

• The anthocyanin content was first qualitatively

identified using ammonia HCl test followinga
previous procedure
• 2 mL of the extract was added with 2 mL of 2N
HCl and ammonia
• The color change from pink-red to blue-violet
indicates the presence of anthocyanin
 Determination of Total Anthocyanin Content

• The total anthocyanin content was determined by the pH differential method

which bases on the structural changes in chemical forms of anthocyanin and
absorbance measurements at pH 1.0 and4.5
• Crude extracts were diluted separately with 0.025 M hydrochloric acid–potassium
chloride buffer(pH=1) and 0.4M sodium acetate buffer (pH=4.5)
• Each sample was diluted with the buffers to give an absorbance reading between
0.2 and 1.4
• The absorbance of the mixture was measured at λvis-max and 700 nm using a UV-
Vis spectrophotometer
• The total anthocyanin content was expressed as cyanidin-3-glucoside equivalents
as in the following equation

• Where A is the absorbance, MW is the molecular weight of cyanidin-3-glucosode

(449.2 g/mol), DF is the dilution factor, V is the solvent volume (mL), a is the molar
absorptivity (26,900 L.mol−1.cm−1), and l is the cell path length (1 cm).
 Lycopene
• Lycopene (from the neo-Latin Lycopersicum, the
tomato species) is a bright red carotenoid
hydrocarbon found in tomatoes and other red
fruits and vegetables, such as red carrots,
watermelons, grapefruits, and papayas
• Not present in strawberries or cherries
• Lycopene is chemically a carotene, but it has no
vitamin A activity
• Foods that are not red may also contain lycopene,
such as asparagus, guava and parsley
 Lycopene
• Like all carotenoids, lycopene is a tetraterpene
• It is insoluble in water
• Eleven conjugated double bonds give
lycopene its deep red color

• Owing to the strong color, lycopene is useful

as a food coloring (registered as E160d)
 Estimation of Lycopene
• If sample contains lipids, add 30% KOH in
methanol and store overnight for saponification
• Filter and wash the cake with water to remove
alkali
• Extract the lycopene using warm acetone
• Evaporate acetone and dissolve residue in
petroleum ether
• Make appropriate dilution of extract and note
down absorbance at 503nm
• Construct calibration curve of lycopene and
calculate amount of lycopene in sample OR
• Calculate lycopene content using following
formula
• mg lycopene per 100 gm =
(3.1206*Aborbance*Volume made
up*Dilution*100) / (1*wt of sample in
gm*1000)
 Non-enzymatic browning
• The browning index (absorbance at 420 nm)
of the clear extracts was determined using a
spectrophotometer