Science and Plants for Schools – Student Project Starter
Colour changes during ripening . . . of fruits and
vegetables
This is a project starter, suitable for Advanced Higher biology investigations or
A-level extended projects. Don’t forget to credit this resource in your
bibliography by including the title, the website, the web address and the date
you accessed it.
How do YOU decide whether a fruit is ripe? . . . squeeze it? . . . smell it? . . .
or does colour give you the most useful guide?
Domesticated plants have been the subject of intensive selective breeding
programmes and, amongst the many features selected for, colour of the fruit
certainly must have been one of the features. The skilful greengrocer
arranges a display of fruits and vegetables to entice customers to purchase
them, and, for many artists and cooks, the aesthetic appeal of fruit and
vegetables depends on their appearance as much as their taste and texture,
especially when they are arranged in a still-life or stir-fry!
We can see that an important biological role of these fruit colours is to appeal
to animals, and these animals then have a role in the distribution of the seeds
within the fruit. It is important that the fruit is eaten only when the seeds are
mature, and presumably ready to face the jaws and digestive systems of the
dispersal animals. It seems logical that most fruits have an unappetising
flavour and insignificant colour until the time is ripe (ouch!) for them to be
eaten. In a more modern context, the colour of a fruit is often used as a
means of determining their shelf life.
The green colour of the unripe fruit is due largely to the presence of
chlorophylls, and the development of different colours during ripening is due
to the disappearance of these pigments and the synthesis of carotenoids - or
just revealing carotenoids that were already there, masked by the chlorophyll.
Anthocyanins also make a contribution to colours in some ripe fruits and
vegetables. It is now known that several of these pigments are valuable items
in the human diet.
You can set up investigations to follow the changes in the concentrations of
these pigments during ripening by trying to answer some of the questions
below.
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
How do the pigment levels change during the ripening process?
How do the conditions of storage affect the colour changes?
An investigation from Science & Plants for Schools, www.saps.org.uk/students

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How do pigment levels change if a fruit / vegetable is allowed to become
over-ripe?
How do pigment levels change if the fruit / vegetable becomes infected
by a fungal or bacterial pathogen?
How do the pigment levels change during food preparation and
cooking?
How many anthocyanin pigments contribute to the colour of a particular
fruit? In what sequence do they appear?
Practical protocols and other suggestions to explore
1. Measurement of the relative amount of red versus green (anthocyanins v
chlorophylls a & b) pigments in plant tissues using a colorimeter
2. Measurement of relative amount of chlorophyll-a, b, and carotene in plant
tissues using a spectrophotometer
3. Thin layer chromatography of plant pigments
Measurement of the relative amount of red versus green pigments
(anthocyanins vs chlorophylls a & b) in plant tissues
With a colorimeter, it is possible to make comparative measurements of green
and red pigments in plant tissues, using some of the broad band colour filters
provided with the colorimeter.
A colorimeter filter passes a beam of light consisting of a whole range of
wavelengths through a solution. Different coloured filters allow different
sections of the visual spectrum (400 nm to 700 nm) to pass through, but the
range of wavelengths might include as much as 20% of the spectrum. Green
pigments get their colour from the fact that they absorb visual spectrum
wavelengths other than green, and, as it happens, chlorophylls absorb red
wavelengths particularly well. Red pigments get their colour from the fact that
they absorb visual spectrum wavelengths other than red, and, as it happens,
anthocyanins absorb blue wavelengths particularly well. Hence, by measuring
the absorbance of a plant pigment extract, using a red and a blue filter, it
should be possible to estimate the relative amounts of the "red-absorbing"
and "blue-absorbing" pigments present.
Chlorophylls

Use a small volume of absolute ethanol to extract chlorophylls from
plant tissues, ground in a mortar with silver sand. Then centrifuge this
solution to remove any solid debris.

Keep the resulting clear supernatant and, using the blue filter and a
green filter in the colorimeter, make two readings of the optical density
of this solution. For each reading, first set the instrument to read zero
An investigation from Science & Plants for Schools, www.saps.org.uk/students
absorbance with a tube containing ethanol alone (the blank). Then
replace this tube with the tube carrying the coloured extract.
If the absorbance reading is off the scale (greater than 1.0), dilute the extract
with ethanol, by a known factor, until a reading can be made. Then use the
dilution factor to calculate the original absorbance value.
The figure for chlorophyll content is calculated by subtracting the value for
absorbance with the green filter from that with the blue filter, as follows:
absorbance with blue filter - absorbance with green filter
Anthocyanins

Use a small volume of acidified methanol (1 part concentrated HCl : 99
parts methanol) to extract anthocyanins from plant tissues, ground in a
mortar with silver sand. Then centrifuge this solution to remove any solid
debris.

Keep the resulting clear supernatant and, using the blue filter, make two
readings of the optical density of this solution. For each reading, first set
the instrument to read zero absorbance with a tube containing acidified
methanol alone (the blank). Then replace this tube with the tube carrying
the coloured extract.

Do the first reading with the original supernatant, which has a pH of
much less than 3. At this pH, anthocyanins are coloured ions.

Then add 1 M NaOH dropwise, until the pH of the extract is greater than
5 (which causes the anthocyanins to become colourless), and measure
the absorbance again.

Note the actual volume of NaOH added, and calculate its dilution effect.
Once this is done, any remaining difference between the absorbance
values at the two pH values is the result of anthocyanin concentration.
Measurement of relative amount of chlorophyll a, chlorophyll b and carotene
in plant tissues
If a spectrophotometer is available, measurements can be taken, at four key
wavelengths, of the absorbance of light by a plant pigment extract. It is then
possible to calculate the concentration of each pigment in mM.

Grind up equal fresh masses of the fruit skin, in measured volumes of
absolute ethanol (say 10 cm3).

Place the ground material in centrifuge tubes (equal volumes in each
tube) and spin at high speed for 5 minutes to remove debris. Decant the
supernatants to clean small capped specimen tubes.
An investigation from Science & Plants for Schools, www.saps.org.uk/students

Take absorbance measurements of the whole ethanolic extract, at the
following crucial wavelengths, using absolute ethanol as the blank for
zeroing the machine.

663 nm, 645 nm, 480 nm

Calculate concentrations of pigments as follows:
Chlorophyll a concentration in mM = 12.7 x A663 - 2.69 x A645
Chlorophyll b concentration in mM = 22.9 x A645 - 4.68 x A663
(where A663 and A645 are the values for absorbance at wavelengths 663
nm and 645 nm respectively)
Carotenoid concentration in mM = (A480 + (0.114 x A663) - (0.638 x
A645))÷112.5
 Once the value in mM is known, this information can then be used to
estimate the total quantity of extracted pigment, by taking account of the
mass of plant material used for extraction and the initial volume of
extract that was made.
It is worth noting that the ratio of carotenoids to chlorophylls can be affected
by environmental stresses experienced by the plant tissues. (Think, for
example, about the changes that happen to colour of the grass on a lawn, if a
paving stone is left in one place on it, say for a week.)
The basis for this protocol is taken from:pp148-152 in Methods in Comparative Plant Ecology - A Laboratory Manual.
edited by GAF Hendry & JP Grime. Chapman & Hall 1993
An investigation from Science & Plants for Schools, www.saps.org.uk/students