Science and Plants for Schools Student Project Starter

Science and Plants for Schools – Student Project Starter Sweet or sour?

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.

When enjoying eating ripe fruits, do you think about their pH (as a measure of sourness) or are you really most interested in the sweetness in terms of sugar content?

Fruits originally evolved as a means of dispersing the offspring of the plant (the seeds) well away from the parent plant. For plants that produce succulent fruit this requires the cooperation of animals (especially birds and mammals) which are tempted to eat the fruit. They then carry the seeds - on their feathers, fur or within their intestines - to new habitats to be colonised. It is the attractive taste that rewards the cooperative animal, as well as the energy rich meal provided by the flesh of the fruit. However, it is essential that the fruit avoids 'premature' consumption. This might lead to the immature seeds being destroyed, or they may be too immature to germinate once they have been dispersed. Avoiding this premature consumption, may be the reason why the attractive flavour of the fruits develops only when the fruit is ripe. In fact, unripe fruit may even carry distasteful chemicals as a deterrent.

Vegetables are mostly storage organs used by plants to provide a source of nutrition during the next growing season. Few are sweet in themselves, though many have large reserves of carbohydrate polymers, such as starch and inulin. During the mobilisation of these reserves, these polymers are broken down to produce mobilisable sugars such as sucrose and fructose. The process of cooking helps to remove some of the chemical deterrents that plants use to deter herbivores consuming these tissues.

Domesticated fruits and vegetables have all been selectively bred so that they contain a particularly attractive taste for humans. Skilful greengrocers and cooks know how to tempt their customers with subtle combinations of flavours, and food manufacturers use panels of tasters to determine which flavours are best. (See http://www.cf.ac.uk/biosi/staffinfo/jacob/ for a tutorial on smell.)

It is reasonable to suppose that the improved flavour of a ripe fruit, compared to an unripe one, is at least partly due to increased levels of sugars. It is also possible that these sugars will have been liberated from storage, in the form of starch granules or some other polysaccharide. Other changes in flavour may result from changes in the pH of the juice of the fruit, linked to changes in

An investigation from Science & Plants for Schools, www.saps.org.uk/students the concentrations of organic acids such as ascorbic acid (vitamin C), and commonly citrate and malate. A desirable sugar-acid balance makes an important contribution to a 'pleasant taste' in ripe fruits. In addition, volatile chemicals released from the fruit indicate its state of ripeness (See http://editweb101v.csa.com/crw/websites.html for more about this) and add to our enjoyment when eating fruits.

Practical protocols and other suggestions to explore

There are many possibilities for investigating these changes during the ripening process, including:

1. Starch grain assay

2. Ascorbic acid (Vitamin C) assay

Protocol 1 – Starch Grain Assay

The following procedure can be used to determine the mass of solid starch grains present in fruit or vegetable tissues. The procedure as described is for analysis of a potato, but it can be applied to any vegetable or fruit. The blender is not essential, and can be replaced with a mortar and pestle, or the latest small hand-blender (usually used for pureeing vegetables for soups) is very effective at disrupting tissues. The violence of the blending process is effective at liberating the maximum number of starch grains from the cells.

The assay

The method depends upon the insolubility of starch in saline and the resulting sediment gives a measure of starch grains present in the material.

 Peel and cut the potato into small pieces (less than 1 cm cubes).  Weigh the pieces, and put them in a blender, adding 2 cm3 1% NaCl for each gram of potato. Blend the mixture, to produce a loose slurry. Try blending for about 30 seconds, or more if needed.  Pour the slurry through two layers of muslin, stretched over the mouth of an appropriate sized beaker. Dispose of the solids left in the muslin.  Leave the filtrate in the beaker to stand for some minutes, to allow the grains of starch to form a solid sediment at the bottom of the beaker.  When the sediment has formed, carefully pour away the liquid above it, and re-suspend the sediment in fresh 1% NaCl. Once again, allow time for the starch grains to settle to the bottom.  Repeat the re-suspension / re-washing procedure twice more.  Finally rinse the starch layer in a similar fashion, using 0.01 M NaOH.  Then assess the 'amount' of sediment (containing the starch grains), either by its volume or by its mass. (Volume could be useful in terms of simplicity.)

An investigation from Science & Plants for Schools, www.saps.org.uk/students To measure volume of sediment, pour a known volume of the starch suspension into a tapered, calibrated centrifuge tube. Allow it to stand vertically for a few minutes. The volume of starch grains can then be directly read off the side of the tube. If you do not have a pre-calibrated tube, then make one by adding small aliquots of water to a suitable tube, and using a marker pen to indicate the volumes on the side of the tube.

As an alternative, to assess the sediment in terms of mass, weigh to find the wet mass, or dry to a constant mass and then weigh to find the dry mass of starch grains obtained.

Remember you can always check the quality of your extract by adding iodine solution, which should colour the grains blue-black, and/or examining a sample under the microscope. You might be able to distinguish the unique features of starch grains belonging to particular species of vegetables. Oh, and don't forget to add a drop of amylase solution to such a sample, and see the effects of amylase on the structure of the grains.

Protocol 2 - Measuring changes in ascorbic acid (vitamin C) concentration in ripening fruit and vegetables

We know a lot about ascorbic acid (vitamin C) in the human diet and are familiar with its role in controlling scurvy and as an antioxidant. But it may come as a surprise to know that only very recently have the functions of this substance within the plant been worked out in detail.

Ascorbic acid may be found in all the compartments in the plant cell, where it plays diverse roles. It is known to be involved in cell division and cell wall synthesis and also acts as an inhibitor of dangerous compounds such as hydrogen peroxide and the dangerous radicals of oxygen generated as a by- product of respiratory and photosynthetic machinery of the cell.

We have recognised the presence of ascorbic acid in ripening fruits, particularly in citrus fruits, but are less certain about its role. Other organic acids, notably citrate and malate, are often present in larger quantities and contribute to the sugar-acid balance. You probably know from experience how this changes (in favour of the sugars) as fruits ripen, but you can use this method to check for yourself whether the ascorbic acid really does disappear during ripening and in storage in your own selection of fruits and vegetables.

The assay

The principle of this method is a titration with dichlorophenolindophenol (or phenol-indo-2:6-dichlorophenol, also known as DCPIP). Ascorbic acid reacts with DCPIP, changing the colour from blue to colourless. They react in a 1:1 fashion, so if a known quantity of DCPIP solution reacts with the plant tissue extract, the quantity of DCPIP used gives a direct measure of the quantity of ascorbic acid present.

An investigation from Science & Plants for Schools, www.saps.org.uk/students  Grind up a measured mass of fruit or vegetable in a known volume of distilled water Try a ratio of perhaps 1g fruit to 5 cm3 water, though the precise ratio of fruit to water may need to be modified if, for instance, the plant material is particularly glutinous. The grinding can be done in a mortar and pestle, or using a blender for larger samples.

 Filter or centrifuge the plant macerate to provide a clear solution (of whatever colour!), then use this solution for the titration reaction.

 Transfer say 1 cm3 of the plant extract solution into a test tube. Place the test tube beneath a burette or vertically mounted 1 cm3 pipette (with pipette-pump attached) containing 0.1% DCPIP solution and add DCPIP dropwise to the extract in the test tube. [When the first drop of DCPIP is added to the extract, the initial blue colour should quickly disappear - as a consequence of the reaction between any ascorbic acid present and the DCPIP. If this does not happen then the ascorbic acid concentration is very low (or even zero!) and a larger volume of extract may be needed in the test tube, if a measurement is needed.]

 Add DCPIP until the blue colour does not disappear, and note the volume of DCPIP used. This volume is equivalent to the amount of ascorbic acid present in the extract, so the concentration of ascorbic acid in the original plant material can be calculated and expressed as units per gram of fresh mass

Here is an example of how you can calculate the concentration of ascorbic acid in your original sample of fruit. Suppose you started with 10 g of tissue and made an extract in 50 cm3 of water and then in the titration a 1 cm3 sample of your extract required 1.5 cm3 of 0.1% DCPIP solution. As each cm3 of 0.1% DCPIP (MWt 290.08) solution is equivalent to 6.071x10-4 g ascorbic acid (MWt 176.12). Therefore 1 cm3 of extract must have contained 9.107x10- 4 g of ascorbic acid. This means the original 50 cm3 extract contained 0.0455 g and this came from 10 g of fruit. So you can work out that each gram of fruit contained 0.00455 g ascorbic acid.

Note that some plant materials may be very acid, in which case the colour of the DCPIP becomes pink / magenta. In this case, look for the appearance of a permanent pink colour during the titration, rather than a blue colour.

(You can find further information in: Smirnoff N. Ascorbic Acid: metabolism and function of a multifaceted molecule. Current Opinion in Plant Biology 2000, 3:229-235.)

An investigation from Science & Plants for Schools, www.saps.org.uk/students