Dean Madden from the National Centre for Biotechnology Education (NCBE), University of Reading, UK, suggests an experiment to make lactose-free milk – useful both for cats and for the 75% of the world’s human population that are intolerant to this type of sugar.
This simple practical investigation introduces students to the principles of digestion and enzyme immobilisation. It can be used as the starting point for other, more advanced activities such as the regulation of lactase production in Escherichia coli (the lac operon), the evolution and social significance of lactose tolerance in humans, and the use of enzymes in food production.
Lactase (beta-galactosidase) catalyses the hydrolysis of lactose to glucose and galactose:
Lactose -> D-glucose + beta-D-galactose
Both of these sugars taste sweeter and are more readily digestible than lactose. Despite their traditional fondness for milk, cats are unable to digest large amounts of lactose. Milk can be treated with the enzyme to make a lactose-reduced milk suitable for cats or for humans who are lactose intolerantw1.
Although the production of a special ‘cat milk’ may seem trivial, an estimated 75?% of the world’s human population are lactose intolerant in adulthood – it is lactose tolerance that is unusual.
Commercially, milk is treated by injecting an enzyme into the carton as UHT milk is packaged, or by using an immobilised enzyme – an enzyme that has been trapped on an inert material so that it can be used repeatedly.
In this activity, students immobilise the lactase in calcium alginate beads held within a small column, over which the milk is passed.
Needed by each person or group:
Note: All solutions must be made using distilled or deionised water. Sodium alginate is not readily soluble, and requires both warm water and stirring to dissolve.
This activity takes about 40 minutes. The sodium alginate takes some time to dissolve, so the solution is best prepared before the lesson. The immobilised enzyme may be prepared in advance if desired: the beads should be refrigerated, although they will not keep for more than a few days.
The enzyme suggested for this work is safe to use, provided it is handled appropriately. Although Novozymes Lactozym® is a food-grade product, milk prepared using it should not be consumed. This is because the enzyme has not been handled aseptically, so it (and the product made using it) may have been contaminated.
Readers are advised to refer to any local safety guidelines and to carry out their own risk assessment for any practical work.
As enzymes are water-soluble, water should always be used for their removal if they are spilt.
If liquid preparations are allowed to dry up, there is a risk of dust formation. In susceptible people, the repeated inhalation of such dust may provoke asthma or a reaction similar to hay fever. Any spillage – on equipment, the floor or the bench – should be rinsed away immediately with water
If enzyme-containing aerosols are formed, there is a risk of inhalation of the enzyme. In susceptible people, the repeated inhalation of such aerosols may provoke asthma or hay fever. For this reason, enzyme preparations should never be sprayed.
If, by accident, you get liquid enzyme on your skin or in your eyes, the remedy is plenty of tap water. The same applies to clothing. In the event of a spill on clothes, rinse with water then wash as usual. This treatment will generally prove sufficient, but if symptoms develop in the respiratory passages, on the skin or in the eyes, consult a doctor immediately.
Some UHT milk will test positive for glucose, probably because the heat treatment hydrolyses some of the lactose. UHT milk should therefore be avoided.
The immobilised enzyme column may also be used to treat whey, producing a sweet whey syrup which is widely used in confectionery (it is usually described on labels as ‘hydrolysed whey syrup’ or just ‘whey syrup’).
Lactase is strongly inhibited by galactose (one of the products of its action on lactose). As a result, the flow rate of the substrate over the column is critical to the rate of the enzyme-catalysed reaction: too fast and there isn’t time for the reaction to occur; too slow and galactose will accumulate and then inhibit the reaction. Students can therefore investigate the effect of the flow rate on the conversion of lactose to glucose and galactose.
The enzyme preparations should be stored, undiluted, at 3-4 °C.