Papain production
Practical Action
A papain sample is prepared by dissolving a known weight of papain in a known volume of acetic
acid solution. This is then added to a fixed amount of milk, which is prepared by dissolving a
known weight of milk powder in a known volume of water, warmed to 30°C in a water bath. The
contents are thoroughly mixed and then observed until the first signs of clotting – the formation
of lumps - are detected. The length of time from when the papain was added to the milk until
clotting began is recorded.
The experiment is repeated using different known amounts of papain solutions. The varying
amounts of papain sample used should give a range of clotting times, between 60 and 300
seconds for optimum results. The activity of the papain samples is then calculated by plotting a
graph, finding the time taken to clot milk at an infinite concentration of papain and then using
that value in a formula to calculate the activity. In order to introduce a measure of
standardisation, the amount of milk can be fixed at a known concentration. This is done by
reacting a known concentration of high-grade papain with the milk. The concentration of milk
powder solution can then be adjusted in order to obtain the desired clotting time under fixed
reaction conditions. The 'activity of pure papain' at this known amount of milk can then be
calculated. Testing the sample papain under the same reaction conditions and same (known)
amount of milk will then give an activity relative to the pure papain.
Method 2
The second method is based on the science of light absorption, or absorptiometry. This
technique measures the amount of radiation (or 'colour' of light) absorbed by a chemical
solution. It is known, for example, that a yellow-coloured solution absorbs blue light. (Blue is
the complementary colour to yellow). The greater the concentration of yellow in the solution, the
more blue light is absorbed. This is a useful technique because the resulting products of some
chemical reactions are coloured. The more intense the colour, the more concentrated the
resulting product. Therefore, by shining the
relevant complementary colour through the sample liquid, the amount of light absorbed can be
related to the concentration of product. Not all 'colours' (or radiations of light) are visible to the
human eye. The technique used when the 'colours' extend beyond the visible spectrum is known
as spectrophotometry, and the instrument used is called a spectrophotometer.
In this second method of determining the amount of activity of papain, a known amount of the
enzyme is mixed with a fixed amount of casein (the protein found in milk). The reaction is
allowed to proceed for 60 minutes at 40°C. After this time the reaction is stopped by adding a
strong acid. The resulting product of the reaction is tyrosine, which is known to absorb ultra-
violet light (invisible to the human eye). The solutions containing the tyrosine are prepared for
analysis using the spectrophotometer. The amount of ultra-violet light absorbed by the solution
can then be compared to the number of tyrosine units produced by the papain sample. Hence,
the greater this number, the greater the activity of the papain sample.
World trade in papain
The principal producers of crude papain are Democratic Republic of Congo (formerly Zaire),
Tanzania, Uganda and Sri Lanka. Most spray-dried papain comes from DRC.
Main importers are USA, Japan, United Kingdom, Belgium and France. Almost all the best
quality papain goes to the United States. Crude papain is used in Britain in the brewing industry
for chill-proofing beer and lager. However, an increasing trend in additive-free beers - initiated
by other European countries - is taking effect in Britain, whose market for papain is therefore
declining. Papain is, however, used in the tenderising of meat and in production of meat
tenderising powders.
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