PREPARATION OF VEGETABLE RENNET FROM WITHANIA
COAGULANS, DUNAL
By NOSHIR. N. DASTUR, K. N. SHAMA SASTRY AND D. VENKATAPPIAH, Indian Dairy Research
Institute, Bangalore
(Received for publication on 18 June 1948)
A N enzyme which can be used in the place of animal rennet in the preparation of cheese will prove
very useful taking into consideration the particular food habits of our population. The berries
of Withania coagulans, Dunal, are known to contain an active material useful for this purpose [Lea,
1883-84]. Recently Kothavalla and Khubchandani [1940] used a saline extract of Withania coagulans
for preparing cheeses. These authors used a temperature of 105°F. for coagulation. Some difficulty
in preparing hard cheeses of satisfactory quality was experienced as they developed open texture.
Narain and Singh [1943] have described the properties of alcohol precipitated enzyme and also used
this material for making Cheddar cheese.
Since Kothavalla and Khubchandani carried out their work at this Institute, further experiments
have been in progress with the object of isolating an enzyme which can be used in place of animal
rennet, under the accepted working conditions. The results of this work are described here.
EXPERIMENTAL
(a) Preparation of extract. Preliminary trials were carried out to find out where the enzyme
material is stored in the plant. Leaves and stems of the plant and seeds from the berries were
found to be devoid of all enzyme material. The pulp in the berries was the most active source of
milk clotting enzyme, and the aqueous extract of the outer husk of the berries had about a third of
the activity of the pulp. For experimental work, an extract of the whole berries was used as it was
very tedious to remove the seeds embedded in the pulp and the weight of husk in relation to the
pulp was very small. For extracting the enzyme, various materials in different concentrations
were tried, viz., water, NaCl (0.3 to 10 per cent), Na 2 CO 3 (5 per cent), glycerine, alcohol (5 per cent),
HCl (1-5 per cent), CaO (1 per cent) and CaCl 2 (0.3-2 per cent). Distilled water and 1 per cent NaCl
proved most suitable.
The extraction was carried out as follows : 1,000 gm. of berries were ground with about 750 ml.
of water to a thin paste. This was centrifuged when a considerable amount of insoluble material
settle down. The top liquid was opaque brown. The residue was washed twice with water and
separated in the manner described. The total quantity of water used was 850 ml. and the yield after
centrifuging was about 700 ml. The extract was quite stable at room temperature even without
any antiseptics. On storage, both in cold and at room temperature, some insoluble material deposited
from the extract. The precipitate formed was completely inactive and the removal of this deposit
by centrifuging had no effect on the activity of the supernatant liquid. Various preservatives were
added to see if these would prevent the formation of the precipitate. The substance tested included
borax (0.5 per cent), boric acid (1 per cent), mustard oil (1 per cent), glycerine (5-10 per cent). NaCl
(5-10 per cent), alcohol (5 per cent) toluence and combinations of some of these. None of these
preservatives was able to prevent the formation of the precipitate.
(b) Determination of activity. Activity was determined using milk substrate at a temperature
of 37°C. Fresh milk, and milk adjusted to an acidity of 0.2 per cent lactic acid by adding a suitable
quantity of 0.25 N pure lactic acid solution, were used. This acidity was adopted as this is the acidity
in milk normally used for cheese making. One ml, of rennet solution under test was added to 50 ml.
of milk and time of clotting when first flakes began to appear was noted in seconds. Each result
given in the subsequent Tables represents the mean of two observations.
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