RAMJI NARAIN AND ATAM SINGH 225
wild in the central and southern Punjab, but owing to the laborious process of
collecting its latex, it did not prove to be an economical source. The search
was, therefore, further extended to Withania somnifora and Withania coagulans.
Both these plants produce berries, different parts of which contain different
amounts of rennet of varying activity but Withania coagulans possesses berries
containing much larger amounts of the active principle. The fact that the
dried berries of Withania coagulans are not only rich in this enzyme but are
also easily obtainable in the market at all times of the year, weighed consider-
ably in the final selection of this material for the preparation of rennet.
This paper describes the different methods of extracting the enzyme,
its characteristic properties and its use in the preparation of cheddar cheese.
EXPERIMENTAL
A. Preparation of vegetable rennet from Withania coagulans
The berries of Withania Coagulans consist of husk, seeds and the resinous
material in which the seeds are embedded. The activity of the extract ob-
tained from the husks and the seeds is almost equal, while that of the extract
from the resinous material is four times high. Separation of the seed is,
however, tedious, hence resin along with the embedded seed, after being freed
from the surrounding husk, was used for the preparation of the enzyme extract.
Kothavalla and Khubchandani [1940] had prepared an extract with 5 per cent
sodium chloride and used it as such for the preparation of cheese. Our ex-
perience had shown, however, that the saline extract so prepared contained a
bitter substance and the resulting curd possessed a similar taste and an un-
pleasant flavour. Consequently, a number of methods was employed to obtain
a preparation free from this defect. As mentioned already by Russel [1937],
we were able to obtain an active preparation capable of coagulating 25000
parts of milk in 40 minutes. The preparation was free of all traces of bitter
taste or of undesirable flavour.
The following methods of extracting the enzyme were used :—
(a) Salting out from the saline extract.—The powdered berries freed from
husks were steeped overnight in four times their weight of 5 per cent sodium
chloride solution and occasionally stirred. The extract was filtered under
suction through a pad of filter paper pulp laid on a Buchner funnel. To the
filtrate, salt was added to saturation. The precipitated enzyme was separated
by centrifuging and dried under vacuum at a temperature below 60° C.
(b) Dialysis of the saline extract.—The saline extract obtained as above
was dialysed in a bag of parchment paper placed in a stream of cold water for
two to three days. During this period most of the salt was removed. To the
contents of the bag a small quantity of dilute acetic acid was added drop by
drop till the enzyme was precipitated at its iso-electric point (4.8 pH). It was
separated and dried as above.
(c) Precipitation with alcohol.—A suspension of the powdered berries was
made in dilute alcohol and kept overnight with occasional stirring, and
filtered the following morning. From this filtrate the enzyme was precipitated
by the addition of commercial alcohol. The precipitated enzyme was separated
by centrifuging and dried.