234
Preparation of Vegetable Rennet
[Vol. XVIII, Part IV
At a later stage it was found that CaCl 2 enhances the activity of the enzyme and so milk was
fortified with Ca by adding 2 ml. of 12.33 per cent CaCl 2 solution to 98 ml. of milk.
(c) Concentration of the enzyme
(i) Concentration under vacuum. Attempts were made to concentrate the enzyme by distilling
off water under vacuum at 40-45°C. A heavy froth was produced during distillation. In the end a
thick pasty mass was obtained resembling molasses in appearance. Attempts were made to dry
this material over sulphuric acid under vacuum without success. Eight hundred and fifty gramms
of the aqueous extract gave nearly 275 gm. of the concentrate after vacuum distillation and drying.
This method was abandoned as it did not promise to give useful results.
(ii) Precipitation with acids and alkalies. When the aqueous extract was treated with dilute
H 2 SO 4 , HCl, NH 4 OH and NaOH, no precipitation occurred.
(iii) Precipitation with salts. Sodium chloride, ammonium sulphate and magnesium sulphate
were used for this purpose 1,000 ml. of aqueous extract required 350 gm. of NaCl for complete precipita-
tion of the precipitable matter. Some precipitate was obtained at 20.8 and 38.4 per cent ammonium
sulphate but the bulk of the precipitate was at 39.0 per cent concentration. Magnesium sulphate
gave precipitates at 22 per cent and 53 per cent concentrations. With each of the above precipitants,
when complete precipitation had occurred the clear supernatant liquid was decanted off and the
solution containing the precipitate was dialyzed after adding some toluence. When the dialysate
was free from the added salt, the bag containing the material was removed and the contents trans-
ferred to a measuring flask of 250 ml. capacity and made up to the mark. During the process of
dialyzing some precipitate was formed which did not go in solution and which was inactive. For the
determination of activity, the whole suspension was used in each case. The results showed that in
no case was a highly active material obtained. As this method of precipitation was likely to prove
tedious under commercial conditions, it was not perused further.
(iv) Precipitation with organic solvents
(a) Precipitation with alcohol. Each 100 ml. of water extract required 250 ml. of absolute alcohol
for precipitation of the active enzyme. First signs of a precipitate occurred after 160 ml. of alcohol
were added. By this method about 9 gm. of dry powder was obtained from 100 ml. of aqueous
extract. Larger yields could be obtained by adding more alcohol to the supernatant liquid but the
precipitate formed was quite inactive. The enzyme powder was not completely soluble in water and
on keeping, insoluble matter settled down. The alcohol precipitate had good milk clotting properties
as shown in the following Table.
4 per cent solution of
Sample I . . . . .
.
.
.
.
.
.
.
.
.
.
Sample II . . . . . . . . . . . . . . .
Animal Rennot . . . . . . . . . .
Clotting time of milk
adjusted to 0.2 per cent
lactic acid
61
63
65
The results show that it is possible to get an active enzyme by this treatment. Similar results
have been reported by Narain and Singh [1943].
(b) Precipitation with acetone. By trials it was found that the optimum amount of acetone to
be added was two volumes to one volume of aqueous extract. Aqueous solution of Withania coagulans
of different strength were made and precipitated with acetone. It was observed that the enzyme
precipitated from dilute solutions was more active. Extraction of Withania coagulans berries with a
5 per cent CaCl 2 solution and adding acetone to this extract gave a completely inactive product.