A "Rennet" Ferment Contained in the Seeds of Withania Coagulans.
(Communicated by Professor M. Foster, Sec. R.S.—From the 'Proceedings of the Royal Society.')
BY SHERIDAN LEA, M. A., Trinity College, Cambridge.
The Report of the Royal Gardens at Kew for 1881 contains abstracts of correspondence in which it was pointed out that, in order to introduce a cheese-making industry in India, some vegetable substitute must be found for the ordinary animal rennet, since cheese made with the latter is unsaleable among the natives. In response to the above "Surgeon-Major Aitchison brought to the notice of the authorities at Kew that the fruit of Puneeria coagulans (The genus Puneeria is now reduced by botanists to Withania.), a shrub common in Afghanistan and Northern India, possesses the properties of coagulating milk;" and experiments showed that an aqueous extract of the seed-capsules of the above plant does somewhat rapidly coagulate milk.
I was recently requested to make some experiments on the seeds of Withania to determine whether they contain a definite ferment with the properties of ordinary rennet, and the applicability of such a ferment to cheese-making purposes.
The material supplied to me consisted of an agglomerated dry mass of seed-capsules and fragments of the stalks of the plant. When crushed in a mortar the whole crumbled down into a coarse powder, in which the seeds were for the most part liberated from the capsules. I picked out the larger pieces of stalk, sifted out the finer particles, chiefly earth and fragments of the capsules, and then by a further sifting I separated the seeds from the other larger particles. The seeds appeared to be each enveloped in a coating of resinous material, presumably the dried juice of the capsules in which they had ripened.
Taking equal weights of the seeds, I extracted them for twenty-four hours with equal volumes of (i) water, (ii) 5 per cent. sodic chloride, (iii) 2 per cent. hydrochloric acid, (iv) 3 per cent. sodic carbonate. Equal volumes of each of the above were added in an acid, alkaline, and neutral condition to equal volumes of milk, and heated in a water-bath at 38 °C. The milk was rapidly coagulated by the salt and sodic carbonate extracts, much less rapidly by the other two; of the four, the salt extract was far the most rapid in its action. All subsequent coloring-matter is scarcely soluble in either ether or alcohol, so that no advantage is gained by a preliminary treatment with these before extraction with the salt solution. I have also endeavored to get rid of the color by treating the seeds as rapidly as possible with successive quantities of water before making the final extract. By using a centrifugal machine I was able to wash the seeds six or seven times with large volumes of water without their being exposed for any considerable time to the action of the water. Each portion of water was highly colored and the seeds were thus freed from adherent coloring-matter. But, apart from the fact that some, though not much, ferment is thus lost, no special advantage is obtained, since the seeds are themselves colored, and even after prolonged treatment with water the final extract is always of a dark brown color.
In order to obviate the disadvantages of this coloring matter, if disadvantage it is, I have found it best to prepare very concentrated active extracts of the purified seeds, so that it should only be necessary to add a very small quantity of the extract in order to coagulate the milk and obtain a colorless curd. This I have done by grinding the dry seeds very finely in a mill and extracting them for twenty-four hours with such a volume of 5 per cent. sodic chloride solution that the mass is still fluid after the absorption of water by the fragments of the seeds as they swell up. From this mass the fluid part may be readily separated by using a centrifugal machine (such as is used in sugar refining), and it can then be easily filtered through filter-paper; without the centrifugal machine the separation of the fluid from the residue of the seeds is tedious and imperfect, 40 grams of the seeds treated as above with 150 cubic centims. of 5 per cent. sodic chloride solution gave an extract of which 0.25 cubic centim. clotted 20 cubic centims. of milk in twenty five minutes, and 0.1 cubic centim. clotted a similar portion of milk in one hour. When added in these proportions the curd formed is quite white. The presence of the coloring-matter is however, perhaps on the whole unimportant, since even if a larger quantity of the ferment extract is added in order to obtain a very rapid coagulation the coloring matter is obtained chiefly in the whey, the curd being white. (It is extremely probable that some stage in the growth or ripening of the seeds of Withania might be found at which the development of coloring-matter is slight, while at the same time the ferment is present in considerable quantity.)
The question of preparing an extract which should be capable of being kept for a considerable time is perhaps of importance. Ordinary commercial rennet usually contains a large amount of sodic chloride and some alcohol. One specimen I analysed contained 19 per cent. of common salt, and 4 per cent. of alcohol. I have, therefore added to the 5 per cent. chloride extract mentioned above, enough salt to raise the percentage of this to 15 per cent., and also alcohol up to 4 per cent. The activity of the extract is not appreciably altered by this, and such a preparation corresponds very closely in activity with a commercial solution of animal rennet with which I compared it. The possibility of making extracts which may be expected to keep is thus indicated, but of course time alone will show whether the activity of the ferment is impaired to any important extent by such keeping.
I may add in conclusion that I have coagulated a considerable volume of milk with an extract such as I have described, and prepared a cheese from the curds. I have also given a portion of the extract to a professional cheese-maker who has used it as a substitute for animal rennet in the preparation of a cheese. The product thus obtained, and the statements of the person who has made the experiment for me, lead me to suppose that extracts of the seeds of Withania can be used as an adequate and successful substitute for animal rennet—Pharm. Jour. Trans., Feb. 2, 1884, p. 606.