SCIENCE 



NEW YORK, NOVEMBER 4, 1892. 



THE ISOLATION OF RENNET FROM BACTERIA CULTURES. 



BY H. W. CONN, WBSLETAN UNIVERSITY. 



Ever since the beginning of the study of micro-organisms it 

 has been a debated question whether fermentations are to be re- 

 garded as biological or purely chemical phenomena. Beginning 

 with the work of Schwann and others in the early part of the 

 century, careful experimentation aided by microscopic study 

 seemed to point to a biological explanation of nearly all forms of 

 fermentation. The work of the third and fourth decades of the 

 century proved beyond question that most fermentations were 

 always intimately associated with the growth of micro-organisms, 

 and the Inference was a natural one that the micro-organisms 

 themselves were the cause of the fermentations. Shortly after 

 the valuable work of Schwann, however, appeared the brilliant 

 investigations and discussions of Liebig upon fermentations 

 in general. By Liebig all fermentations were regarded as purely 

 chemical phenomena and the presence of micro-organisms was 

 regarded only as a concomitant incident. According to him all 

 albuminous matter was thought to tend spontaneously toward 

 decomposition, and such decomposition was the basis of fermen- 

 tation and decay. This, the chemical theory, was for twenty 

 years the favorite theory. The biological and chemical theories 

 were very rigidly opposed to each other and supposed to be con- 

 tradictory. With the work of Pasteur and the great development 

 of the study of micro-organisms thereby inaugurated, the biologi- 

 cal theories of fermentations again came to the front, rapidly 

 gained the ascendency, and soon displaced almost entirely the 

 chemical theory as advanced by Liebig. For the next twenty 

 years it was regarded as an almost settled fact that most natural 

 fermentations were biological phenomena, and the theory of 

 Liebig was at last practically abandoned. 



Within still more recent times there has been a partial swing- 

 ing back of the pendulum toward a chemical explanation of many 

 forms of fermentation at least. This has not, however, been in 

 the direction of Liehig's theory, but rather toward a theory of 

 the action which unites together a chemical and biological ex- 

 planation. It has been recognized for more than half a century 

 that there are forms of ferments, such as pepsin, trypsin, etc., 

 which do act in a purely chemical manner. These ferments, it 

 is true, are produced originally by living organisms, but when 

 once produced they are not living themselves, at least in any 

 proper sense, and their action is not dependent upon growth or 

 multiplication, for they are not organisms. So far as can be de- 

 termined their action is purely chemical. On the other hand, a 

 large number of fermentations, such as the alcoholic fermenta- 

 tions, the souring of milk, etc., have been traced with certainty, 

 not only to living organisms in the form of bacteria and yeasts, 

 but to the actual growth and multiplication of these organisms. 

 These fermentations occur only when micro-organisms are present 

 and only when these micro-organisms grow and multiply. The 

 amount of growth of the organism is a measure of the amount of 

 fermentation. Undoubtedly these biological fermentations are 

 of a different nature from the other class. We are, however, 

 learning now to look upon some of the biological fermentations as 

 chemical in their immediate nature. 



In the first place, the bacteriologist has been learning that germ 

 diseases, which are caused primarily by the growth of micro- 

 organisms in the body, are caused immediately by certain pois- 

 onous bodies which these organisms produce. He has called 



these bodies ptomaines, and at present biologists are very rapidly 

 becoming convinced that it is the direct action of these poisonous 

 materialswhichproducesthesymptomsand disturbances associated 

 with most germ diseases. It is not the simple growth of bacteria 

 which produces disease, but the poisonous products of their 

 growth; and thus a chemical explanation is added to the biologi- 

 cal. 



Not only in germ diseases but in other forms of fermentations, 

 not associated with disease, bacteriologists are learning of the pro- 

 duction of chemical ferments by the micro-organisms. Many 

 organisms have been found to produce dyastase, sucrase, ^lucase, 

 etc. Within the last year or two it has been demonstrated that 

 many bacteria produce a chemical ferment very similar in its 

 character to trypsin. The general class of bacteria which 

 liquefy gelatin have long been known to have an action quite 

 similar to that of pancreatic juice. Indeed, it is this peptonizing 

 action which is the cause of the liquefaction of the gelatin. Re- 

 cently Brunton and Macfadyen and especially Fremi have suc- 

 ceeded in actually isolating from bacteria cultures a chemical 

 ferment which has this power even when acting in sterilized 

 media. 



Some recent work in the bacteriological laboratory at Wesleyan 

 University has isolated another chemical ferment from bacteria 

 cultures. A large class of bacteria have the following actions on 

 milk: They first curdle the milk rendering it slightly alkaline; 

 subsequently the curd is slowly dissolved into a more or less 

 watery liquid. Chemical study shows that this last action is a 

 simple digestion and peptonization of the curd and that it is due 

 to the trypsin-like ferment above mentioned. The curdling, which 

 precedes the digestion, however, must be due to a different action, 

 inasmuch as trypsin produces no curdling of the milk. It has 

 been suspected for some time that this curdling is really due to a 

 " rennet-like " ferment which is produced by the bacteria. It 

 has been my good fortune recently to demonstrate the truth of 

 this supposition. My method of work has been as foUows : — 



The bacteria in question are cultivated in milk for several days, 

 in some cases for two weeks. By this time the curd is precipitated 

 and at least partially dissolved, and the result is a somewhat 

 thick liquid containing, of course, immense numbers of bacteria. 

 This liquid is filtered through a porcelain filter to remove organ- 

 isms, and a clear, usually amber-colored, filtrate is thus obtained. 

 The filtrate, of course, contains in solution all of the soluble 

 chemical ferments which may have been formed by the bacteria. 

 This filtrate is now acidified with HjSO^ and then common salt 

 is added to a state of super-saturation. When this condition is 

 reached there appears on the surface of the liquid a considerable 

 quantity of snow-white scum. This scum is removed from the 

 liquid, purified if necessary by reprecipitation, and then dried. 

 It produces a snow-white powder, which upon experiment is 

 found to be active in its curdling action upon milk and to have 

 all of the essential characters of rennet. The ferment which is 

 thus obtained is not chemically pure, containing, besides the 

 rennet ferment, a varying amount of the tryptic ferment formed 

 at the same time. But the rennet ferment is most abundant and 

 is very active. This ferment can be kept indefinitely, is killed 

 by heat, acts best at a temperature of 30°-3o° C, and curdles 

 sterilized milk under proper conditions in half an hour. Experi- 

 ment shows that no organisms are present in the curdled milk, 

 and there is thus no doubt left that we are dealing with a chemi- 

 cal ferment similar to rennet, and which is produced by the 

 growth of these micro-organisms in milk. The ferment does not 

 appear to be exactly identical with rennet, some of its chemical 

 tests being different. This maybe due to the impurities which, 

 are present or to an actual difference in the ferment. 



