Fig. 11.—Section through a Kephir Grain.
Contrary to what one would expect in an organism existing in keffir grains, this streptococcus is as little able to withstand desiccation as the above-mentioned yeast. Streptococcus b forms smaller cells as well as smaller colonies than Streptococcus a, but produces more lactic acid and more gas, and retains its vitality after desiccation. The relation of these four organisms is, according to E. von Freudenreich, as follows: Sacch. keffir is unable to ferment directly milk or lactose, so that its growth must be preceded by that of Streptococcus b. Streptococcus a does not seem to play this part, but, unlike Streptococcus b, is able to coagulate milk on its own account. By the combined action of the yeast and the two streptococci, then, milk can be coagulated, milk sugar inverted, acid and gas produced by the streptococci, while gas and alcohol are formed by the activity of the yeast. The rôle of Bacillus caucasicus is unknown, but it would seem to play a part in the formation of the keffir grain itself. By means of mixed cultures of the above organisms Freudenreich was successful in obtaining a fermented product possessing in all respects the characteristic properties of normal keffir. On the other hand, experiments to induce the formation of keffir grains gave negative results, but in this respect the cultural characteristics of Lactobacillus keffir would seem to give promise of success in the synthesis of the keffir grain. Fig. 11 is a photo-micrograph of an extremely thin section through a keffir grain, after a preceding treatment with saffranin. The matrix is composed entirely of long thin bacilli (Bacillus caucasicus), while the peripheral portions, which are more deeply stained, consist to a large extent of dense masses of yeast cells with occasional streptococci. In a normal grain the latter organisms are present on the surface or in the cavities and grooves of the grain, and only to a less extent in the matrix. Nikolaiewa[57] claimed to have isolated a hitherto unknown bacillus capable of coagulating milk by acid production, Bacterium caucasicum, not identical with, but related to Freudenreich's Bacillus caucasicus, and also a torula. Although no experiments were carried out, Nikolaiewa asserts that this organism forms the matrix of the grains. He was able to produce a beverage resembling keffir, just as Freudenreich and Essaulow did with entirely different organisms, but his product would appear to have been slightly too acid and to have lacked the characteristic aroma of the normal product. In the course of an extensive series of experiments Kuntze[58] found the following organisms:
(a) True lactic acid forming bacteria, Streptococcus acidi lactici (Grotenfeldt).
(b) Bacteria of the group Bacterium acidi lactici (Hueppe) and Bacterium lactis aërogenes.
(c) Various torula and yeast species.
(d) Two species of butyric acid bacteria, Bacillus esterificans and Bacillus keffir (Kuntze).
His conclusions are: 1. In any case the presence of a yeast capable of directly fermenting milk sugar is not essential. 2. The significance of the presence of yeast lies in the fact that stimulation of the lactic bacteria occurs; further, the yeast exerts a regulating influence upon the rapidity of the fermentation proper. The variety is of minor importance, provided always that the yeast does not produce an unpleasant flavour. By the use of mixed cultures of Bacillus esterificans, Bacillus keffir, and Streptococcus acidi lactici, and a keffir yeast, Kuntze obtained a product that possessed to the fullest degree all the characteristic properties of a normal keffir. In such cultures he was successful in obtaining the formation of keffir-like grains. Keffir fermentation is, according to Kuntze, the result of the action of various organisms. During the initial stage butyric acid fermentation takes place, but is prevented from becoming predominant by the action of the keffir yeast. Simultaneously a true lactic acid fermentation proceeds and eventually gives place to a subsequent secondary production of butyric acid. Finally, then, we have a certain amount of unison in the results obtained by Freudenreich, Essaulow, Nikolaiewa, and Kuntze. These show that, for the production of a characteristic keffir, specific organisms are not essential, provided always that those used possess, either individually or collectively, the essential capacity of acidifying, coagulating, and fermenting the milk. For the growth of normal grains the presence of a matrix-forming organism, such as Bacillus keffir, is indispensable.