Leben Raïb or Leben (Laban.)—This is a beverage prepared largely by the Egyptians, and differs from keffir, as does matzoon, in possessing a characteristic aroma and taste. It differs also from the former by having only a very weak alcoholic fermentation, and by the coagulum being coarse and lumpy instead of being extremely fine. It is made from buffaloes', goats', or cows' milk by the addition of roba (or old leben) to the previously boiled and cooled fresh milk. The use of leben is many centuries old, and it is used in Egypt as in Arabia for medicinal purposes, although that of the Syrians and Arabians is said to differ from that of the Egyptians and Algerians. The fermentative changes occurring in the formation of the Egyptian leben have been investigated by Rist and Khoury,[64] and also by Guerbet,[65] who found that five organisms were normally present. These comprised a chain-forming bacillus (Streptobacillus), a second smaller bacillus (Bacillus lebenis), a diplococcus, a saccharomyces, and a mycoderma. Of these five organisms, it would appear that four live in metabiosis, the streptobacilli and bacilli hydrolyse the milk sugar, the components of which are split up by the yeast to alcohol and carbon-dioxide. The alcohol thus formed, together with the glucose formed by hydrolysis, are eventually converted to acid or combusted by the mycoderma species. The leben thereby assumes the sharp, unpleasant flavour met with in old samples. The diplococcus merely produces acidification and coagulation of the milk. Rist and Khoury were able, by the use of these organisms, to produce normal leben, especially when the true yeast was allowed to grow in the milk for some time before inoculation with the other organisms was made.
Some of the half-civilised tribes of Siberia, the Tartars and the Burgaten, prepare a strong alcoholic beverage, arakà or ojràn, from fermented milk. This is really a product of distillation, and contains seven to eight per cent. of alcohol and volatile fatty acids.
Fig. 13—Photo-micrograph of preparation from Armenian soured milk (Matzoon). This is related to Yoghourt, and contains, as will be seen from the above photo, yeasts, streptococci, diplococci, and a bacillus with the morphology of Bacillus bulgaricus. This, and similar foods, owe their peculiar properties primarily to the presence of Bacillus bulgaricus (type A, White and Avery), and only in a lesser degree to the yeasts and lactic streptococci.
Matzoon.—This is a drink used largely in Western Asia, and is similar in character to keffir, but has a peculiar taste which distinguishes it from all other fermented milks. According to Weigmann,[66] it is prepared from buffaloes', goats', or cows' milk, and is used partly as a means of souring milk for butter-making and also as a lactic food, eaten with spoons. In the same way buttermilk produced from milk which has been previously ripened by matzoon is used as a beverage. Finally, the coagulum (than) of such buttermilk is strained off, and, after being pressed, is mixed with meal and dried by exposure to the sun's rays. The preparation of matzoon is in many respects very similar to that of keffir and koumiss, but differs by inducing a comparatively weak alcohol fermentation. In common, too, with yoghourt, the prevailing temperature is much higher than is required for keffir and koumiss.
In regard to the biology of matzoon, the occurrence of various organisms has been recorded. Emmerling[67] isolated, in addition to a yellow pigment-forming organism, Bacillus subtilis, Bacillus lactis acidi, and several fungi, a small micrococcus capable of hydrolysing milk- and cane-sugar. The organism produces and without gas formation, or peptonisation of the medium. Of the nine yeasts isolated from matzoon by Lindner[68] and Kalantharianz,[69] three were able to ferment milk sugar without previous hydrolysis, while two others, by the simultaneous production of lactic acid and fruit esters, gave to the matzoon its characteristic taste and aroma.
Yoghourt and Soured Milk.—Yoghourt is another fermented milk, and is related to the matzoon of Armenia, the gioddu of Sardinia, and the leben of Egypt. After a preceding boiling and reduction of the volume of the milk, inoculation of the mass is made by the addition of a small quantity of old culture, and it is then allowed to sour at a comparatively high temperature. A moderately compact, jelly-like coagulum is thus formed, while keffir and koumiss possess a liquid consistency. The fermentation necessary for the two latter products only proceeds, too, at a much lower temperature, at which yeasts play an important part. According to Guerbet, yoghourt incubated for ten hours at 113° F. contained 0.34 per cent. lactic acid and 0.012 per cent. alcohol. Luerssen and Kühn[70] came to the conclusion that yoghourt contained chiefly a mixture of Bacillus bulgaricus, diplostreptococci, and a "granule" bacillus, so called on account of its granulated appearance after treatment with methylene blue. According to these authors, the first two organisms were found in each of eight samples of maya (young yoghourt) and of yoghourt itself, but the occurrence of the "granule" bacillus in plate cultures was by no means regular. In addition, yeasts were found in almost every sample examined, but were regarded more as accidental infections rather than as essential to the formation of a typical product. The combined action of the three organisms already mentioned gave rise to a product closely resembling normal yoghourt. Piorkowski[71] subjected Bulgarian maya to examination and associated himself with Metchnikoff[72] in finding three species, a streptococcus, a diplococcus, and a specific organism to which he gave the name Yoghourt bacillus. Similar results were also obtained by Grigoroff.[73] Piorkowski's Yoghourt bacillus is similar in form to Bacillus subtilis, but does not sporulate, nor does it liquefy gelatine. Young individuals are stained by Gram's method; older individuals are, however, Gram negative. The optimum temperature is 112° F. Kuntze attempted to isolate the organisms mentioned by Luerssen and Kühn, and by plate culture procured growth of a spore-forming bacillus similar to Weigmann's Bacillus matzoon. To this organism is attributed the power to impart a specific taste to the matzoon, but as growth is comparatively slow, it can only be of significance in determining the quality of the curd and cheese prepared from this product. Cultures were also obtained which resembled in general character those of the organism described by Luerssen and Kühn as Bacillus bulgaricus and named by Kuntze Bacterium W. Granule formation was transient in this culture, and the organisms eventually became inactive. Further analysis of maya gave cultures of the "granule" bacillus, but these passed over from the type forming irregular colonies (see Figs. 14, 15, 16) to that producing smooth colonies. Further, although the granule formation persists largely in milk, the organisms soon revert to the non-granular type if cultivated on agar. By the use of the Gram-Weigert stain organisms from a several-days-old culture on beer-wort-agar gave an interesting reaction. The bacillar threads are in places Gram-negative, in others Gram-positive, and bear small club-like swellings (see Fig. 14). Results similar to these were also obtained with cultures of Bacillus matzoon (Weigmann and Grübner) and also with Bacillus acidophilus.
Neisser's method of staining failed to give such good effects by the examination of fresh maya, as did an alcoholic aqueous solution of methylene blue in showing up the granules of the organisms. Again, Grixoni[74] found, but did not isolate, a similar granule-forming organism (Bacterium sardous) in Sardinian gioddu. As already mentioned in the description of leben, Rist and Khoury found a long bacillar lactic ferment (Streptobacillus lebenis) which also exhibited the irregular greyish white hairy colonies and high optimum temperature characteristic of this group. On account of the similarity in form, staining reactions, temperature requirements, and cultural growth of the organisms described by Emmerling, Düggeli, Weigmann, Grixoni, and Rist and Khoury, Kuntze is inclined to regard them as belonging to one single group of lactic ferments. According to him the granule formation is rather variable, and may be induced or suppressed by cultural methods. Not only do organisms of this group produce far more acid than the normal lactic bacteria; they are also more resistant to acid, and are able to develop in milk to which 0.5 per cent. hydrochloric acid has been added. A comparatively high percentage of alcohol seems to encourage growth, and this was obtained in milk containing 4 per cent. alcohol. This would no doubt tend to explain the phenomenon observed by Kuntze that milk is not so rapidly fermented by organisms of this group as when cultures of diplococci and yeasts are added. Since organisms of this group would seem to be widely distributed, the question of their natural habitat arises. Luerssen and Kühn were unsuccessful in their search for such organisms in Königsberg milk, but Leichmann records the occurrence of a long bacillus (Bacillus lactis acidi) in milk that had spontaneously soured at 112° to 120° F. This organism, too, showed characteristic growth on agar media, and produces lævo-rotatory lactic acid. The examination of calves' stomachs showed, according to Kuntze, only occasional long bacilli, but inoculation of sterile milk and incubation at 100° F. with repeated over-inoculation gave a culture showing the characteristic granule reaction (see Figs. 18 and 20). Although plate cultures made direct from calves' stomachs do not exhibit the regular contours generally shown by the granule bacillus, yet this growth may be induced by preceding cultivation in lactose bouillon to which 0.5 per cent. acetic acid has been added. A similar organism, Bacillus acidophilus, was isolated from calves' manure by means of this acetic bouillon, as was also a diplostreptococcus which resembled very closely the typical lactic acid streptococcus. This resemblance was made all the more striking by the fact that they were capable of coagulating milk at a temperature of 99° to 104° F. Since these organisms are present in large numbers in manure and also in the digestive tract of ruminants, it would seem probable that their occurrence is not without significance for the operations of cheese manufacture. According to Jensen, the practice of applying farmyard manure to Swiss meadows has been regarded as absolutely essential to the production of cheese of the best quality; while, on the other hand, the application of artificial manures would seem to have been responsible for an increase in abnormal cheese. Küntze found further that by the combined inoculation of sterile milk with the diplostreptococcus and the "granule" bacillus from calves' stomachs, together with a yoghourt yeast, he was able to obtain a product possessing a taste and aroma little different from normal yoghourt. During their investigations upon the ripening of Swiss hard cheese, Freudenreich and Jensen[75] isolated five varieties of lactic acid bacilli, and were able to show that one of these, especially Bacillus casei ε, was of the greatest importance for the production of good cheese. This organism has been found by Thöni to be present in rennet tablets, while a related variety, Bacillus casei δ, was found in fresh calves' stomachs. Unfortunately, staining tests with these organisms were not carried out, so that no data are available in regard to the presence of granules. The photo-micrographs of these organisms show the small clubs and true-branched forms. The presence of these diplococci and bacillar lactic ferments in the intestinal tract of ruminants and horses might possess some importance for the preparation of yoghourt in bags or tubes made from the stomachs of these animals. Finally, Moro[76] has isolated an acidophilic organism from the dejecta of infants which resembles closely, both in manner of growth, resistance to acids, true branching, and temperature optimum, the granule bacillus and related forms.
[This group of sixteen illustrations (Figs. 14 to 29), showing various aspects of the Yoghourt bacillus and others of a cognate nature, is taken from the Centralblatt für Bakteriologie of Jena.—L. M. D.]