CHAPTER V
THE BACTERIOLOGY OF FERMENTED OR SOURED MILK
A CHAPTER FOR STUDENTS
During the last few years much work has been done in investigating the action of various classes of organisms—bacteria, yeasts, and moulds—upon milk and its products. While, however, the attention of the dairyman has been chiefly directed to the propagation of acid-producing organisms and the use of pure cultures of lactic acid bacteria in their relation to butter and cheese making, a new sphere in micro-biology has been disclosed by the study of the effects caused by the combined growth of two or more different classes of organisms in milk and the consequent production of lactic, alcoholic, and gaseous fermentations. The simultaneous occurrence of these fermentative changes is responsible for the formation of such beverages as keffir, koumiss, milk-wine, etc. It has therefore become essential, in connection with the study of new developments in the milk industry, that we should make a more intimate acquaintance with the bacteriology of the ferments involved.
Keffir (kephir, kifyr, kiafyr, kephor, kyppe) is the name given to an acid, slightly alcoholic drink, which for many centuries has been prepared by the nomadic tribes in the Caucasus. The characteristic fermentation is induced by the addition of so-called keffir grains. These are yellow or golden-yellow, warty, and furrowed flakes or nodules, the former varying in size from that of a rice grain to that of a bean, while the latter are often about an inch across and one eighth of an inch thick. Bearing in mind the fact that the preparation of keffir has been carried on for many centuries, it is not surprising that the origin of these grains should be surrounded by myths.
The belief is prevalent among the Mohammedan tribes of the Caucasus that keffir grains were, in the first instance, presented by Allah, as a sign of immortality, to one preferred tribe. Others hold that, in past ages, they were found by shepherds growing on a shrub in the Caucasian highlands; while, according to Skolotowski,[47] they were originally found adhering to the walls of an oaken vessel used for the preparation of airam. This is a soured milk beverage similar to keffir, but possessing a weaker alcoholic fermentation, and prepared from goats' milk by the addition of pieces of calf's stomach. This would undoubtedly serve to introduce various species of lactic acid bacteria, and will be referred to in the portion dealing with soured milks. Keffir is prepared by the Caucasians from cows', sheep's, or goats' milk, and the operation is carried on in large leathern tubes or bottles. After the addition of the grains or seeds to the milk the vessel is placed in a cool chamber, and the fermentation is allowed to proceed for one or two days, by the end of which time the normal fermentation is at an end. During this period the keffir grains have increased enormously in size, assume a bright yellow colour, and lose their sour buttery smell.
Previous to the removal of the fermented liquid, a portion of the bottle is firmly bound from the rest by a stout cord, and the greater portion of the remaining keffir is quickly removed for use, thus avoiding, as far as practicable, any outside infection. After the addition of fresh warm milk the cord round the end of the bottle is removed, and the old and new milk thoroughly mixed for a time in order to ensure uniform inoculation of the new milk for the next fermentation. During the winter months the leathern vessels are often placed in the sunshine, so that the temperature remains at 61° to 65° F.
The necessary agitation of the vessel is said to be supplied in the form of kicks by passers-by or by the children during their play.
The beverage prepared in this way is so gaseous in character that it is often blown forcibly from the vessel during removal, and possesses, according to Podowyssozki,[48] a very acid taste.
During any interruption in the preparation of keffir in the above manner, the grains are taken out, and after having been well washed in clean water, are spread out on a clean cloth to dry in the sunshine. They thereby assume a characteristic cheesy or buttery odour and become rather darker in colour. Thorough desiccation is essential in order to prevent subsequent mouldiness or disease of the grain.
In European countries the grains are subjected to a preliminary soaking in water for five to six hours and then placed in four to five changes of milk, each change having a duration of two to three hours. As soon as the grains commence to rise to the surface of the milk, they may be used for the actual preparation. To this end, a small quantity of the grain is added to freshly boiled milk and allowed to stand for eight to twelve hours at a temperature of 55°-62° F. with agitation of the flask every two hours. By this time the milk, now known as Sakwaska, has become abundantly inoculated with the organisms essential to the fermentation, and after the removal of the grains, may be poured into well-corked flasks for the secondary brew. The flasks should be kept at a lower temperature for twenty-four to forty-eight hours, by which time the product is ready for consumption.
According to the temperature and length of period to which this subsequent fermentation is allowed to proceed, the resultant keffir is more or less acid and gaseous. The grains may again be used for starting a fresh portion of milk, and a regular supply obtained in this manner. Well-fermented forty-eight-hours-old keffir should be an effervescent beverage with prickling and acid taste and a consistency and smell similar to sour cream. Large, persistent bubbles should form on the surface of the liquid and the casein be present as an extremely fine flocculent precipitate which remains suspended for a considerable time.
From the third day there ensues a gradual peptonisation of the casein. If the temperature at which the secondary fermentation has occurred should be higher than 72° F., or if the milk has not been sufficiently agitated, then the casein will be present in the form of porous small flakes, which on shaking form a fine emulsion.
The chemical changes undergone by the milk during the preparation of keffir are confined almost exclusively to the milk sugar. As already stated, a slight peptonisation occurs in old samples, but this depends very largely upon the method of preparation and purity of the culture. Hammersten[49] and Essaulow[50] show, however, that this is not a concomitant of normal fermentation. According to Hammersten, normal keffir contains—
| Per cent. | |
| Water | 88.26 |
| Fat | 3.35 |
| Casein | 2.98 |
| Lactalbumen | 0.28 |
| Peptones | 0.05 |
| Milk sugar | 2.78 |
| Lactic acid | 0.81 |
| Alcohol | 0.70 |
| Ash | 0.79 |
In no case should the acid be higher than 1.0 per cent., and the alcohol more than 0.75 per cent.
Biology of the Keffir Grain.—The first communication on the biology of the keffir grain seems to have been made by Kern.[51] He regarded the grain as a zoöglœa composed of bacilli and yeasts, the latter being regarded as the ordinary beer yeast (Saccharomyces cerevisseæ), while to the former he gave the name of Dispora caucasica. As the name indicates, this bacillus possesses two polar spores, and germination of these proceeded in the same manner as with Bac. subtilis. As, however, pure cultures of the organisms were not made, and the descriptions and illustrations made by Kern fail to show any distinctive characteristics, it seems probable that accidental confusion with other organisms must have occurred.
A Milk Filling Apparatus
Fig. 10—Where soured milk is handled on the large scale, a special filling apparatus for bottles is desirable, and the soured milk supply should be under cover as shown. This apparatus is made by the Dairy Machinery and Construction Company.
Krannhals[52] succeeded in isolating ten different keffir bacteria among which were several sporulating bacteria. Here too it is impossible to attach any importance to the results, as the artificial preparation of keffir, by means of these bacteria, was not attempted. Beijerinck[53] studied the organisms constituting keffir grains and attached prime importance to the occurrence of two organisms, viz., (a) a yeast, Saccharomyces kefir, which was capable of inverting milk sugar by means of an enzyme (lactase) and afterwards fermented the products with the formation of alcohol and carbon dioxide, and also (b) a non-motile non-sporulating bacterium, afterwards Lactobac. caucasicus. The latter, when cultivated on gelatine, gave rise to tough warty colonies about 1/40 in. diameter, and was regarded as one of the lactic acid bacteria found in milk which has been incubated at 77° to 90° F. and afterwards incubated at a higher temperature, 100° to 104° F. Scholl[54] isolated three different organisms, of which a yeast inverted milk sugar for the lactic acid bacteria, while Dispora peptonised the albuminoid matters.
Adametz[55] failed to isolate Dispora, and came to the conclusion that ordinary lactic bacteria and yeasts played the most important part in the fermentation.
Essaulow found in keffir grains six different organisms—yeast cells, cocci, short thick bacilli, bent bacilli, long threads, and motile bacteria. The two latter would seem to be Bacillus subtilis, while the others may be regarded as Bacterium acidi lactici (Hueppe), Bacterium aërogenes, and Streptococcus lacticus (Grotenfeldt). Pure cultures were insufficient to produce keffir, while mixed cultures of Bacterium acidi lactici and yeasts were effective.
Freudenreich,[56] to whom we owe a record of very carefully executed experiments, could not arrive at a satisfactory explanation of the rôle of Bacillus caucasicus. This organism is described as being 5-6 µ long and 1 µ thick, slightly motile, and possessing bright refractive spots at the poles of the bacilli. It is extremely difficult to cultivate, and forms flat, small greyish colonies of irregular outline. The bright refractive spots above referred to are, however, granules taking the usual stains quite readily, and not spores as supposed by Kern.
Freudenreich also found three other organisms—a yeast and two streptococci. The yeast, to which he gave the name Saccharomyces (Torula) keffir, forms small oval or roundish cells 2-3 µ wide and 3-5 µ long. The optimum temperature would seem to be about 72° F.; the maximum 82° F. This organism is unable to ferment milk directly, but is able to decompose maltose and glucose with gas production. It does not coagulate milk, but imparts to it a characteristic taste and is unable to withstand desiccation for more than a few days.
Of the two streptococci isolated, Streptococcus a resembles organisms of the group Streptococcus lacticus in appearance, but is able to ferment milk, with weak acid and gas production, and is capable of inducing coagulation.
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.
Fig. 12—Streptococcus lacticus (Grotenfeldt) growing on lactose-agar, stained by Gram's method. ( ✕ 900 diams.)
Diseases of Keffir Grains.—According to the age and the previous treatment to which keffir grains have been subjected, the vitality of one or more of the organisms constituting the grain may have been impaired. The results of Freudenreich have shown that Saccharomyces keffir and Streptococcus a are unable to withstand desiccation for more than a few days, and this is sufficient to account for the frequent failures to obtain normal keffir from the grain. Further, grains succumb to a mucilaginous disease; the cavities become filled with a slimy fluid, and the grains are covered with mucilaginous matter. They lose their elasticity and become brittle or mealy, but large grains appear to be more subject to this fault than do the small ones. Such grains should be disinfected by immersion for a short time in two per cent. salicylic acid solution, followed by drying in the sun, whereby they are completely regenerated.
Another disease consists in the predominance of certain butyric acid bacteria which impart an unpleasant rancid taste to the keffir (Podowyssozki). This is generally attributed to the use of rich milk, or too high a temperature during preparation.
Koumiss.—Another product of the combined action of lactic acid and alcohol-producing organisms is called koumiss, kumys, milk-wine, lac fermentation, or vinum lactis. In the steppes of Southern Russia and Asia, as we have seen,[59] it is prepared chiefly from mares' milk, but occasionally from that of camels and jennets. The name is said to be derived from that of a tribe mentioned by Xenophon and Pliny, viz., the Kumanen, by whom its preparation was practised. After the war with the Tartars in 1215 its use was adopted by the latter people, and eventually spread to the Turkomanen, Kalmucks, Khirgiz, Mongolians, etc.
Rubruck, in 1253, records the use of a fermented drink—kosmos—prepared from mares' milk, and about the same time Marco Polo mentions the occurrence of a milk-wine, chumis or chemius, among the Tartars. The fact that the Tartars were seldom ill, and were almost invariably free from lung troubles, led to an influx of visitors from surrounding countries, until finally its use spread to Russia, Austria, and Germany. At the present time the best koumiss is that produced in the province of Orenburg; but specially equipped koumiss establishments, under the control of physicians, exist in Odessa, Samara, Ufa in the Urals, and other districts. The curative properties of koumiss have long been recognised and its use is indicated in cases of indigestion, chlorosis, scurvy, tuberculosis, etc.
Rubinsky states that, among the nomadic tribe, of Khirgiz and Kalmucks, a special leathern bottle (Turssuk, Orroth, or Soaba) is used for the preparation of koumiss, while wooden tubs (Tschiljak) similar in shape to the old-fashioned churn are used by the Bashkirs, and in koumiss establishments.
The fermentation is induced by the addition of koumiss to fresh mares' milk, in proportions which vary according to the cleanliness observed in the actual preparation. Where the process is carefully controlled, one part of koumiss to ten parts of milk is often used, but where gross infection from outside sources takes place one part of koumiss to three parts of milk is taken. The mixture is stirred at frequent intervals, and stored at a temperature of 73°-90° F. Weak koumiss is obtained after twenty to twenty-four hours in winter and twelve to fourteen hours in summer, but is scarcely ever consumed immediately, as it possesses a strong purgative action.
It is generally poured into bottles (bottled koumiss); or allowed to remain in the tubs (tschiljak koumiss); in the former case the fermentation is anaërobic, in the second it is aërobic.
Storage of the koumiss upon ice or in a cellar is necessary since medium koumiss is converted to strong koumiss in twelve to sixteen hours at ordinary temperatures, while at the lower temperature this occurs only in two to four days.[60]
According to Biel,[61] either old koumiss or the dried sediment from old koumiss may be used for the initial inoculation. It may also be prepared by the repeated inoculation of mares' milk with soured cows' milk until a fermenting product is obtained. Koumiss may be prepared by a method stated by Allik[62] to be in general use in the Caucasian health-resorts. One part of beer-yeast is added to four to ten parts of fresh mares' milk (according to the strength of product required), and after thorough mixture of the two liquids the whole is allowed to ferment at a temperature of 70° to 72° F. for two days. One part of this first product is then added to five parts of fresh cold milk, and allowed to stand three to four hours at 75° to 77° F. It is then poured into bottles, and after the expiration of another three to four hours is stored away in a cellar at about 45° F. This koumiss may be used at any time from one to five days (generally two to three) after bottling according to the strength desired or prescribed in each individual case.
The changes undergone during fermentation consist in a vigorous gas and acid production accompanied by alcohol formation and coagulation of the milk. The coagulum exists in an extremely fine state of division, and the liquid froths violently on the bottle being opened. It has a full pleasant acid taste, but should not contain more than one per cent. acid and two per cent. alcohol. The specific gravity of koumiss is 1.008 to 1.020 at 60° F. Appended is an analysis of two different samples of koumiss:
| Prepared from | ||
| Mares' Milk. | Separated Cows' Milk. | |
| Per Cent. | Per Cent. | |
| Water | 91.535 | 88.933 |
| Fat | 1.274 | 0.854 |
| Nitrogenous bodies | 1.913 | 2.025 |
| Sugar | 1.253 | 3.108 |
| Ash | 0.293 | 0.444 |
| Carbon dioxide | 0.876 | 1.027 |
| Alcohol | 1.850 | 2.647 |
| Lactic acid | 1.006 | 0.796 |
| Glycerine | ... | 0.166 |
Fleischmann[63] gives a formula for preparing an artificial koumiss from separated cows' milk, water, cane sugar, and milk sugar, with the addition of distillery yeast. Needless to say, this product must possess some of the characteristic by-flavour of the yeast employed, and is less suitable than koumiss prepared by the aid of a lactic yeast. Schipin investigated the fermentation of koumiss and found three distinct organisms.
Rubinsky in a recent article threw much light on the phenomena of koumiss fermentation. According to him, koumiss contains almost invariably four different organisms, viz., koumiss yeast, koumiss bacterium (Lactobacillus), Streptococcus lactis (Lister), Bacterium aërogenes, and occasionally Bact. caucasicum (Nikolajewa). For the preparation of normal koumiss only the two former organisms are required; they exceed in number any of the other organisms whose presence in the dairy is unavoidable. The presence of the two latter organisms is favourable to the production of good koumiss, as, by inducing a preliminary lactic fermentation, they tend to inhibit the growth of undesirable extraneous bacteria, etc. In medium and strong koumiss they die out on account of the amount of lactic acid formed (1%).
Koumiss yeast possesses strongly differentiated protoplasm, but lacks any cultural characteristics. Abundant growth occurs in milk, and lactic acid (0.3%), alcohol, carbon dioxide, albumens and peptones, volatile acids, and aromatic substances are formed.
Koumiss bacterium is related to the Lactobacillus of various other fermented milks, and is similar to Bac. acidophilus, and possesses like these a distinct polymorphism (branched cells, long and short bacilli, etc.). It is non-sporogenous, has an optimum temperature of 90° to 97° F., and possesses cultural characteristics similar to those of the rest of the Lactobacilli.
The by-products of koumiss yeast appear to favour the growth of the koumiss bacterium, as this organism, like the other Lactobacilli, is favourably influenced by the presence of small quantities of peptone, alcohol, and acid.
The organisms found by Schipin consisted of a species of Saccharomyces and two bacilli, Bacillus acidi lactici and a non-sporulating bacillus. The latter organisms coagulate milk at 98° F., but not at room temperature, and although a minute description of cultural characteristics is not given it would seem to be related to Bacillus or Lactobacillus caucasicus.
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.]
In a review of the literature of the subject of soured milks, Makrinoff suggests the adoption of the two names, Streptobac. lebenis viscosus and Streptobac. lebenis non-viscosus, for the organisms of the so-called Bulgaricus group, and known at present as Bacillus-bulgaricus, Streptobac. lebenis, Bacillus of Massol, Granule bacillus, Bact. Mazun, Bac. lactis acidi, etc.[77]
White and Avery[78] have made a comparative study of a large number of varieties and species of lactic acid bacteria of the above type obtained from various fermented milks and milk tabloids. Their descriptions are so detailed and their conclusions are so important that we give them at length. According to this work, the whole of the thermophilic lactic acid bacilli of the so-called Bulgaricus type may be divided into two sub-types, A and B.
The Cultural Characteristics of the Bacillus Bulgaricus Group
The cultural characteristics of all the strains of Bacillus bulgaricus (granule bacillus) are as follows:
In Whey Agar.—All strains exhibit wide variation in size, 2 µ to 50 µ long and about 1 µ broad. Almost all individuals are intensely Gram-positive, and show regularity of outline. All strains show involution form, exhibiting vacuoles, and often show empty cell membranes. The latter are Gram-negative, and vary greatly in both dimensions as well as in form. All strains show tendency to chain formation, some being arranged in chains of six to twenty-five segments, which may contain both Gram-positive and Gram-negative individuals. Type B exhibits Gram-negative spherical bodies varying from 0.25 µ to 1 µ in size, adhering to the sides of some of the Gram-negative individuals.
In Whey.—In this medium there is a marked tendency toward degeneration and involution. In the early stages of incubation, at 100° to 112° F., the bacilli are uniform in size and intensely Gram-positive; in succeeding stages the irregular, vacuolated, inflated, and ruptured forms predominate. Between the eighteenth and twenty-fourth hours of incubation at 112° F. the strains of type A develop oval to kidney-shaped nodules attached to a stem extending from the cell substance. As the incubation is prolonged these nodules increase in size, often measuring 1 µ to 2 µ in length; this nodule formation occurs at the expense of the cell protoplasm, and appears to be a marked characteristic of growth in whey. Cultures of type B do not form nodules or clubs, but small spherical bodies more or less securely attached to the cell wall are seen. Again, type A assumes the form of small bacilli in chains, while type B strains develop to a greater length and exist almost exclusively as single isolated forms. True branching has been observed in strains of type B.
In Milk.—In milk there is a tendency to thread-formation consisting of four to ten segments in the case of type A, while type B shows longer and more curved forms. With increasing age of the culture there also appears to be increase in the length of the organisms. All strains are non-motile, non-sporogenous, and non-capsule-forming.
Staining Reactions.—All strains are readily stained by the usual aniline dyes.
A. Gram's Method.—Young individuals give an intense reaction with this stain; old bacilli are easily decolourised, and degenerate forms are always Gram-negative, while single individuals have been observed which showed gradation from one pole of the cell to the other.
B. Loeffler's Methylene Blue.—According to the behaviour of the organisms studied, a separation into two types appears possible, type A being uniformly impregnated, while type B shows distinct differentiation. The cell body is seen to contain a varying number of round to oval bodies or granules. This is the appearance already mentioned by Düggeli, Luerssen and Kühn, and Kuntze, and from which the granule bacillus derives its name. In opposition to the observations of Kuntze, the occurrence of granules was not found to be variable; it was, indeed, so constant as to constitute a distinguishing characteristic between the two types. The organisms of this group are difficult to cultivate, and freshly isolated growth is obtainable only on media containing whey, malt, or in milk. They grow equally well under aërobic or anaërobic conditions. The optimum temperature for growth is 113° to 115° F.; growth is fair at 85° F., slight at 75° F., and does not take place at 68° F.
Colonies on whey agar are round to irregular, greyish white, curled and filamentous, often streaming, and in a few cases smooth and even in structure. Gelatine is not liquefied. There is no surface growth on gelatine stab-cultures. Along the stab the growth is filiform, beaded, with subsequent horizontally projecting ramifications. Milk is coagulated in eight to eighteen hours at 112° F., and is the most favourable medium for growth.
[I am indebted for this group of illustrations (seventeen in number) to the editor of Bacteriotherapy, New York, U.S.A.—L.M.D.]
Fig. 30—Photo micrograph of preparation made from Yoghourt, showing yeast cells, large lactic diplococci, small slender bacilli and many large bacilli possessing the morphology of Bacillus bulgaricus. Yeast cells are almost invariably found in native Yoghourt, but do not appear to be essential to the production of a tropical beverage. Indeed, they would seem to be responsible for the unpleasant astringent taste often met with in old samples of this product.
Fig. 31—Photo micrograph of smear from Greek Curdled Milk called "Giaourti," and showing yeast cells, long bacilli and a mould (Oidium lactis), possessing very large elongate cells. The presence of the latter is very undesirable, as it rapidly combusts the lactic acid, digests the casein, and imparts a strong unpleasant cheesy flavour to the beverage.
Type A produces 2.7 per cent. to 3.7 per cent. inactive lactic acid in milk, while type B produces only 1.2 per cent. to 1.6 per cent. lævo-rotatory lactic acid in milk. There is a small quantity of acetic, formic, and succinic acids formed. The conclusions of White and Avery are:
I. A review of the morphological culture and biochemical features of the lactic acid producing bacilli from yoghourt, matzoon, and leben, appears to justify their classification as a single group.
II. This group would seem to be identical with Bacterium caucasicum (Kern).
III. The significant variations exhibited by these bacilli in regard to the presence or absence of granules demonstrable by differential stains, the degree of lactic acid production, and the nature of the acid produced, suggest a division into two different types—the true type A, and the paratype B.
Quite recently Hastings and Hammer[79] recorded the isolation from milk of an organism producing more acid than either Bacterium coli commune or Bacillus lactis acidi. It is characterised by possessing a high optimum temperature, and by the limited conditions under which it grows on nutrient media. On this account these investigators suppose it to be related to those described in the paragraphs on fermented milks, leben, matzoon, etc., and which are regarded by Kuntze as being identical.
Similarly Boutroux[80] found 1.5 per cent. acidity produced in a solution containing albuminous matter and glucose; while Richet[81] states that with the addition of gastric juice to milk as much as four per cent. acidity may be formed. After storing samples of milk for eight days at 100° F., Koning[82] found 2.35 per cent. and 2.5 acid; while similar samples stored at 60° to 62° F. for the same period only developed 0.9 per cent. Heinemann[83] records the production of 3.0 per cent. acid in milk incubated at 100° F.; and Jensen states that Bacillus casei ε is capable of developing 2.7 per cent. lactic acid.
Dr. H. B. Hutchinson, bacteriologist at Rothamsted Experimental Station, has also been successful in isolating a bacillus from English market milk resembling in every particular those classified by White and Avery as type A.
It will thus be seen that organisms related to those of Oriental and Occidental milk beverages are present in conditions where it is impossible for them to attain to any active growth. The same class of organism has also been found in many cases in butter and cheese throughout the United States.
Of recent years the consumption of milk fermented by these organisms has been introduced more or less successfully into all European countries. This custom is due, as we have seen,[84] to a very great extent to the announcement of Metchnikoff[85] that the action of such organisms in the alimentary tract conduce to a prolongation of life. Moro found that the dejecta of children contain large numbers of Bac. lacidophilus and Bac. bifidus, but, as age advances, the bacterial flora of the intestines tends to change. The number of acid-producing organisms gradually becomes less, and other bacteria capable of producing far-reaching decomposition of albuminoid matter tend to increase.
Working on the assumption that senility is partially due to the absorption of by-products formed from albuminoid food by the decomposing or putrefactive bacteria mentioned, Metchnikoff instituted a search for organisms capable of suppressing the growth of the putrefactive bacteria.
It has long been known that milk allowed to become sour will keep for a considerably longer period in hot weather than if lactic bacteria had not grown. This preservative action of lactic acid also comes into play in the manufacture of sauerkraut and in the preservation of meat by immersion in sour milk.
Fig. 34.—Photo-micrograph of smear of culture of Bacillus bulgaricus, recommended by Metchnikoff for use in cases of intestinal auto-intoxication. Unlike the ferments of normally soured milk, which are sometimes indifferent, or even injurious, in their action, this bacillus is capable of growth at blood heat, and, by producing much larger quantities of lactic acid than such organisms as Streptococcus lacticus, Bacillus coli commune, or Bacteria lacticus aerogenes, inhibits the multiplication of bacteria responsible for the putrefaction of albuminoid food in the intestines.
Bienstock has shown that the growth of Bac. putrificus is inhibited by the action of Bact. coli commune, which is capable of setting up a slight lactic acid fermentation. Bact. coli commune, however, gives rise to substances of an injurious character, and, although present very abundantly in the intestinal tract, it may by reduced almost entirely by the active growth of lactic acid bacteria. This fact is of great value to the cheese-maker, since by the addition of a lactic acid culture (starter) to milk before renneting, gas-producing bacteria such as Bact. coli may be checked in growth. Since the ordinary lactic acid bacteria such as Streptococcus lacticus, Bac. lactis acidi, and others, are incapable of growth at blood temperature, it appeared necessary to procure cultures of lactic bacteria able to grow at temperatures of 100° F. to 112° F. Such an organism was found in Bulgarian soured milk (yoghourt), and was considered pre-eminently adapted to this purpose. As has been shown in the preceding paragraphs, this organism is merely one of a large group of bacteria found distributed in the intestinal canal of many domestic animals, in manure, and in ordinary market milk. It is then not surprising that the introduction into the intestinal tract of bacteria of the type Bulgaricus in the form of tabloids has not met with any decided success. Although it was considered to be merely necessary to introduce the desired type of organism into the body, and the amount of lactic acid taken into the system by the administration of soured milks was looked upon as of secondary importance, yet, it would seem, in the light of recent investigations, that benefits derived from a soured milk regimen are attributable in part to a chemical as well as a purely bacterial action. This receives support from the fact that soured milk beverages prepared by the use of ordinary lactic bacteria, distinct from those of the Bulgaricus type, often exert a beneficial influence upon human beings even although the organisms responsible for the fermentation are incapable of growth at blood temperature.
It may be mentioned in conclusion that cultures prepared by the use of organisms of the type Streptococcus lacticus combined with Bac. bulgaricus possess a more agreeable flavour and aroma than those prepared from a pure culture of Bulgaricus alone.
Fig. 35—Bacillus bulgaricus, showing the cultures in English cow's milk. (Magnified 450 diams.)
Fig. 36—Photo-micrograph of pure culture of Bacillus bulgaricus. The administration of cultures of this organism is indicated in all cases of intestinal ailments, caused by the excessive growth of proteolytic bacteria, and consequent putrefaction of foods in the alimentary tract. By the formation of large quantities of lactic acid, a state of asepsis is ensured, which is particularly valuable in cases of operations on the abdomen and intestines.
Fig. 37.—Photo-micrograph of smear of combined culture of Bacillus bulgaricus and Bacteria paralacticus. This double culture possesses an advantage over single cultures in that, while the characteristic disinfecting action of the former is retained, any secondary action of the growth of this organism upon the milk-fat is checked by the growth of Bacillus paralacticus, thus ensuring the production of a more palatable product.