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 zoögloea 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.
[Illustration: 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.
[Illustration: FIG. 11.--Section through a Kephir
Grain--highly magnified.]
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.
[Illustration: FIG. 12--_Streptococcus lacticus_
(Grotenfeldt) growing on lactose-agar, stained by Gram's
method. ( X 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.
[Illustration: 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. Kuntze 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 [Greek: e]_, 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 [Greek: d]_, 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.]
[Illustration: FIG. 14.--Granule Bacillus from Yoghourt.
Shredded preparation of a fresh skim-milk culture at 37°
C. for six hours. Stain: aqueous methylene blue. (Enlarged
1:500.) In Figs. 15 and 17 will be noticed the chain
arrangement of the bacillus, which, in spite of the
supposed data of Luerssen and Kuhn, will be generally
noticed in the granule bacillus.]
[Illustration: FIG. 15.--Granule Bacillus from Yoghourt,
cultivated after the usual Agar method, for twenty-four
hours at 37° C. Stain: aqueous methylene blue. (Enlarged
1:500.)]
[Illustration: FIG. 16.--Granule Bacillus from Yoghourt.
Agar Milk Sugar Culture cultivated for forty-eight hours
at 37° C. Below is the true branching, above, the
distorted involution form. This production of involution
forms occurs chiefly in old cultures, and is an indication
of degeneration. Stain: aqueous methylene blue. (Enlarged
1:700.)]
[Illustration: FIG. 17.--_Bacteria W._ from Milk,
cultivated twenty-four hours at 37° C. Methylene blue.
(Enlarged 1:500.) The similarity in the pictures ought to
serve as a proof of the near relation of the granule form
and non-granule varieties.]
[Illustration: FIG. 18.--Agar Milk Sugar Culture. From the
original Bulgarian Yoghourt. In the centre, and beneath,
the characteristic hairy irregular colonies of the granule
bacillus (_Bacillus bulgaricus_ group), to the left, the
smooth contoured yeast colonies. The colonies of the
former organism always remain microscopic in size.
(Incubated several days at 20° to 25° C. Magnified X 10.)]
[Illustration: FIG. 19.--Agar Milk Sugar Culture. Surface
colony of granule bacillus from calf's stomach. The great
resemblance this colony bears to those formed by the
granule bacillus from Yoghourt will be apparent. This
fact, as well as close agreement in other cultured
features, induced Kuntze to place these organisms in one
group. (Incubated two days at 37° C. Magnified X 100.)]
[Illustration: FIG. 20.--Agar Milk Sugar Culture.
Deep-lying colony of granule bacillus from calf's stomach.
The form of the colony is often determined by the relative
presence or absence of air. (Two days at 37° C. Enlarged
about 1:50.)]
[Illustration: FIG. 21.--Agar Milk Sugar. Colony of
_Bacterium W._ from Yoghourt (non-granular variety of the
granule bacteria, as far as possible identical with
Luersen and Kühn's _Bacillus bulgaricus_), of a cubical
branching-out form.
According to Kuntze, the granule formation of this and
related organisms is variable, while White and Avery
regard it as a constant characteristic. (Incubated two
days at 37° C. Magnified x 50.)]
[Illustration: FIG. 22.--Two colonies of _Bacillus
acidophilus_ from calf's manure. Agar Milk Sugar Culture.
With this organism, also, we have conformation to one type
of colony, while, in other respects, temperature
requirements and production, etc., we have close agreement
with the granule bacillus (_Bacillus bulgaricus_). (Two
days at 37° C. Enlarged about 1:50.)]
[Illustration: FIG. 23.--Beer-wort Gelatine.
Fourteen-days-old colony of Yoghourt yeast. (Enlarged
about 1:50.)]
[Illustration: FIG. 24.--Shredded preparation of the
Bulgarian original Yoghourt. Stain: aqueous methylene
blue. Granule bacillus, diplostreptococci, and yeast. (See
also other photo-micrographs of Yoghourt. Enlarged
1:70.)]
[Illustration: FIG. 25.--Granule Bacillus from Yoghourt.
Cultivated in skim milk in twenty-four hours at 37° C.
Stain: aqueous methylene blue. (Enlarged 1:50.)
By means of this staining treatment the presence of
granules (not spores) can be easily detected. Treatment
with fuchsine fails to bring out these formations.]
[Illustration: FIG. 26.--_Bacteria W._, Agar Milk Sugar
Culture. Cultivated twenty-four hours (knobs, clubs).
Stain: Gram's method coloured with aqueous fuchsine
afterwards. (Enlarged 1:600.)]
[Illustration: FIG. 27.--_Bacteria acidophilus_ from
calves' manure, isolated by means of bouillon as acid as
vinegar. Shredded out of the usual Agar culture.
Twenty-four hours at 37° C. Stain: aqueous methylene blue.
(Enlarged 1:700.)]
[Illustration: FIG. 28.--Mucus from calf's stomach
inoculated into milk after eight transferrings. Shredded
preparation cultivated in milk for twenty-four hours at
37° C. Diplostreptococci and granule bacillus. Stain:
aqueous methylene blue. (Enlarged 1:500.)]
[Illustration: FIG. 29.--Diplostreptococcus from Yoghourt.
Pure culture in skim milk. A comparison of the
illustrations will show how close a resemblance exists
between bacteria found in the mucous membrane of calf's
stomach and those occurring in Yoghourt. In fact, by the
combined action of granule bacilli, and of
diplostreptococci from calf's stomach, together with a
Yoghourt yeast, it is possible to prepare normal
Yoghourt.]
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.]
[Illustration: 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.]
[Illustration: 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 [Greek: e]_ 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.
[Illustration: FIG. 32 is a photo-micrograph of soured
milk inoculated with a tablet containing viable and pure
cultures of _Bacillus bulgaricus_, and incubated for
seventy-two hours. These tablets constitute a valuable
means of preparing soured milk for therapeutic purposes.]
[Illustration: FIG. 33 is a photo-micrograph of milk
inoculated with a ferment tablet in which _Bacillus
bulgaricus_ is no longer viable, and the only growth
obtained is that of an organism allied to the _Bacillus
subtilis_ (Hay bacillus) group. Such milk would be
absolutely without value.]
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.
[Illustration: 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
aërogenes_, 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.
[Illustration: FIG. 35--_Bacillus bulgaricus_, showing the
cultures in English cow's milk. (Magnified 450 diams.)]
[Illustration: 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.]
[Illustration: 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.]