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Peg aad a) MASTER OF HOME ECONOMICS BY LYDIA ZAE NORTHRUP i 1913
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THKH UTILITY OF LACTIC BACTERIA.
Thesis for the derree of
MASTER OF HOME EBFCOWOMICS .
by
Lydia zoe Northrup
=a on ww we 4 oe OR
1913
THESIS
THE UTILITY OF LACTIC BACTERIA.
Pernaps no other single group of micro6r- ganisms has such a wide distribution in nature and
in addition, such a range of usefulness sas has the
group of lactic acid forming bacteria which sare re~
sponsible for the cheracteristic fermentative
change in milk known as curdling. These bacteris
are also responsible for the characteristic fer-
mentation in other types of food as well, ssauer~
kraut and dill pickles being well known examples: ensilage also derives its peculiar odor and flavor
from the growth of these micro6rganisms.
The most common phenomenon produced by the
lactic bacteria, the curdling of milk, has been
recorded from time immemorisl. "Butter", the
translation of the Hebrew word “hemah" given in the
Fnglish authorized version of the Old Testament,
means "curds" or curdled milk (9). In Genesis 18:
8, we ere told that Abraham placed curds before his guests (2250 = 2000 B.C.); in Deuteronomy 32: 14 (1750 B.C.), Moses told the Israelites that
curdled milk was one of the blessings which Jeho- ve 103196 van had given to his chosen people (46). In Isaiah
7; 15 and 22, butter or curds is mentioned as one of the few foods left to the Judeans during their war with the Syrians and Ephraimites (735 B.C.) (10.).
For centuries soured milk has been used as a food by the peoples of Eastern Europe, Western and
Middle Asia and parts of Africa. Fermented milk, a soured milk beverage, has also been used from time immemorial by the wandering tribes of Arabia.
Those of the ancient fermented milk bever-~ ages best known today are koumiss, kefir, leben, yoghurt and mazun, dadhi, gioddu, khilat, oxygala, chiston, rayet, huslanka and taettamoelk being less commonly known. Each of these milk drinks is the result of the concerted action of one or more species of lactic bacteria with other bacteria and yeasts.
Koumiss (kumyss, kumiss, etc.) is a drink originally prepared from mare's milk by the Kumanes
@ Russian trive which lived along the river Kuma. The milk of, the mare was placed in leather bags
and a small amount of old koumiss added as a star-
ter. This beverage is strongly effervescent, due to the evolution of gas (carbon dioxide) produced
Curing the alcoholic fermentation by the yeasts present. Kefir (kephir, kepfir, kefyr, kefr, etc.),
the fermented milk of goats, sheep or cows wes Oo-
riginelly made end used by the inhabitents of the
Caucasus Mountains and wos prepared by adding "“ke-
fir grains" to the milk. The origin of these
grains is unknown but the natives believe that
they were the gift of Mahomet and are carefully
preserved by them. The natives prepared this drink by placing the dried kefir grains in milk in a goat
Bkin vag and vy shaking occasionally until fermen-
tation commenced, when the grains were removed and
dried, thus preserving them for future use. The
fermentation was also often sterted by adding some
of the freshly fermented kefir to a new lot of milk.
In kefir, as with koumiss, alcoholic fermentetion
with the accompanying effervescence is more desir-~ able than marked acid production. Four different
organisms were found in the Kefir grains, yeasts
and three different types of bacteria; the combined
action of all four seem to be necessary to the pro-
Guction of good kefir.
Leben or leben raib is the name given by
the Eeyptians to their fermented milk drink. This
oo,
~4~
beverage which they have used for centuries, is pre- pared from the milk of cows, buffaloes and goats.
The fermentation process is very similar to that of other fermented milk drinks. Yoghurt (yoghourt, yogurt, etc.) is a sim- ljlar fermented milk prepared by the Bulgarians.
This differs from the preceding beverages in that the acid fermentation is allowed to predominate.
It is made in skin bags in much the same way that the Russians prepare koumiss. Mazun (matzoon, matzun, madzoon, etc.) is an
Armenian drink almost identical with yoghurt.
Dadhi and khilat are fermented milks used in India, Calcutta and Bengal respectively, very similar to yoghurt, leben, etc.(17).*
Gioddu is a Sardinian fermented milk and is produced by micro@8rganisms similar to those found in the more studied fermented-milk beverages.(31).
The oxygala and chiston of Rome and Greece, the rayet of Algeria and the huslanka of the Ruth- enians are other known varieties of curdied milk drinks (17}( 44).
* Chatterjee notes that the type of fermented milk "“khilat" used in Bengal alone, has no prototype to his knowledge. "The production of the curd depends on the action of the products of a bacillus and not on the living bacillus itself****there are very few Bengalee householcs in which food prepared from this curd is not in daily use ond 2 large trade is cerried on in curd" (17).
There are two types of Tactic bvacteris which ore capable of producing the characteristic cos¢g- ulation of milk; these are for convenience often differentiated by the terms "short" and "long" lac-~
tic bacteria. The former is the type met with in
ordinary sour milk, comprising over ninety nine ner cent of its microflora. These ordinary lactic bacteria, Bacterium lactis acidi, sre very small, being from 0.6 micron to 1 micron long and O.5 micron wide, are oval in form and from the fact
that some of the shorter cells appear nearly spher-
ical and of ten oceur in chains, hes led to the ap- plication of the name Streptococcus lacticus to
the group.
The “long” lactic bacteria present in the
Eastern fermented milks, sare true lactic bacteria
also as they produce almost exclusively lactic
acid from the sugar fermented, only very small
quantities of formic, acteic and propionic acids
being formed. These differ sreatly from Bact.
lactis acidi in size, being large rod-shaped forms
~ microns to 3 microns long and 0.5 micron to 0.75
,
“~~? TT =a6G=
micron wide. The bacteria belonging to this latter group are known as Bacterium bulgaricum, socalled because first isolated and studied from Bulgarian soured milk.
Both types of organisms are alike in that they do not form spores and consequently are easi- ly killed by heat, have no organs of locomotion and form lactic acid from certain sugars as a main product of their metabolic activities. They dif- fer physically however in their respective temper- ature requirements, Bact. lactis acidi requiring a temperature between 30° and 35°C. for its @pti- mum growth while that for Bact. bulgaricum is from
40° to 45°C; they also differ physiologically, in their acid production, Bact. lactis acidi producing in normal milk a maximum acidity of from 0.9 to
1.25 percent only while with the long lactic bac- teria the maximum acidity varies from 1.25 to 4.0 percent.
It is this physiological property, that of acid formation, /which depends the importance of these lactic bacteria in their many phases of use- fulness. Milk does not lose any of its valuable properties by undergoing acid fermentation, in fact its benefits as a food are generally augmented a7 =
by the presence of the acid produced by the activity of the lactic bacteria. The longevity of the Bastern peoples is attributed by Metchnikoff to the use of acid milk as their staple article of diet. He offers this explanation of its action: lactic bacteria when introduced into the intestine transform its carbohye drate contents into lactic acid; this acts as an in- testinal antiseptic, destroying or inhibiting the putre- factive organisms (which work in an alkaline medi- um), thus preventing the formation and subsequent absorption of toxic substances which have a degen- erative influence on the body tissues.
To Metchnikoff is due the great credit for having insisted on the importance of introducing these lactic bacteria into the intestines for the purpose of controlling the type of fermantation therein. As a result of his teachings many lead- ing physicians today are recommending acid milk Ber- erages for use in intestinal therapy, and several large laboratories have been organized solely for the preparation and distribution of concentrated forms of starter for these curdled milk drinks.
Two of such are the “Pasteur Laboratories of Amer- ica" and the "Ferment Company" both located at New
York City. Other commercial firms put out start- ers for fermented milk or artificial buttermilk for therapeutic purposes. Such are the “Lactone" and
"Bacillus bulgaricus” tablets prepared by Parke
Davis and Company of Detroit. H. K. Mulford Com- pany of Philadelphia also have a similar preparat- ion on the market. Within the last few years a number of the fermented milk beverages themselves have also been placed on the market under such trade names as zoolak, vitalac, yogurt, bacillac, lactobvacilline, fermentlactyl, bulgarzoon, etc. The flora of these seems to be practically the same as that of the original yoghurt.
As has been previously mentioned, the lac- tic bacteria of the Bact. bulgaricum type are pre- sent in the Eastern fermented milks in which they dominate the fermentation. This property can be explained by the physical influence of temperature upon the metabolic activity of this group of micro- organisms (see p. 6), climatic conditions favoring the optimum growth of the long lactic bacteria rath- er than that of the Bact. lactis acidi type. This is a partial explanation also of the greater thera- peutic value of milk soured by the Bact. bulgari- cum type of lactic bacteria, body temperature offer- ing the optimum conditions for growth; the greater
=Q ua
amount of acid produced, adding materially to its beneficial action in intestinal therapy by acting as a strong antiseptic, completes the explanation.
After having hed the centenarians of Bul-~ garie and of other sour milk drinking countries held up a8 a living example of the efficiency of acid milk beverages as an intestinal sntiseptic, many investigators were led to carry on experi-= ments simulating natural conditions as far as pos-~
Bible for obtaining corroboration or refutation of
the theories advanced by Metchnikoff and his co- workers. Quite widely vurying results were 8s0 ob» tained. Helen Baldwin (2) studied the influence of lactic acid ferments upon intestinal putrefact=~ ion in a healthy man forty three years of age, feeding him upon lactobacilline malt, bacillac, zoolak and plain milk and concluded that in a mixed diet containing meat, the patient's condition was better when lactic acid ferments were not added to
the diet. She found however that when the main proteid portion of the diet was furnished by the milk, intestinal putrefaction was seemingly checked
to quite an extent during the use of the fermented milks but in only one experiment was the reoction
of the feces acid. Grekoff (30) to the contrary, found an acid reaction in the feces after the fourth or fifth day of treatment with fermented milk.
Boas and Oppler (11) and Kaufmann (42) found a Bact. bulgericum-like organism constantly pre-~ sent in carcinoma of the stomach but Straus and
Bialacour (77) found this organism in normal gas- tric fluid; Heinemann and Hefferan (35) also con- clude that it is normally present in small numbers in the digestive tract of many animals since it is so widely distributed in nature, they having found it in ordinary milk, feces of cows, horses and man, in soil, grains and pickles. The inference may be drawn from their work that since this organism is so widely distributed, it must be taken into the digestive system almost continuously, and if it were adapted to growth in the intestines it would become established there in the natural course of events. Rogers (65) states that an acid condition of the intestines is carefully guarded against by the provision for the neutralization of the acid juices of the stomach as they enter the intestine and he presumes that this action is automatic, i.e. en increasing acidity in the food coming into the intestine would be followed by a corresponding in- cre:sed flow of alkaline secretion. *** It seems allie
improbable that the acid contained in any form of fermented milk would be great enough to affect the activities of intestinal bacteria even along with the heavy inoculation of the acid forming organ- isms. However, Cohemdy (18) wes able to establish
Bact. bulgaricum ‘in the intestinal tract by long continued feeding. Grtnbaum (32), in discussing lactic acid therapy, describes the value of Bact. bulgaricum as not being due to its section as a phagocyte, i.e. ingesting harmful organisms, but rather that it produces lactic acid and further, forms it where it is of value, i.e., in the contents of the lower intestine which otherwise are subject to continued putrefaction. There are two advantages in the use of Bact. bulgaricum, first that its purity is readily determined by simple microscopic examin«t- ion and second thet it is a more active acid pro- ducer than the ordinary type of lactic bacteria.
In its use in therapy the patient should be given a carbohydrate diet, limiting the proteids and fat, at least four days before beginning treatment.*
* Cohendy(18) found that the disinfecting action of the lactic acid culture was not appreciably in-« fluenced by variations in the amount of sugar eaten, wlpea
indicating that the ordinary diet contains sufficient | suger to support the growth in the intestines of ' the lactic bacteria.
Grfnbaum in conclusion says that although many patients have been benefited by the use of these fermented milk drinks he is inclined to think that the ailment in many cases was only "maladie imaginaire“. This view is also held by Dr. Har- ley (33) of the University College of London; Rob- ert Hutchinson (41), Assistant Physician to the
London Hospital, is also skeptical of results.
However, most physicians who have given the lactic bacteria a thorough trial in intestinal therapy, heartily endorse their use. Herscnhnell
(37) sags, that with reasonable use, lactic bac- teria heave excellent therapeutical value for abnor- mal proteid putrefaction in the intestine or for some forms of constipation. Fresh (24 hours old) cultures of lactic bacteria, preferably Bact. bul- garicum, should be used for colonization in the intestines, about two and one half months being necessary to obtain a complete transformation of the intestinal flora. -13 of patients dieting on sour milk preparations; in health, when B. coli ( a common intestinal organ- ism) predominates, the intestinal flora are Gram- negative (37). Previous to abdominal operations he advises rendering the sastro-intestinal canal aseptic by feeding the patient fresh liquid cul- tures of the "Bulgarian" bacterium (38). Herter and Kendall (39) investigated the fate of Bact. bulgaricum in the digestive tract of a monkey and found that an acid reaction could be maintained in all but the Lleoececal region or large intestine. A frozen milk preparation "Lacto" contain- ing lactic bacteria of the Bact. lactis acidi type has been concocted by Mortensen and Gordon of the Iowa Agricultural Experiment Station (54). The purpose of this product has been to supply the lactic bacteria in an attractive form to people to whom loppered milk or buttermilk is objectionable. Lacto is made from loppered whole or skim milk with the addition of eggs, sugar, lemons and flav- oring material; it has ao more pleasing flavor than sherbets and ices and contains considerably more nutriment, its protein content equsling that of ice cream but containing less fat and more acid; ~l4@ the main argument however, is that the lactic or- ganisms in this frozen seur milk preparation remain alive for some time after the milk is frozen thus allowing these bacteria to be supplied in large numbers to replace the putrefactive forms present in the intestinal contents. The largest percentage of persons who sampled lacto preferred it to vanil- la ice cream or sherbet; 30 it seems that if this type of lactic bacteria ss. as beheficial as the long lactic type, this frozen |jsour milk product may become a wide spread benefit, especially so in the Summer months when gastrosintestinal diseases are predominant. Many physicians, Hutchinson, Harley, Grfinbauwm, Klotz, etc., are skeptical as to the therapeutic value of lactic acid preparations but the greater number who have employed them in one form or anoth- er are convinced of their utility in intestinal and other forms of therapy. Dr. J. W. H. Byre (25) successfully treated pathdgenic conditions of the colon with his pure culture of lactic bacteria. Hertz (40) and Grtinbaum (32) treated colitis sucessfully. Dunn (24), upon feeding children suf< fering with intestinal catarrhn, upon pasteurized 2a. aw ———-- a buttermilk inoculated with a known species af Jac- tic bacteria caused the flora and the character of the stools also to change to normal, the toxic symp- toms passed away and the children gained in weight; practically the same effect as is produced by yog- hurt. Allen (1) effected a cure of South African typhus fever and dysentery by the use of lactic acid bacteria, Cummins, Fawcus and Kennedy (21) on the contrary, found no cessation of the excreting of B. typhosus in the feces nor in the urine by treating with Bact. bulgaricum. Other pathological conditions have been successfully treated with liquid cultures of lactic bacteria. Dr. W. Langdon Brown (15) successfully applied this form of therapy to indicanuria. Grfin- baum (32) treated uremia and diabetes mellitus and found that the lactic acid is excreted as a carbon- ate which tends to increase the alkalinity of the blood and to defer acidosis; the lactic bacteria by changing the carbohydrates in the food into lactic acid, tend to diminish gitgcemia. Sahli (72) has had good results from the use of lactic acid pre- parations in some cases in conditions of anacidosis in diabetes also and in pernicious anemia but says that the presence of Bact. bulgaricum is not essen- ~l6u tial as the same benefits were derived in most cas- es from using the acid alone. Liquid cultures of lactic bacteria have been used also as a spray or wash for the disinfect- ion of the mucous membrane of the nasal, oral and genito-urinary cavities. Dr. Charles BE. North (55) (56) of New York City was the first in the United States to use suspensions of living lactic bacteria for the disinfection of bvody cavities. Many cases, chronic rhinitis, ethmoiditis, frontal sinusitis, otitis, antritis, tuberculous sinuses, gonorrhea, cystitis, leucorrhea, diarrhoea, acute and chronic suppurating wounds and sinuses, inflammation of the maxillary bones and palate, gonorrheal ophthalmia and conjunctivitis, numbering three hundred in all, were treated with injections of dextrose broth cul- tures of Bact. bulgaricum. Of these, sixty three were permanently cured, one hundred sixty two showed marked improvement while with only eighty six was there no result from the treatment. Dr. North con- cluded by stating that the use of these cultures as a wash or spray on inflamed surfaces or cavitiés often diminishes odor caused by putrefaction; that both acute and chronic inflammations caused by in- fections sometimes appear to be checked by inject- -17~ ing these cultures at the seat of infection; and that the use of the cultures seems to be accompanied by no special danger and that they cause no irrita- tion. The extensive work and favorable conclusions of North suggested to Giltner (29) the possibility of the therapeutic use of lactic acid cultures in treating the purulent metritis following abortion in cattle. Whey cultures of Bact. bulgaricum were injected into the uterus and vagina after abortion with the result that the discharges soon ceased and portions of the retained placenta passed off without any decomposition. No evidence has been noted of sterility having been produced by the use of this method, in fact, it is being used to combat steril- ity or failure to breed in cattle with encouraging results. In three year's application, no instance of failure to restore a case of utero-vaginal trouble to an apparently normal condition has been recorded, @ large number of cases indeed indicating the value of this treatment in assisting toward the restor- ation of fertility to sterile cattle. Since Dr. North's work in 1908 - 1909, oth- er investigators have taken up the lactic acid treatment of mucous membrane infections, especially in connection with the genito-urinary tract. Dr. Persson (62) of Mount Clemens treated thirty four cases of chronic specific urethritis with the fol- lowing conclusions: secretions in the infected ur- ethra inhibited common lactic acid organisms within 15 minutes but when the lactic organism was grown on nucleic acid media the organisms could be recove ered in an active state after 10 hours sojourn in the urethral cavity. The disappearance of the gono- coccus bore a constant relation to the length of time that the lactic bacteria retained their “virulence" in the urethra. Dr. F. S. Mason (47) in his article on the ‘Application of Lactic Ferments tn Genito-Urinary Work' finds that the lactic ferments inhibit the growth of the pathogenic organisms in the genito- urinal passages. Dr. Watson (78) treated gonorrheal and mixed infections of the female genital tract with a fil- trate of sour milk containing large numbers of lac- tic bacteria. This liquid culture was introduced into the vagina after a thorough disinfection and a subsequent washing of the infected parts with sterile water. This treatment caused a stimulation of the secretions which gradually changed to normal in ete stihl ~l19u character, then the amount of secretion also dimin: ished to normal. This type of infection seems to respond encouragingly and with rapidity to this form of treatment. The use of liquid cultures of | lactic bacteria in gonorrheal infections seems to have a most promising future. Affections of the nose, ear and throat have been successfully treated also to a large extent by liquid cultures of these bacteria. North's work stimulated investigations of the therapeutic value of these cultures as applied to the respiratory passages. Dr. H. Holbrook Curtis (22) of New York City began experiments along this line with a cer- tain amount of skepticism but obtained such markedly good results in certain cases that he concluded that a very valuable advance had been made in the treatment of pathogenic conditions of the nose and accessory cavities. He examined the secretions from the nasal fossae before the lactic bacteria were introduced and again after successive appli- cations of the culture. In some cases after a limit- ed number of injections, not only were the pyogenic bacteria destroyed but there was a total absence of all bacteria in the secretions. In an acute case of frontal sinusitis of over a year's duration, the pro- =20— fuse discharge was caused to diminish ninety per cent after the second dose of a cubic centimeter of the culture, Other types of cases were cured or greatly relieved by this treatment. Dr. Curtis says “Should future experimentation substantiate the evidence of results obtained thus far, it would seem that a new era in the treatment of pathogenic bone cavities has been instituted, and that the theories of Metchni- koff admit of a anch wider application than he has claimed. Berthelot (7) used the lactic culture suc- cessfully on three cases of “Nasenrachenraum" and freed a nine year old child of adenoids and "Fieber-~ anfallen". In another publication (8) he tried the action of Bact. bulgaricum on the meningococcus in vitro and found that the destruction of the patho- gen followed in a short time the strong acid pro- duction of the former. This lactic culture was to be used as a nose and throat spray for annihilating the meningococcus from "carriers". Practical ex- periments had not been carried out at this time. Dr. Francis Bond (12) used a whey culture of Bact. bulgaricum as a throat BpraAy with fair success in the treatment for Bact. diphtheriae. Gilpatrick (28) however says that although the pure culture of Bact. bulgaricum is sometimes ae)» of benefit as a lecal antiseptic it must ve |used with caution, especially in the oral cavity because the action of the lactic acid on the enamel of the teeth may give rise to caries, In Pyle's Bersonal Hygiene (63) this statement has been fefuted by investigations carried on by Black of Indiana who subjected a large number of extracted teeth to a great variety of tests including the action of chemicals and the attacks of bacteria and found that strong and feeble teeth were affected alike. Miller (52) sides with Gilpatrick in believ~ ing that caries is the result of lactic acid proe duced by certain bacteria; Hopkins (63) of Harvard has shown that of the one fifth of the fifty varie eties of bacteria in diseased teeth which produce lactic acid, none formed over 0.5 per cent. He then subjected good and poor teeth alike to the action of 0.5 per cent lactic acid for four months wbthout any carious action taking place , and stated further that caries is often accompanied by a free secretion of saliva, i.e., that the mouth is alkaline. Other miscellaneous conditions of widely different nature have been benefited by treatment with lactic bacteria. Dr. C. H. Miller (52) treat- mooe ed rheumatoid arthritis with but little benefit to the arthritis, the patient's health was greatly im- proved however. McCarrison (48) in treating endemic goiter stated that the administration of Bact. bulgaricum as an intestinal antiseptic had a favorable healing | action when used when goiter was just beginning. McLaughlin used a powder prepared from s&ter~ ilized and dried milk solids inoculated with Bact. bulgaricum for dusting into suppurative wounds such as carcinoma, chancroid, purulent ulcers and septic wounds with good results (49), Giltner (29) had equally good results in packing large abscesses in cattle with sterile cote. ton soaked in a lactic culture; such abscesses were healed completely in a few days, characterized by the absence of discharge. [In all cases the healing was rapid and complete, Much work has been done upon the effects of Bact. bulgaricum and Bact. lactis acidi upon differ- ent organisms in vitro. As early as 19035, Bonska (13) determined that there is a marked antagonism between bacteria of the B. subtilis group (some of which are pathogenic) and lactic bacteria, the lat- ter showing their specific energy in media whether —ee = a --. -. \ . yf” eee sugar-containing or not. Tne proliferation of the subtilis bacteria soon ceases and their number rap- | idly diminishes. Bonska does not attribute this phen- omenon to the accumulation of lactic acid alone as this diminution in numbers occurs in eugar-free or in strongly alkaline solutions. These results taken generally are contrary to those obtained by other investigators, who claim that the antiseptic power of lactic cultures is due alone to the sacid pro- duced. Crithari (23) in studying the symbiosis of Bact. bulgericum with B. butyricus (non-pathogenic) determined that when the acidity of the medium was maintained at a constyunt amount that the phenomenon of fermentation was reduced to a minimum. Very Similar are the results obtained With pathogenic and lactic organisms in vitro, by other scientists. T. von Kern (43) attributes the inhibitive action of Bact. bulgaricum upon B. coli to the nas- cent lactic.acid. In the intestine this is followed by a diminution of undesirable intestinal products. He advises the use of “lactobacilline" to prevent the accumulation of these products. G. Rosenthal (67)(68)(69)(70)(71) has car- ried out extensive experiments on the associative —— an +e action of Bact. bulgaricum upon a number of organ- isms, B. perfringens, the gas-bacillus found in lung gangrene and ear-disease; B. pyocyaneus, the bacillus of blue or green pus; "l'anhemobacille enterocogene du rhumatisme"; B. diphtheriae and bacteria of the B. typhosus group with almost uni- form conclusions, that the destruction of the path- ogen is soon effected by the introduction of Bact. bulgaricum into the culture, even if the former has oeen allowed to gain a start. The delay in the destruction is inversely proportional to the quantity of lactic culture superadded and if the reaction of the medium is carefully kept neutral, hoth organisms grow. In the case of B. typhosus he concludes that the acid is essential to and per- haps the only cause for the death of the pathogen. In my own investigations (57) on the influ- ence of the products of lactic organisms upon B. typhosus, the results obtained led to the conclusion that, of equivalent amounts of acid formed by Bact. lactis acidi and Bact. bulgaricum respectively, the acid produced by the former was twice as ef- ficient in antiseptic properties as that of the Kni¢ch sold producing organism. Whether these same results and conclusions would be derived from a SS er 9 » See + wom parallel set of experiments with other strains of the different organisms is a matter of question. The most marked results were obtained with another high acid producing lactic bacterium. This par- ticular organism resembles Bact. bulgaricum in nearly all respects, but produces acid in larger ymounts and more quickly at room temperature, has a much greater vitality and resistance to its own products and produces a color reaction in litmus milk culture similar to that produced by the or- dinary sour milk bacteria. This bacterium, at 60° acid (Fuller's scale), caused the destruction of the typhoid organism within 24 hours and at 72° acid within 2 hours. The acid produced by this lactic organism however was not as efficient in small quantities as that of Bact. lactis acidi. Rodenwaldt (64) in his paper given at the German Tropical Medicine Convention in Hamburg, April, 1912, said that in vitro, Bact. bulgaricum inhibits B. typhosus and B. dysenteriae principal- ly in the first twelve hours of their development, the typhoid bacilli still growing in culture after 6 hours growth with Bact. bulgaricun. rrom these many examples of the influence in vitro and in vivo of the different types of lactic Cae aCe... @26- orsanisms, it is evident that these bacteria already have an extensive use in therapy and from the prom- ising results obtained thus far in the various cases, it is not altogether presumptuous to predict a much wider range in this phase of their useful- ness. From the type of references just cited, one might be led perhaps to entertain the erroneous i- dea that the lactic bacteria find their greatest utility in therapy. A much older and more firmly established application however is the use of these organisms for controlling the type of fermentation in the butter-making industry. Lactic bacteria of the Bact. lactis acidi type are employed almost ex- clusively for this purpose. A quarter of a century ago the use of pure culture starters for butter-making was unknown. The buttermaker prior to this time possessed lit- tle or no control over the growth of the organisms that produce cream ripening. The cream that was received from a wide territory by the creameries necessarily contained a numerous flora both as to species and number, and if it chanced that the proper species predominated in such mixed crean, the ripening would go on in a normal fashion and a — —_—_— 227 the results obteines would be satisfactory. If it chanced however that the cream brought to the crean- ery contsined in great numbers, species of bacteris which were unfsevorable to good flavor production, the butter would be likely to be poor in quality and no care which the buttermaker could take in the subsequent preparation of the butter could remedy this particular defect, and consequent financial losses would ensue. (19). The revolution which had been produced in the brewing industry by the use of pure yesst cul- tures led to the suggestion that a similar control might be possible in cream ripening by the intro- duction into the cream of the organisms responsible for the ripening process. It was Professor Storch (76) of Copenhagen who first (about 1890) conceived tne idea of obtaining from cream those species of bacteria which produce the best results and then furnishing them to the buttermeker for the purpose of ripening the cresm, somewhat as yeast is fure- nished to the brewer. This plan as suggested vy Professor Storch was put into practical use in Denmark, and later in other countries in northern Europe. In one sense this plan was not new. For many years pre- f= a vious, buttermakers had added what they called a "starter" to hasten the ripening of the cream in winter weather. A: starter was simply a lot of good-flavored already-soured milk or cream contain- ing of course the lactic bacteria in large numbers in an actively growing stute. The new feature of the method suggested vy Storch was the artificial preparation of such a starter by the use of pure bacterial cultures obtained by bacteriological methods. This method soon distributed itself more or less in the various butter-making countries. Nowhere however has this method been used to a very great extent except in the countries of Northern Europe and anly then at times when the butter of a creamery shows an off flavor or aroma as a result of improper ripening of the cream. The analogy may be drawn between this off-fermentation in cream and butter and disease, the lactic bacteria added in pure culture acting as a preventive of disease largely through the agency of the lactic acid pro- duced. This illustration holds true for all phas- es of the usefulness of these micro68rganisms. In the use of Professor Storch's method of cream ripening the pure cultures of lactic organ- isms can be obtained only by bacteriological methods. =O Gu To obtain such a culture bacteriologists have nature ally had to select the type of bacteria found most often in ripened cream which produced a high grade butter. There is no difficulty in obtaining from such cream tie lactic bacteria that are present in the greatest abundance as they form over ninety nine per cent of the flora. The problem of a proper culture for cream ripening is quite different in different countries Since the public taste for butter is so dissimilar; no type of ripening which is satisfactory in one country will meet the demands of another neccssar- ily. The cemand in the United States fs for a highly flavored butter while Denmark and the rest of North- ern Europe prefer a very mild flavored product. By a happy provision of nature, many quite differ- ent flavors are produced by the different varieties of lactic bacteria so that these varied demands may be wet without difficulty. The lactic bacteria as obtained either dir- ectly by isolation or in a powdered or liquid form as furnished by commercial firms are not added di- rectly to the cream to be ripened, for although the organisms in thése commercial or newly isolated cultures amount to many billions, still the number is —— jee on ee” Ce "OC 7 =r =-30~ qguite insufficient to inoculate properly a large quantity of cream. The culture must of necessity be "built up", i.e., the number of lactic bacteria must be greatly increased. This is done simply by adding the small emount of culture to a quart or so of cream or milk which has been heated previous-~ ly to the temperature of boiling, covering such inoculated cream or milk to keep out the dust and placing it where it is moderately wurm for a period of 48 hours. During this time the bacterie increase enormously in numbers, thoroughly impregnating the railk or cream, This large amount of culture is then added to several gallons of pasteurized milk or creem, stirred thoroughly, covered and set aside for the further development of the starter orgen- isms. In the course of two days the "starter" is ready for inoculeting the body of the cream which is to be ripened. Altnough this process is simple, it is some- what troublesome ond for this reason the wider ex- tension of the use of pure cultures in butteremak~ ing has been prevented. Several commercisl firms heve attempted to put on the market s form of pure culture that could be added directly to the cream without previous cultivation. The tests of such —_— ae re 8 re C- cultures however heve not been satisfactory; the bacteria are not present in sufficient abundance to accomplish the best results. This is true of all comumercigl pure cultures of lactic bacteria of eith- er type; the organisms succumb either to the dry- ing as in preperations in powdered or in tablet form, or to the antiseptic influence of their own products, lactic acid mainly, in the liguid form. At the present time there is no reason for believing that such pure cultures for direct use are of any practice#l value and the use of pure cultures in buttermaking still depends upon the "building- up" process. The starter prepared by the building=up process is added to the cream to be ripened in the proportion of about 10 per cent. After the cresm has ripened and is ready for churning, a few gal- lons are removed to be used as a starter for the next day's churning. In this way the starters sre carried from day to day for several days. About once a week however, it is commonly found desir- able to build up 3 new starter from the commercial pure culture, The more common method of utilizing the lactic bacteria for buttermaking is vy the'natural' ow On bun starter prepared by the buttermaker himself without the necessity of purchasing a commercial product. This natural starter is nothing more than ae lot of good-flavored, naturally-soured milk or cream and was used for many yeers previous to Storch's methe ocd. By the use of this method the dairyman hes no method of knowing what hacterila sre present or of how to control them. Logically then and theo-« retically too, the method of the use of natural starters is incorrect end practical experience a- mong dairymen goes to show that although natural starters are as a rule, quite successful and pros« duce a satisfactory ripening of the cream, they are not uniform and occasionally are not reliable. Even though produced under similar conditions they do not always give rise to the tyre of ripening that is desired. The fact however, that such ir- regularities seldom occur, in contrast with the trouble and attention necessary to prepare a pure culture starter from commercial products has led to the use of the natural starter almost exclusive-~ ly vy dairymen who wish to make o high grade of butter. Experimental tests of the value of butter os Oe ee - made with and without pure cultures have shown that the best quality of butter made with pure cultures is apparently not quite equal to the best quality of butter meade by spontaneous ripening. Thses exceptionally fine grades of butter however, are not the ordinary yield but have been made in an experiment station or unon an individual farm where special cere has been taken to produce cream in the best condition, Under these circumstances 4 pure culture moy be of no advantage, but in general dairying and especially in cregmeries, 911 sorts of cream both good and bad are accepted. Here, pure culture starters should always be used, and while these cultures cannot correct entirely the imperfections resulting from the miscellaneous organisms in the mixed cream, they act as an effective check upon the development of a heterogeneous microflora and their use is thus clearly of benefit. The use of pure culture starters kas an ad- vantage along three distinct lines: the buttermaker can handle his cream more uniformly, being able to regulate the ripening, the grade of ripeness and the amount of starter to add to a certain amount of cream; the product posses 3 greater uniformity, -d4ua Fluctuations in quality due to 6 miscellaneous flora or to seasonal variations tend to dissppear. This seems to be the most distinct edvantsege of the pure culture method, being the one which gradually brought about the introduction of this method of buttermaking throughout northern Europe. Ag to the third advantage, on the average, the flavor of butter is somewhat improved by such cultures although it is difficult to obtain any proof of this owing to the uncertainty in greding flavors. Markets that demand a clean, mild-acid-flavored butter prefer that made from pure culture starters. It is not essential that the butter fat be present during the acid |fermentetion in order to impart flavor to the butter. Sweet cream may be mixed with sour cream or milk and churned at once, the flevoring compounds being absorbed from the soured milk by the fat, and the butter will have much the same flavor both as to intensity and kind a8 though the fat had been present during the fer- mentation. This method is identical with those employed by the manufacturers of oleomargarine and renovated butter. The flavor of these butter sub.» stitutes is identical with and derived from the same source as the flavor of butter, “35 — The question may now be asked, what becomes of the immense number of lactic bacteria necessarily introduced into the butter in the process of churn- ing? and do they play any further part in the bute ter after it is resdy for consumption? A large part of the bacteria are removed from the butter with the buttermilk, also by the ywober in thé processes of washing and working the butter, but the completed product still contains them in great numbers. Freshly made butter contains millions of bacteria per gram, but es they do not find favorable conditions for growth on account of the compactness of the butter and the comparative- ly small amount of moisture and the lack of oxygen penetration, they begin to die qtickly. Conn gives the figures for the decline of bacteria in a same ple of butter, starting out with 50,000,000 bacteriz. per gram the nunber is reduced 50 per cent in one day, down to 4 per cent of the original number in four days and 0.6 per cent inside of thirty days. This may be looked upon as the normal history of tha bacteria contained in butter. The salting of butter has some influence upon checking the growth of lactic bacteris, and other microSrganisms contained therein and thus, — Eee - ee <————" A oe -J36< within certsin limits, hos a preservative action. The lactic acid contained in the butter, contrary to what might be supposed, is not present in sufficient quantity to serve as an important fac- tor in its keeping quality, the total acid in fresh butter titrating from 1.5 per cent to 2.8 per cent (73). In fact, Rogers, Thompson and Keithley (66) state that butter of as much superior keeping qual« ity was almost invariably produced by limiting the acidity to the region of 0.3 per cent and below. The presence of larger quantities seems to sid mater= ially in the decomposition of the butter proteins and fat, resulting in aff flevor and rancidity, i.e€., butter made from overripened cream, which is high in acidity, possesses much poorer keeping qualities than butter from properly ripened cream, However, Hastings (46) says that sweet cream butter is morked by the rapidity with which it undergoes decomposition chenges, especially when it is made from raw creem, and atates that as a rule, sour- cream butter has much better keeping qualities than those of sweet cream butter. Lectic bacteria of the Bact. bulgaricum type have been used instead of the ordinary Bact. lactis acidi as a starter for cream ripening and buttermak- - ~ re” _. i ing with good results es to flavor and aroma of the finished product. The dairymen finds difficulty however in maintaining this starter at a, suffici-~- ently high temperature to insure the curdling of the milk within 18 to 24 hours; es 2 consequence, this type of lactic organism seems to hseve no prospect of being utilized in this way on a large scale. Cheese is another of the important cairy products in whose manufacture the lactic bacteria play an importeunt part. In the process of making cheese it is necessary to curdle the milk, that the cheese constituents may be separated from the whey and soluble elements of the milk. Two methods are employed to accomplish this purpose, and as a result two types of cheeses are produced, tne “acid- curd" and the "rennet-curd" cheeses, in both of which the lactic acid forming organisms are neces- sary for producing the principal flavor. In the acid-curd cheeses, the curdling is accomplished by allowing either spontaneous acid formation through the agency of the normal flora of the milk, or by the addition of a pure lactic culture os a starter. These cheeses which are eat- en fresh, owe their flavor to the products of the acid fermentation, especially lactic acid. — -- en 8 - In the preparation of the second type of cheese, rennet is made use of for curdling the milk. Rennet-curd cheeses undergo a process of ripening, in contrast to the sacid-curd cheeses and if harm- ful forms of micro§rgeanisms are present in the milk they will pass into the cheese and there produce their harmful effects. If the milk is inoculated with » pure culture of lactic bacteria before the addition of rennet, the proportion of desirable Organisms can be increased and a partiel control of the fermentation thus secured. The proper ripening of rennet-curd cheeses can only be accomplished in the presence of a con- siderable number of lactic bacteris. In the eorli- er process of cheese-making the curd is cut into small cubes which enclose 95 to 99 percent of the bacteria in the milk. The development of the acid in the curd is rapid, due to tke concentration of large numbers of bacteria in a small volume and to the favorable environment. This elaboration of «cid in the curd through the agency of the lactic baec- terise is an important step in the process of mak- ing cheddar cheese as it brings about the condi- tion known as “matting". The curd particles at e first show little tendency to cohere, but as the _ we eee ———"— ae acidity increases, the nature of the curd changes and this “matting or fusion of the curd particles takes place. The nature of this change though not well understood, is probably due to a combination between the peracasein and the lectic acid, the’ resulting compounds differing from the parsacasein in physical properties and solubilities. In regard to the further reletion of the lactic bacteria to cheese, i.e., in the ripening process, de Freudenreich, se Swiss microbiologist, ascribed an important role to the acid-forming bacteria in the ripening of Swiss cheese for the reason that great numbers were present in fresh cheese, and when cheese was made from milk drawn under aseptic conditions, no ripening Occurred; also, that lactic bacteria of the Bact. bulgaricum type predominating in Swiss cheese have a solvent effect on the casein of milk. It is generally conceded that acid forming bacteria are essential factors in the ripening of cheddar and probably all kinds of rennet-curd chees- es. At this point it is pertinent to mention the possibilities of dairy products as carriers of infectious diseases. This would naturally raise ~40- the question as to whether the lactic bacteria which occupy such a pertinent place in therapy vill not exercise the same functions in this con- nection also. As naturally soured milk is not commonly used in this country as a food or bever- age, the chances for diseases like typhoid fever, diphtheria, scarlet fever or tuberculosis being transmitted by this means is very slight. Sour milk and cream products however, such as buttermilk, cottage cheese, milk and cream cheeses and butter hold an important place in the diet of most people in the United States and should a type of pathogenic organism find its way into one of these products, the question arises immediate- ly ‘how long will this organism retain its vital- ity and virulence under the conditions existing in ‘this product?! Obermttller (58)(59) found that both the tubercle and the typhoid bacillus are capable of living for many days in butter and therefore may possibly be a source of these two diseases in the consumer. Various investigators have found the limit of life of the tubercle bacilli in butter to vary from 12 days to 8 months, and although both certainly cannot be right as the difference in the ee ———— oA} « respective limits of time is too great, one or the other must approach accuracy. Most investigations show that the organisms of tuberculosis are not killed in ordinary salted butter in four to five months even though the butter has become rancid and moldy and unfit for human consumption.(74). If we take the figures of European investigators we may conclude that butter is more frequently in- fected with tubvercle bacilli than milk, in fact,. from the individual investigations of Herr (36), Cornet (20) and Bro@érs (14) tubercle bacilli could be found thirteen times in butter for every ten times in milk. It is a simplé matter to destroy tubercle bacilli in milk and cream by pasteurization, but just how to make infected butter safe is a more difficult problem. As it is a well known fact that these organisms are unaffected in milk or cream by the presence of the acid produced by active lactic acid producing bacteria, the only safe-guard left is pasteurization. Butter apparently contains no- thing except salt which has an inhibiting influ- ence upon the vitality and virulence of the organ- jams of tuberculosis, but the germicidal value of salt in the proportion in which it is used in com« - - —~ —_ mercial butter is very low (74). As to the effects of the products of lactic bacteria upon the organisms of typhoid fever when contained in butter, Barthel (3) states that in but- ter which is strongly acid, typhoid bacteria may be found after ten days, the strong brine enclosed in such butter being a good nourishing medium. On the other hand, Bassenge (5) has attempted to show that during the process of cream ripening, the lac- tic acid produced by fermentation organisms serves to diminish the number of typhoid bacteria present in the cream, and after the cream is thoroughly ripened and churned, the butter after being worked will contain no living typhoid organisms. Most investigators agree that the lactic fermentation in milk has a deleterious effect upon the vitality and longevity of the typhoid bacillus and when the acidity in milk or its products has reached 0.4 per cent the typhoid bacilli are killed within 24 hours (5)(6) although Fr&nkel and Kister (27) found them living even after 48 hours. How- ever if one wishes to insure the safety of butter for human consumption, the efficient pasteurization of mixed lots of cream before being manufactured into butter, or of the butter after having been ~43- made from such cream, should be accomplished. Cheese made from raw unpasteurized milk Should also be considered as a possible carrier of infection, is the conclusion drawn from several experiments cited by Mohler, Washburn and Doane (53). Harrison (34) also recommends the pasteurization of the milk in order to make cheese absolutely safe. I believe that the conclusions in my pub- lication on the "Influence of the Products of Lac- tic Organisms upon Bacillus Typhosus® may be applied to all forms of dairy products: the acid (and oth- er products) formed by lactic bacteria is efficient a8 an antiseptic in amounts of 0.7 per cent and a- bove in pure culture, but if certain species of micro8§rganisms are present in milk or its products they may either by checking the production of acid or perhaps by living in symbiosis with the patho - gen allow of its existence and proliferation there- in. While butter and cheese allow of comparatively little multiplication of pathogenic and other bac- teria on account of their physical nature, still, conditions exist which permit of the continuance of these organisms in a virulent state for surpris- ingly long periods of time; the many factors enter- ing under natural conditions prevent any definite - — OW E_Ce ~44- conclusions being deducted wnen these natural condi- tions exist. Bacteria of the two lactic acid producing types are responsible for the principal fermenta- tion and flavor in other food substances besides in milk and its products. The best known of these finished food products are sauerkraut and dill pickles. In general practice these are produced Ay spontaneous fermentation. Although many differ- ent species of bacteria of an indifferent or harm- ful type are initially present in the food sub- stance, when thé process is successful the lactic bacteria multiply rapidly from the first and quick- ly produce enough acid to restrain growth of the micro8rganisms which would otherwise injure the final product. The Russians produce "“barszcz" composed of red beets which have undergone a lactic fermentation. Other vegetables such as beans, string beans, corn, asparagus, etc., sourec 3pontaneously vy lactic bacterla are popular articles of diet in some coun- tries. Fresh meat is often submerged in sour or buttermilk for keeping it from putrefactive changes for a short time; its flavor is improved consicer- y a45 = ably by this treatment. Sour dough bread is a German product made from rye flour, and although the fermantation is not always the same, bacteria of both the Bact. lactis acidi and the Bact. bulgaricum type are re- sponsible for its high acid content. A certain amount of lactic fermentation is sought in the preparation of some beers. This is true in the so-called ginger beer prepared from gin- - ger, sugar and water inoculated with “ginger beer plant" an impure culture of yeasts and lactic bac« teria preserved in much the same way as are grains of kefir for the fermentation of milk. Formerly,a lactic acid fermentation consti- tuted an early step in the preparation of tomatoes for ketchup. Ensilage, composed of shredded corn, alfal- fa, sorghum, beets or other similar materials, un- dergoes a lactic fermentation and owes its preserv- ation largely to the presence of the acid. Still another phase of the usefulness of lactic bacteria has been determined by Barthel and Rhodin (4). Their experiments have ascertained @ the utility of pure cultures of lactic bacteria for the conservation of the fertilizing efficiency * ~A6< of manure. They added from fifty to one hundred liters of whey to one thousand kilograms of manure and found that the loss of ammonia was greatly re- duced thereby; the untreated manure possessed 3 fertilizing efficiency only 59 per cent as great as the treated manure. This addition of whey fur-~ nished the culture medium for the lactic organisms which quickly taking advantage of the favorable conditions, began active acid production, thus ef- fectively checking the over-growth of the organisms of the putrefactive type which other-wise would soon have broken up the complex proteins largely in- to their volatile gaseous constituents, NHs, HS, PH3, COg, Hg0, N, etc. Thus it is evident that the lactic bacteria of both types have not only a wide distribution in nature but have a number of diverse and important uses each of which covers a wide field in itself, the action in each case seemingly dependent mainly on the physiological property of acid production. These different phases of the utility of the lactic organisms show no signs of undergoing a decline and bid fair to retain their plece in the order of things although some are but fads at the present time, It is also not impossible that new phases — will be discovered ere long. Nature has placed everything here for a purpose; sometimes this is very evident, many times however, the purpose and application have to be attained either by a process of reasoning or by accident. When man thoroughly understands the intricate workings of the lowest forms of life he will have made large strides to- wards attaining the millenium, oA. BIBLIOGRAPHY. Allen, R. W. Observations on Lactic Bacilli. British Medical Journal. Nov. 28, 1908. ae Baldwin, Helen. Observations on the Influence of Lactic Acid Ferments upon Intestinal Putrefaction in a Healthy Individual. Journal of Biological Chemistry, Vol. 7, 1909. Barthel, C. Bakteriologie des Meiereiwesens, Barthel, C. and Rhodin, S. A Biological Meth- od for Preserving Manure. Deut. Landw. Presse. Bd. 39, 1912. Bassenge, R. Das Verhalten der Typhusbacillen der Milch und deren Produkten. Deutsche Med. Wochenschr. Bd. 29, 1903. Behla, Handbuch d. Techn. Mykologie, Lafar, Bd. Ze Ber thelot,A. 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