'URTHER OBSERVATIONS ON THE METABOLIC REQUIREMENTSOF LACTOBACILLUS BIFIDUS VAR. PENNSYLVANICUS1

PAUL GYORGY AND CATHARINE S. ROSE

Department of Pediatrics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Received for publication September 3, 1954

During the past few years a large number ofstrains of Lactobacillus bifidus var. pennsylvanicuahave been isolated in our laboratory from thestools of breast-fed and bottle-fed infants(Gy6rgy, 1953) and from the vaginal secretionsof pregnant women (Harrison et al., 1953). Thisspecific variant showed only scant or undetectablegrowth in the regular semisynthetic medium.However, it could easily be propagated in thebasal medium after the addition of human milkor of nitrogen containing oligo- and polysac-sacharide fractions isolated from human milk(Gauhe et al., 1954), of gastric mucin (Tomarelliet al., 1954), or of other sources of such nitrogencontaining carbohydrates.The original basal medium contained in addi-

tion to chemically well defined constituents, suchas amino acids, purines, vitamins, and salts, anenzymatic digest of casein. When acid hydrolyzedcasein was substituted for the enzymatic caseindigest, growth in the presence of human milk wasmuch reduced. Furthermore, such sources of the"bifidus factor" as purified fractions from humanmilk and gastric mucin, highly active as supple-ments to the original medium, were not able tosupport growth of strains of L. bifidus var.pensylvanicus in the medium containing acidhydrolyzate of casein.The present report deals with the analysis of

these observations and with the continued studyof the metabolic requirements of L. bifidus var.pennsylvanicu under various experimental con-ditions. Particular attention was paid to the be-havior of substrains within the specific group ofL. bifidus var. penneylvanicu as well as to theresponse of the variant to supplements autoclavedwith the medium or added to it aseptically.

MATERIALS AD METHODS

Microbiological assays were carried out aspreviously described (Gyorgy et al., 1954). The

I Supported by a grant of Wyeth Laboratories,Division of American Home Products Corpora-tion.

basal medium (Norris et al., 1950; Hassinen et al.,1951) was used either with an enzymatic digest ofcasein ("NZ case peptone", Sheffield) or with anacid hydrolyzed casein (G.B.I., "vitamin-free")in equal concentration of 2.5 per cent. The originalmedium contained 400 Ag of calcium panto-thenate per liter. This was omitted in later experi-ments. The pantethine content of the mediumcontaining enzymatically digested casein wasadequate for maximum growth of the organisms;the medium containing acid hydrolyzed caseinwas supplemented with 50 ,ug of pantethine perliter.Growth of the organisms was estimated by

titrimetric assay of the acid produced. Thespecific growth promoting activity for L. bifidu8var. penneylvanicu was expressed in microbio-logical units related to a particular sample ofhuman milk as reference standard.Two strains of L. bifidus var. penneylvanius

were studied: the original strain (212A) and oneof its mutants (5S). Each of these was representa-tive of several other strains. A strain (105C) ofL. bifidus which did not require the specific"bifidus factor" essential for the growth of L.bifidus var. pennmylvanicus and a strain (Jackson,Be) of L. parabifidus (Norris et al., 1950; Williamset al., 1953) were included in the study for com-parison. In a previous publication (Gy6rgy et al.,1954) strains of L. bifidus which require the"bifidus factor" were given the variety namePenn; however, the variety name pennsylvanicusis better from a nomenclatural standpoint.The supplements used included skimmed

human milk, skimmed cow's milk, charcoaleluates from human milk (Gauhe et al., 1954),hog gastric mucin (Wilson and Bruno, 1950),4 - 0 - , - galactopyranosyl - N - acetyl -D - glu-cosamine2 (Tomarelli et al., 1954), methyl andethyl N-acetyl-,3-D-glucosaminides,3 N-acetyl-D-

2Kindly furnished by R. M. Tomarelli, WyethLaboratories, Mason, Michigan.

3 Prepared by Dr. F. Zilliken in our laboratory.483

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TABLE 1The requirement of Lactobacillus bifidus var.

penn8ylvanicus for pantethineAcid Production(ml of 0.1 N)

Supplements to Basal MediumMedium MediumA B

None 0.7 0

Human milk (skimmed) 0.02 ml 7.3 3.10.06 ml 12.2 7.50.2 ml 14.7 11.7

Charcoal eluate from hu- 0.1 mg 2.8 0.3man milk 0.3 mg 10.7 0.5

1 mg 11.7 0.3

Pantethine, 0.1 ,ug 0.3

Pantethine, 0.1 pg + 0.1 mg 2.0Charcoal eluate from hu- 0.3 mg 8.2man milk 1 mg 10.4

3 mg 10.9

All amounts are per 10 ml tube.Medium B differed from medium A only in

containing acid hydrolyzed rather than enzy-matically hydrolyzed casein. Both media con-tained 4 pg of calcium pantothenate per tube.

glucosamine, pantethine,4 pancreatin (ViobinCorp.), and various fractions obtained from cow'smilk by precipitation."

RESULTS

The pantethine requiremen of L. bifidus var.penn8ylvanicu. Substitution of acid hydrolyzedcasein for the enzymatic digest in the mediumconsiderably reduced the growth promoting effectof human milk for L. bifidus var. pennsylvanicusand completely obviated the effect of purifiedfractions of the bifidus factor, such as eluates pre-pared from human milk, hog gastric mucin,N-acetyl-D-glucosamine, 4-0-(3-galactopyranosyl-N-acetyl-D-glucosamine and the alkyl N-acetyl-j3-D-glucosaminides. These results indicated thepresence of a particular growth factor present inthe enzymatic casein digest and, to a lesser ex-tent, in human milk.

4Kindly furnished by Boehringer and Son,Ingelheim/Rhine, Germany, and Parke Davis,Detroit, Michigan.

' Prepared by G. Braun in our laboratory.

It has been shown (Tomarelli et al., 1954) thatvarious strains of L. bifidus respond better topantethine than to pantothenic acid. In the lightof this observation, the assumption was made thatfor L. bifidus var. penneylvanicu both the specificbifidus factor and pantethine represent essentialnutrients. This hypothesis was borne out by ex-periments in which pantethine was added invarious amounts to the basal medium containingacid hydrolyzed casein. Whereas supplements ofcharcoal eluate of human milk did not supportgrowth of L. bi.fidus var. penneylvanicu in thismedium, a good growth response was obtainedwhen 0.1 ,ug of pantethine was added with themilk eluate (table 1). Pantethine in the absenceof bifidus factor did not stimulate growth.The original basal medium contained 4 jig of

calcium pantothenate per tube (10 ml). Twoquestions arose: (a) whether the calcium panto-thenate present enhanced the effect of pantethine,and (b) whether a larger amount of calciumpantothenate could replace pantethine. Thesequestions have been put to test using a strain ofL. bifidus var. pennsylvanicus, a regular strain ofL. bifidus, and a strain of L. parabifidus. Theresults obtained are summarized in table 2. Itis apparent that the strains of L. bi,fidus var.penneylvanicus and of the regular L. bifidus testedwere unable to utilize calcium pantothenate evenwhen it was given in very large doses and re-quired pantethine for growth. The pantethine re-quirement was not reduced when calcium panto-thenate, up to 20 Mg per tube, was present in themedium. In contrast the strain of L. parabifidustested required a much larger amount of pan-tethine than of calcium pantothenate. Strainslike L. bifidus var. penneylvanicus 212A and regu-lar L. bifidus 105C may be used for the micro-biological assay of pantethine, even in the pres-ence of free or bound pantothenic acid. In anumber of food products used as supplements inour study, pantethine determinations were madewith strain 105C as test organism. One-tenth,Mg of pantethine was found in about 0.33 ml ofskimmed human milk or skimmed cow's milk, in50 mg of hog gastric mucin, in 10 mglof "NZ casepeptone", and in 12.5 mg of pancreatin. Charcoaleluates obtained from human milk containedonly an insignificant trace of pantethine in 10 mg.

Requirement for supplmentary factors by L.bifidus var. pennsylvanicwus. In the presence ofsufficient amounts of pantethine, the medium

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TABLE 2Pantethine and calcium pantothenate requirement of variants of Lactobacillus bijidus

Ratio ofActivity

Organim Pantethine Calcium Pantothenate Pantethine/Calcium

Pantothenate

L. bifidus (strain 105C) 3 pg maximum growth 1,000 ,g; no effect >100,0000.3 pg almost maximum 20 pg; no potentiating effectgrowth with pantethine

0.01 ,ug perceptible effect

L. bifidus var. pennsyl- 0.3 pAg maximum growth 1,000 ,ug; no effect >10,000vanicus (strains 212A 0.1 pg less than maximum 20 pg; no potentiating effectand 5S) growth with pantethine

L. parabifidus (strain 10 pug maximum growth 0.2 pug maximum growth 0.02Jackson)

containing acid hydrolyzed casein will support

satisfactory growth of strain 212A of L. bifidusvar. pennylvanicus provided the essential micro-biological growth factor is present in the mediumin adequate amount. However, the growthachieved with human milk or with eluates fromhuman milk given in graded doses is still below

that observed with the same supplements whenthe enzymatic casein digest is used in the medium.In addition, the growth curve obtained with themilk eluates shows a distinctly lower slope in themedium with acid hydrolyzed casein than in themedium containing enzymatic casein digest. Amutant, 5S, of strain 212A shows even greater

TABLE 3The requirement of strains of Lactobacillus bijidus var. pennsylvanicus for supplementary factors in

addition to "biffdus factor" and pantethineAcid Production (ml of 0.1 N)

Supplements to Basal Medium Medium A Medium B

Strain Strain Strain Strain212A 5S 212A 5S

None 1.0 1.1 0.3 0.5

Human milk (skimmed) 0.02 ml 5.8 3.1 3.4 1.30.06 ml 13.6 8.5 9.1 2.60.2 ml 19.2 19.0 15.2 5.0

Cow's milk (skimmed) 0.06 ml 1.9 1.4 0.6 0.60.2 ml 2.3 1.4 1.0 1.10.6 ml 4.9 2.2 3.1 2.3

Eluate from human milk 0.1 mg 4.9 2.0 2.3 1.30.3 mg 11.8 6.0 8.0 1.81.0 mg 18.3 17.0 10.3 2.4

Cow's milk, 0.2 ml + 0.3 mg 11.9 3.2Eluate from human milk 1.0 mg 17.1 4.6

The data are expressed as amount per 10 ml tube.Medium A contained enzymatically digested casein, medium B, acid hydrolyzed casein. Both media

contained 0.5 pg pantethine per tube.

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dependence on the supplementary factor. Inmedium containing acid hydrolyzed casein sup-

plemented with human milk eluate there ispractically no growth, and even with supplementsof skimmed human milk, the acid production ismuch lower than in the control tubes inoculatedwith the original strain, 212A. Thus it appears

that both strains, and particularly strain 5S, re-

quire, in addition to pantethine and the specificbifidus factor, a supplementary factor (factors)which is present in the enzymatic casein hy-drolyzate and, to some extent, also in skimmedhuman milk but is absent from the acid hy-drolyzed casein. This supplementary factor isindependent from the specific bifidus factor. Thisconclusion is supported by the fact that humanmilk eluate was found to be free from the supple-mentary factor and, on the other hand, skimmedcow's milk containing only a negligible quantityof bifidus factor may exert a very pronouncedeffect as supplementary factor. The relevant dataof one experiment are summarized in table 3.

The bifidus factor activity of a given substanceis unspecifically enhanced in the medium con-

taining NZ case peptone as the supplementaryfactor. It is of interest that the enhancing effectis not of the same magnitude for various sub-stances. Charcoal eluate of human milk, hoggastric mucin, and the disaccharide of galactoseand N-acetyl-D-glucosamine isolated from gastricmucin have shown, in general, a relatively higheractivity in the medium containing acid hydroly-zate of casein than have N-acetyl-D-glucosamineand alkyl N-acetyl-fl-D-glucosaminides (table 4).The former group of compounds may be regardedas more closely related to the naturally occurring

form of the bifidus factor. In the same experi-ments it was demonstrated that autoclaving, par-

ticularly in the presence of the medium, may alterthe growth promoting activity of substances forL. bijidus var. pennsylvanics. N-Acetyl-D-glu-cosamine decreased markedly in activity when itwas autoclaved with the medium. A smaller, butconsistent, increase of activity was observed with

TABLE 4Differences in the activity of various sub8tances as bifidus factor, with replacement of enzymatically digested

casein in the medium by acid hydrolyzed caseinUnit Ratio of

Substance Treatment Activity in theEnzymatic Acid Two Mediahydrolyzate hydrolyzate

mg mg

Charcoal eluate from human Autoclaved with medium 0.30* 0.30* 1*milk Not autoclaved 0.30 0.36

Autoclaved separately 0.25 0.26

Hog gastric mucin Autoclaved with medium 0.40 0.36 1Not autoclaved 0.47 0.46Autoclaved separately 0.40 0.45

4-O-p-D-Galactopyranosyl-N- Autoclaved with medium 0.091 0.11 1acetyl-D-glucosamine Not autoclaved 0.075 0.075

Autoclaved separately 0.097 0.090

N-Aoetyl-D-glucosamine Autoclaved with medium 2.2 8.0 3.5Not autoclaved 1.2 4.3Autoclaved separately 1.3 5.5

Methyl N-acetyl-P-D-glucosami- Autoclaved with medium 0.15 0.37 2.5nide Not autoclaved 0.17 0.37

Autoclaved separately 0.15 0.33

* By definition, 0.3 mg of the charcoal eluate represents one unit of activity as bifidus factor ineach medium, and the activity of the other substances is related to this unit. As shown in previoustables the unit represents a considerably smaller production of acid in the medium containing acidhydrolyzed casein than in that containing NZ case.

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4 - 0 - , - galactopyranosyl - N - acetyl - D -glu-cosamine when it was sterilized by Seitz filtrationand added to the medium without autoclaving.Both human milk and cow's milk contain the

supplementary factor which enhances the growthof L. bifidus var. penn8ylvanicus strain 212A in thepresence of the specific bifidus factor. For assay

of this supplementary factor in the milk of thetwo species we used the medium containing acidhydrolyzed casein, with eluate from human milkas specific source of the bifidus factor. Whenhuman milk was used as a supplement, theamount of milk eluate was decreased in order tokeep the total bifidus factor activity constant.The results are summarized in table 5. As a sourceof the supplementary factor, human milk appearsto be less active than cow's milk: The effect of thesupplementary factor becomes apparent with0.06 ml of cows's milk and with 0.2 ml of humanmilk.The medium containing enzymatic digest of

casein does not contain the optimal amount ofsupplementary faetor. The addition of skimmedcow's milk may enhance the activity of thespecific bifidus factor, but only when the lattersubstance is present in suboptimal concentration.Conversely, in the medium with acid hydrolyzateof casein the enhancing effect of cow's milk isdemonstrable throughout the whole range of theconcentrations used.The activity of-skimmed cow's milk as supple-

mentary factor remained the same whether it wasautoclaved with the medium, autoclaved sepa-rately, or sterilized with ethylene oxide (Wilsonand Bruno, 1950) and added to the mediumaseptically.Attempts were made to fractionate and isolate

the supplementary factor in milk. A preparationof crude casein showed some enhancing effect ongrowth at levels of 3 to 10 mg per tube. Charcoaleluate from human milk was used as bifidusfactor. The effect of the casein was more apparentas the amount of milk eluate approached thatrequired for maximum growth. Mild acid hy-drolysis of casein (Kodicek and Mistry, 1952) con-siderably increased its effect. In contrast, com-

plete acid hydrolysis destroyed the supplementaryfactor in crude casein: even 100 mg had no ac-tivity. Vitamin-free casein (Laboo) was lesseffective than crude casein. Considerable increasein activity of the supplementary factor in caseinwas also achieved by tryptic digestion.

TABLE 5Growth of Lactobacillus bifidus var. penn8ylvanicue,

as measured by production of acid, wsnth humanmilk and cow's milk a8 sources of the supple-mentary factors.

Supplementary FactorBifidusFactor* 0.02 ml 0.06 ml 0.2 ml 0.6 mlUnits None

SHM SCM SHM SCM SHM SCM SHM SCM

0.33 2.2 2.1 2.91.3 7.7 7.9 7.4 6.9 9.53.3 11.3 10.7 11.3 11.0 12.4 14.1 15.113.0 12.4110.812.3112.014.0 14.7 15.917.217.3

SHM: skimmed human milk; SCM: skimmedcow's milk.

Acid produced is expressed as ml of 0.1 N per10 ml tube.

The medium contained acid hydrolyzed caseinand was supplemented with 0.5 pg of pantethineper tube.

* Human milk has a high level of bifidus factor(0.045 ml 1 unit); cow's milk contains almostnone. Charcoal eluate of human milk (0.3 mg =1 unit) was added to give the desired level ofbifidus factor for each amount of milk tested.

The presence of casein is not essential for theactivit.v of milk as supplementary factor. Casein-free whey was as active as the whole milk. Theprecipitate obtained from whey, after half or fullsaturation with sodium sulfate, showed good ac-tivity as supplementary factor, even after ex-haustive dialysis (4 times 24 hours against 10volumes of distilled water). Five to 10 mg of thisprecipitate had just as high activity as 0.6 ml ofcow's milk. Not all the supplementary factor inmilk is nondialyzable. Approximately 50 per centof the activity of both human and cow's milkmay be recovered in the dialyzate after prolongeddialysis.The distribution of the supplementary factor in

milk, and in particular the activation of casein bymild acid hydrolysis or by trypic digestion,placed the supplementary factor in the categoryof strepogenin. This conclusion has been bomeout by observations with crystalline insulin(Lilly) which, especially after mild acid hydroly-sis, was found to have high activity for strain212A. Very satisfactory enhancement was ob-served with 1.5 mg of insulin and even betterwith the same amount of mildly acid hydrolyzedinsulin.

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Yeast extract (Difco) and crude liver extract(Wyeth) showed only weak activity as supple-mentary factor, 30 mg of the dry residue beingequivalent to about 0.2 ml of cow's milk. Purifiedliver IGet (Parke Davis) and rice bran extract(Nopco) in doses up to 30 mg were inactive.The supplementary factor activity of the

medium containing enzymatic casein digest is duenot only to the presence of the hydrolyzed caseinbut also to that of pancreatic extract. Pancreatin(Viobin) has been found highly active as a sourceof the supplementary factor for L. bifidu var.pennsylvanwus, 20 to 25 mg being equivalent to0.6 ml of cow's milk. As in milk, the supplemen-tary factor in pancreatin is present in dialyzableand in high molecular, nondialyzable form. Whena solution of pancreatin, from which the smallamount of protein had been removed, wasdialyzed against 10 volumes of water for twotwenty-four hour periods in the cold, approxi-mately 50 per cent of the solids dialyzed outduring the first twenty-four hours, 35 per centduring the second twenty-four hours while 15 percent was nondialyzable. The nondialyzable frac-tion was the most active, 6 mg being equivalent

TABLE 6The relative activities of pancreatic extract and ofcow's milk as supplementary factor for strain5S of Lactobacillus bifidus var. pennsylvanicus.

Bifidus AcidFactor Supplementary Factor ProductionUnits ~~~~~~~~~~~0.1N)

0 0.23.3 1.1

3.3 Cow's milk (skimmed), 0.2 ml 2.63.6 0.6 ml 3.74.0 2.0 ml 5.2

3.3 Dialyzate of pancreatin, 5 mg 6.74.0 15 mg 13.33.0 50 mg 16.5

Supplements and acid production are expressedas amount per 10 ml tube.

The basal medium contained acid hydrolyzedcasein and 0.5 pg of pantethine per tube.

Bifidus factor was supplied as a charcoal eluateof human milk. The bifidus factor in the pan-creatic extract and the small amount in the cow'smilk were taken into account in estimating thelevel of bifidus factor.

to 0.6 ml of skimmed cow's milk. The correspond-ing values for the two dialyzates were 20 and15 mg.Milk, insulin, pancreatin, and various fractions

prepared from these products may be used inter-changeably as the supplementary factor for strain212A. The supplementary factor acts only as anaccelerator of growth and is not an essentialnutrient. In contrast, for the mutant 5S of L.bifidus var. pennyltvaniuos, the supplementaryfactor is indispensable for growth. This strainshows full response only to pancreatic extract,not to human or cow's milk or to insulin. Cor-responding observations are sunmmarized intable 6. The preparation of pancreatin used inthis experiment was a dialyzate of pancreatic ex-tract. The results obtained with milk and withpancreatic extract appear to present the conclu-sion that the mutant 5S responds more fully tothe pancreatic extract as the supplementaryfactor than to skimmed cow's milk. Even withincreased amounts of bifidus factor, skimmedcow's milk was unable to bring about full growthor even acceleration of growth beyond a limitedlevel. In contrast, maximal growth was supportedby pancreatic extract as the supplementaryfactor, when an optimal amount of bifidus factorwas present. Thus, it seems plausible to assumethat the mutant 5S requires more than one sup-plementary factor. Whereas the pancreatic ex-tract contains all necessary supplementaryfactors, skimmed cow's milk must be deficient inat least one of the factors required by strain 5S.No supplementary factor was needed by the

regular strain of L. bifidus or for the strain of L.parabifdus when they were grown in medium con-taining acid hydrolyzate of casein.

DISCUSSION

Strains of lactobacilli preferring pantethine topantothenic acid have been reported (McRorieet al., 1950; McRorie and Williams, 1951; Craigand Snell, 1951; Tomarelli et al., 1954). In general,strains with a preference for pantethine mayutilize pantothenic acid when it is offered in themedium in large amounts (McRorie et at., 1950;Craig and Snell, 1951). The strains of regular L.bifidus and of L. bijidus var. pennsylvanicu,selected for the present study, appear to be whollydependent on pantethine and are unable to acceptpantothenic acid as a substitute.

In contrast to the strains of regular L. bifidus

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or L. parabifidus, the strains of L. bifidus var.pennsylvanicu that were studied required foroptimal growth the presence of a supplementaryfactor (or factors) in addition to the specificbifidus factor. The supplementary factor wasfound in appreciable quantity in milk, in whey, incasein, in casein hydrolyzed enzymatically, ormildly, by acid, in insulin, and in pancreatin. Forthe original strain of L. bifidus var. pennsylvanicusthe supplementary factor fulfills only the role ofa growth accelerator. A more specific role of thesupplementary factor (or factors) was exhibitedby the mutant 58 of L. bifidus var. pennsylvanicus.In the absence of the supplementary factor thisstrain showed practically no growth even with anexcess of the specific bifidus factor present. Incontrast to the original strain of L. bifidus var.pennsylvanis, human milk and cow's milkalone were unable to assure optimal growth ofthe mutant 5S. Full growth was achieved onlywith a preparation obtained from pancreaticextract, whereas the effect of cow's milk or insulinremained partial, reaching a suboptimal plateau.In consequence, it appears that this mutant re-quires several supplementary factors or else itwill respond fully only to the specific present inpancreatic extract.The distribution and growth promoting proper-

ties of the supplementary factor place it in thecategory of strepogenin (e.g., Sprince and Woolley,1944) and, from the chemical point of view, in theclass of peptides and of protein, with all theknown difficulties of its further chemical charac-terization. The good activity of exhaustivelydialyzed, high molecular material obtained bysalt precipitation from whey is of special interest.It is reminiscent of recent observations by Sloaneand McKee (1952a,b) on the growth promotingactivity of a protein for Staphylococcus albus andof the report by Williams and Grady (1954) on athermolabile protein, "lactein", from milk re-quired for the growth of L. butgaricus. In contrastto the latter, the protein-like substance requiredfor L. bifidus var. pennsylvanicus has been foundto be thermostable.The requirement for the supplementary factor

varies with the chemical nature of the bifidusfactor used. For N-acetyl-D-glucosamine and foralkyl N-acetyl-j3-D-glucosaminides the effect ofthe supplementary factor is more marked thanfor crude charcoal eluates prepared from humanmilk, hog gastric mucin, or the disaccharide ob-

tained from hog gastric mucin. Thus, the closerthe bifidus factor is chemically to the naturallyoccurring form, the less it is dependent for agrowth promoting effect on the supplementaryfactor.The supplementary factor is present both in

human milk and cow's milk, but its concentrationis slightly higher in cow's milk. Recently, a strainif L. bifidus not requiring the bifidus factor hasbeen described (Gyllenberg et at., 1953), whichhas shown optimal growth only in the presence ofhuarmn milk or cow's milk or of various proteindigests. This effect was ascribed to the presence ofstrepogenin. It should be noted, however, thatthe medium (Hassinen et al., 1951) used byGyllenberg et al. was deficient in many othernutrients possibly required by the organism. Theeffect observed, therefore, was not necessarilydue to a strepogenin-like substance.The interaction of the supplementary factor (or

factors) represents an important possible sourceof error in assaying for the specific bifidus factor.For exact microbiological values of bifidus factoractivity, it is necessary to enrich the mediumwith the supplementary factor as pancreatic ex-tract or skimmed cow's milk depending upon thestrain of L. bifidus var. pennylvanicus used. Forsubstances known to be free from supplementaryfactor, relative comparative values for thespecific bifidus factor may be obtained by usingthe medium containing the enzymatic digest ofcasein. However, the level of the factor in thismedium is below the optimum.

N-Acetyl-D-glucosamine and the disaccharide,4 - 0 - , - galactopyranosyl - N - acetyl- D - glu-cosamine, had much less activity as bifidus factorwhen they were autoclaved with the culturemedium than when they were added aseptically(after Seitz filtration) to autoclaved medium. Incontrast, crude charcoal eluates obtained fromhuman milk, mucin, and purified blood substanceshave shown no change in activity when they wereautoclaved with the medium. It appears that freeor open N-acetyl-D-glucosamine may interactwith constituents of the basal meduim and thatthis reaction reduces the amount of N-acetyl-D-glucosamine available as precursor of the bifidusfactor.

STUMMARY

Strains of regular Lactobacillus bifidus and L.bifidus var. pennsylvanicus were found to be able

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to utilize only preformed pantethine but notpantothenic acid, even when the latter wasoffered in great excess. Such strains may be usedfor the microbiological assay of pantethine.Two strains of L. bifidus var. pennsylvanic

studied required, in addition to pantethine andthe specific "bifidus factor", a supplementaryfactor present in human milk, cow's milk, insulin,and pancreatic extract. This supplementaryfactor may be utilized as a high molecular, non-dialyzable compound, probably a protein.Casein was found active, but its activity was con-siderably increased after mild acid hydrolysis ortryptic digestion. Complete acid hydrolysis led toloss of activity. The chemical characteristics anddistribution of the supplementary factor areclosely related to those of strepogenin.For a mutant (5S) of L. bifidu4s var. pennsyl-

vanic the supplementary factor proved to beessential for growth. Optimal growth of thismutant was only reached with pancreatic extractas the supplementary factor. In contrast to theoriginal strain of L. bifidus var. penn8ylvanicus,this mutant responded only partially to cow'smilk, human milk, or insulin as supplementaryfactor.The closer the bifidus factor is chemically to its

natural form, the less it is dependent for itsgrowth promoting effect on the supplementaryfactor.

REFERENCESCRMG, J. A., AND SNELL, E. E. 1951 The

comparative activities of pantethine, panto-thenic acid, and coenzyme A for variousmicro-organisms. J. Bacteriol., 61, 283-291.

GAuHI, A., GYORGY, P., HoOVzR, J. R. E., KUHN,R., RosE, C. S., RUELIUs, H. W., AND ZIL-LIKEN, F. 1954 Bifidus Factor. IV. Prepa-rations obtained from human milk. Arch.Biochem. and Biophys., 48, 214-224.

GYLLENBERG, H., ROSSANDER, M., AND ROINE, P.1953 A strain of Lactobacilluz bifidus whichrequires Streptogenin. J. Gen. Microbiol.,9, 190-198.

GY6RGY, P. 1953 A hitherto unrecognized bio-chemical difference between human milk andcow's milk. Pediatrics, 11, 98-108.

GYORGY, P., NoRns, R. F., AND RosE, C. S.1954 Bifidus Factor. I. A variant of Lacto-bacillus bifidu8 requiring a special growthfactor. Arch. Biochem. and Biophys., 48,193-201.

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1953 The incidence of Lactobacillus bifidusin vaginal secretions of pregnant and non-pregnant women. Am. J. Obstet. Gynecol.,65, 352-357.

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