Agric. Biol Chern., 49 (1), 13-19, 1985 13

Comparative Studies on Synthesis of Water-soluble Vitaminsamong HumanSpecies of Bifidobacteria

Yoriko Deguchi, Takashi Morishita and Masahiko Mutai

Yakult Central Institute for Microbiological Research,1796 Yaho, Kunitachi, Tokyo 186, Japan

Received March 2, 1984

The ability of bifidobacteria to synthesize six water-soluble vitamins (thiamine, folic acid,nicotinic acid, pyridoxine, vitamin B12 and riboflavin) was systematically investigated with twenty-four strains of five species derived from human feces. The vitamins synthesized were determined asthose accumulated in cultures grown in a semi-synthetic medium. For the vitamins other thanvitamin B12 and riboflavin, extracellular liberation was also examined with the supernatant fluidsobtained after removing cells from the cultures. Manystrains of the bifidobacteria investigatedcould indeed synthesize five of the vitamins, the exception being riboflavin. A large portion of eachof the vitamins synthesized was excreted into the medium.Theconcentrations of the vitamins,especially thiamine, nicotinic acid and folic acid, accumulated varied widely among different speciesor strains. On the basis of the results, these bifidobacteria could be divided into three general typesaccording to the abilities to accumulate thiamine, nicotinic acid and folic acid. These three vitaminswere accumulated in all the strains ofB. bifidum and B. infantis as well as in manystrains of B. breveand B. longum but the vitamin concentrations were significantly higher in the former species

(higher-accumulators) than in the latter species (lower-accumulators). On the other hand, none ofthese three vitamins were detected in moststrains of B. adolescentis or in somestrains of B. breveand B. longum (non-accumulators). Non-accumulator strains of B. adolescentis required thiamineand nicotinic acid for maximal growth in a complete synthetic medium. Further studies on

thiamine synthesis revealed that the addition of exogenous thiamine to the mediumsignificantlyreduced the level of thiamine accumulated in a culture of a lower-accumulator strain of B. longumbut did not affect the vitamin level in a higher-accumulator strain ofB. bifidum. These findings werediscussed in relation to the vitamin biosynthesis and its regulation in bacteria.

The bifidobacteria are generally believed tosynthesize and liberate extracellularly manykinds of vitamins, such as thiamine, folic acid,nicotinic acid, pyridoxine and vitamin B12} ~3)This characteristic of the bifidobacteria has

been expected to be important for the vitaminsupply in humans,4~7) because these bacteriaconstitute a major part of the humanintestinalflora.8~9) However, information on the vi-

tamin productivity of these bacteria and theutilization of the bifidobacteria-synthesized vi-tamins by humans was mostly gathered in theten years during which the bifidobacteria wereincluded in the genus Lactobacillus as onespecies, L. bifidus. These bacteria are classifiedat present in the separate genus Bifido-bacterium and strains derived from human

feces are divided into five species.10) There-

fore, it would now be meaningful to clarifywhether the ability to produce vitamins is ageneral characteristic of all five human spe-cies. Furthermore, it is also an interestingquestion as to whether the productivity ofvitamins in bifidobacteria is affected by exo-genous vitamins and related compounds suchas precursors, since bifidobacteria would becontinuously exposed to these compoundsin the humanintestinal tract.In the course of an investigation on vitaminsynthesis and its regulation in bifidobacteria,we first examined systematically the ability tosynthesize six water-soluble vitamins, thia-mine, riboflavin, pyridoxine, nicotinic acid,folic acid and vitamin B12, in twenty-four

14 Y. Deguchi, T. Morishita and M. Mutai

strains of the five species derived from humanfeces. As shown below, many Bifidobacteriumstrains could indeed synthesize five of thesevitamins when grown in a semi-synthetic me-dium. However, the concentrations of the vi-

tamins accumulated were different dependingon the species or strains tested. The results offurther work on synthesis of thiamine showedthat the addition of exogenous thiamine to themediumsignificantly decreased the level of thisvitamin accumulated in a culture of a lower-accumulator strain but did not affect the vi-tamin level in a higher-accumulator strain,

suggesting different responses of thiamine bio-synthesis to end-product regulation amongdifferent strains or species.

MATERIALS AND METHODS

Bacterial strains. Twenty-four strains isolated from hu-man feces were used in this study (Table I). Eight of thesestrains, i.e., B. bifidum E-319, B. infantis S-12, 659 and S-76e, B. breve S-l and As-50, B. adolescentis E-194a, and B.longum E-194b, were kindly supplied by Dr. T. Mitsuoka(Department of Biomedical Science, Faculty of

Agriculture, University of Tokyo, Japan). The remainingsixteen strains were isolated and identified in our labo-ratory (R. Tanaka, personal communication).

Anaerobic techniques. Preparation of medium, washingof cells, inoculation and cultivation were anaerobicallyperformed by the modified Hungate methodll) using 100%

carbon dioxide.

Media. VL medium12) with minor modification of thecomposition was used for preparing the inoculum ofBifidobacterium strains, it consisted of (per liter):Trypticase (BBL), lOg; yeast extract (Difco), 5g; NaCl,0.45g; beef extract (Difco), 2g; KH2PO4, 0.225g;K2HPO4, 0.225g; (NH4)2SO4, 0.225g; MgSO4-7H2O,

0.0225g; CaCl2-2H2O, 0.0225g; L-cysteine-HCl, 0.5g;D-lactose, 10g; Na2CO3, 3.75g; hemin, 7.0mg; and

resazurine, 1.0 mg.The basal medium for vitamin determinations was

essentially the same as that described by Tanaka andMutai,13) it consisted of (per liter): (NH4)2SO4, 5g;KH2PO4, 3g; K2HPO4, 4g; MgSO4-7H2O, 0.125g;

FeSO4-7H2O, 6.0mg; MnSO4-7H2O, 4.0mg; pyruvic

acid, 0.1 g; L-cysteine-HCl, 0.5g; riboflavin, 10mg; cal-cium pantothenate, 1.0mg; biotin, 0.01mg; adenine,lOmg; uracil, lOmg; Tween 80, 1.0g; Na2CO3, 4g;

resazurine, 1.0 mg; and D-lactose, 20 g.The semi-synthetic mediumfor vitamin determinations

Table I. Strains of Bifidobacterium Used

S p ec ie s S tra in s S o u rc es"

B. bi fi dum E -3 19 A d u lt

G a -1 8B o ttled -fe d

in fa n t

X a -3 1B o ttle d -fe d

in fa n t

1-2 A d u lt

B. infa ntis S -1 2 In fa n t

6 5 9 Tn fa n t

S -7 6 e In fa n t

L a -1 0B r ea st-fe d

in fa n t

U a -6 0B r ea st-fe d

in fan t

B . b re ve A s-5 0 In fa n t

S - 1 In fan t

K a-2B rea st-fe d

in fa n t

V a -S NB rea st-fe d

in fa n t

N a -5B rea st-fe d

in fa n t

B. adole.scentis E - 19 4 a A d u lt

G o -2 8 In fa n t

S l -3 0 A d u lt

Y J -9 A d u lt

S B -1 9B re a st-fed

in fa n t

B. l on g u m E -19 4 b A d u lt

5a - 15B re a st-fed

in fa n t

1-3 A d u lt

T M -2 5 A d u lt

5 C -1B o ttle d -fe d

i nfant

Derived from the feces of the indicated human origin.

was prepared by mixing 500ml of "Proteoliquifase"-

digested skim milk with 500ml of the double-strengthbasal medium. The "Proteoliquifase" -digested (proteo-

liquifased) skim milk was prepared as follows. The re-action mixture (pH 8.5) consisting of60 g of skim milk and0.36 g of "Proteoliquifase" (Ueda Chemical Industry) perliter was incubated at 50°C for 2hr. The reaction was

stopped by heating at 100°C for lOmin. After immediatechilling, the supernatant was collected by nitration andused for preparation of the semi-synthetic medium. Thefreshly prepared semi-synthetic medium contained smallquantities of six vitamins, that is, 38.3ng of thiamine,

ll.1 ng of pyridoxine, 0.023ng of vitamin B12, 290ng ofnicotinic acid, 0.24ng of folic acid and 17 ng of riboflavinper milliliter, when these vitamins were determined bythe methods described below.The buffer solution (pH 6.8) used for washing of cells

Vitamin Synthesis in Bifidobacteria 15

consisted of (per liter): KH2PO4, 0.225g; K2HPO4,0.225g; (NH4)2SO4, 0.225g; NaCl, 0.45g; MgSO4à"7H2O,0.0225g; CaCl2-2H2O, 0.0225g; L-cysteine-HCl, 0,5g;Na2CO3, 30g; and resazurine, 1.0mg.

Growth conditions. Cells of cultures grown overnight at376C in VL mediumwere collected, washed twice in buffersolution and inoculated into the semi-synthetic medium(about 107 cells/ml). After incubation at 37°C for 48 hr, theturbidity was measured with a Klett-Summersoncol-orimeter with a No. 66 filter. In the semi-synthetic medium,some strains required 48 hr incubation to exhibit maximalgrowth, although many other strains only required 24hrto reach the stationary growth phase. Therefore, extrac-tion and determination of vitamins synthesized were per-formed with cultures grown in this medium for 48hr.

Under these growth conditions, the numbers of survivingcells of all 24 strains used reached about 109 cells/ml.

For thiamine, nicotinic acid, folic acid and pyridoxine, thesupernatant fluids of the cultures freed from cells bycentrifugation were also used to determine extracellularvitamins.

Extraction and determination of vitamins. Thiamine wasextracted and determined by the thiochrome method, asdescribed by Glick.14) Riboflavin was extracted and de-termined by the lumiflavin fluorescence method, as de-scribed by Iwao.15) Pyridoxine,16) nicotinic acid,17) folicacid18) and vitamin B1219) were determined by micro-

biological assay methods using Saccharomyces carlesber-genesis ATCC 9080, Lactobacillus plantarum ATCC 8014,Streptococcus faecalis ATCC 8043 and L. leichmaniiATCC7830, respectively, as assay organisms. These fourvitamins were extracted by the respective described vi-

tamin determination methods. The corrected value of avitamin concentration was obtained by subtracting the

value for the freshly prepared semi-synthetic medium fromthe value for the test culture grown in this medium.

RESULTS

Abilities of bifidobacteria to synthesize sixwater-soluble vitamins

The abilities of twenty-four strains of bifi-dobacteria to produce six water-soluble vi-tamins were examined using cultures grownfor 48hr in a semi-synthetic medium. Forvitamin B12 synthesis, the medium was sup-plemented with 1 /ig/ml of cobalt. If a partic-ular vitamin was detected, it was regarded ashaving been synthesized and slightly or ex-cessively accumulated. In the case of failure todetect a vitamin, it was regarded as eitherhaving been synthesized but not accumulated,

J0.3-

nlllwluB.bifldum B.lnfanttsB.breve B.tongufnB.adolescentU(4) (5) (5) (5) (5)

Fig. 1. Accumulation of Thiamine in Bifidobacteria.Each bar represents the average value obtained on de-terminations with several test strains, whose numbers areindicated in parentheses. Striped bars represent the con-centrations of the vitamin in the cultures grown for 48 hrin the semi-synthetic medium,whereas open bars representtlje concentrations of the vitamins in the supernatant fluidsfreed from cells by centrifugation of the cultures. The lineabove each bar represents the standard deviation.

or as not having been synthesized. To de-termine the concentrations of the vitaminsliberated extracellularly, supernatant fluids ob-tained after removing cells from cultures bycentrifugation were also subjected to vitamindeterminations.

The concentrations of thiamine, nicotinicacid, folic acid and pyridoxine accumulated

and excreted into the mediumare shown inFigs. 1, 2, 3 and 4, respectively. The con-

centrations of vitamin B12 in the cultures areshown in Fig. 5. In these figures, the values areexpressed in fig or ng per ml of culture or

supernatant fluid and as averages of the valuesobtained on determinations with test strains ineach of the five species.All the strains ofB. bifidum and B. infantiswere found to well accumulate thiamine witha relatively invariable quantity among teststrains. B. breve and B. longum also accumu-lated thiamine but the averages of the con-centrations were significantly lower in thesetwo species than in B. bifidum and B. infantis

16 Y. Deguchi, T. Morishita and M. Mutai

à"1.6 -

"!iil

å llllllllmB.bifidumB.irifantisB.breve B.longum B.adolescentis(4) (6) (5) (5) (5)

Fig. 2. Accumulation of Nicotinic Acid in Bifido-bacteria.

The striped and open bars, the line above each bar and thenumbers in parentheses are the same as in Fig. 1.

(Fig. 1). The thiamine-productivity of B.adolescentis was considerably weak as com-

pared with that of the other four species andfour strains of B. adolescentis other than Sl-30 did not accumulate an appreciable quantityof this vitamin. In connection with this, thesefour B. adolescentis strains were found to re-quire thiamine for maximal growth in thebasal medium supplemented with 19 aminoacids and other nutrients (data not shown).All the strains of B. bifidum and B. infantiswell accumulated nicotinic acid with a rel-atively invariable quantity among differentstrains (Fig. 2). Nicotinic acid was also accu-mulated in all the strains ofB. longum and twostrains (As-50, Va-SN) of B. breve. The re-maining strains of B. breve and four strains ofB. adolescentis other than strain SI-30 failed toaccumulate a detectable quantity of nicotinicacid. These four B. adolescentis strains re-quired this vitamin for maximal growth in thecomplete synthetic medium(data not shown).The nicotinic acid-accumulating abilities aswell as the thiamine-accumulating abilitieswere appreciably weak in B. breve, B. longumand B. adolescentis as compared to those in B.bifidum and B. infantis (Fig. 2).A similar difference pattern of the vitamin-

«0.07-

à"0-06å  I,

5 |[?l006"^l T

^ II1!

B .btfidum B.ihfantis b.breve B.lohgiim B.adoldscentisC4) (5) (5) (5f (5)

Fig. 3. Accumulation of Folic Acid in Bifidobacteria.The striped and open bars, the line above each bar and thenumbers in parentheses are the same as in Fig. 1.

i

i

iti

l

i.bifldum B.infantis b.breve(4) (5) /5)B.longufnB.adoles

(5) (5)

Fig. 4. Accumulation of Pyridoxine in Bifidobacteria.The striped and open bars, the line above each bar and thenumbers in parentheses are the same as in Fig. 1.

accumulating abilities among different specieswas also seen for the synthesis of folic acid.

The concentrations of folic acid accumulatedwere significantly higher in B. bifidum and B.infantis than in B. breve, B. longum and B.adolescentis (Fig. 3).

Pyridoxine was accumulated in all twenty-four strains, although relatively little variancein the content was seen amongthe strains orspecies (Fig. 4).

Vitamin Synthesis in Bifidobacteria 17

I

i

II

B.bifidumB.ihfantisB.br&vfe B.ldnfium b.adolescents(4) (5) (5) (5) (5)

Fig. 5. Accumulation of Vitamin B12 in Bifidobacteria.The striped bars, the line above each bar and the num-bers in parentheses are the same as in Fig. 1.

Vitamin B12 was found only in a smallquantity in all the strains and therefore theproductivities for this vitamin were indis-tinguishable among the five species (Fig. 5).Riboflavin could not be detected in any ofthe strains and was either required or stimu-latory for their normal growth in the completesynthetic medium (data not shown).The greater parts of thiamine, nicotinic acid,folic acid and pyridoxine accumulated wereexcreted into the medium (Figs. 1 to 4).Whether vitamin B12 formed was liberated

into the mediumhas not so far been examined.The results in Figs. 1 to 5 were essentiallysimilar whenrespective values were calculatedas values per viable cell number or turbidity ofcultures for 48 hr (data not shown).

Effect of the addition ofexogenous thiamine onthe synthesis of thiamine

As one approach to determine whether aregulatory mechanism, such as feed-back re-pression, similar to that well-known in aminoacid biosynthesis of other bacteria,20) is alsoinvolved in the vitamin synthesis in bifidobac-teria, the effect of the addition of exogenousthiamine to the mediumon the abilities tosynthesize thiamine was first investigated com-paratively in both a higher-accumulator and alower-accumulator strain. The formation of

thiamine in the culture during growth for 10 hrin the semi-synthetic medium with or withoutaddition of exogenous thiamine (0.2/ig/ml)was examined using B. bifidum E-319 and B.

20.20

2 4 6 8Time of Incubation (hr.)

(b)

n ^-f2 4 6 8^

Tifne of Incubation (hi\)

Fig. 6. Effect of the Addition of Exogenous Thiamineon the Synthesis of Thiamine in Bifidobacteria.

Cultures were grown in the semi-synthetic mediumwith(#) or without (O) addition of 0.2^g/ml of thiamine,during which thiamine accumulated in the cultures wasdetermined at the times indicated. The concentrations ofthiamine synthesized when the bacteria were grown withthe addition of thiamine were obtained after substractingthe 0.2/ig/ml of thiamine added, (a), B. longum 5C-1; (b),B. bifidum E-319.

longum 5C-1, respectively, as representativehigher- and lower-accumulator strains. In themedium without the addition of exogenousthiamine, both strains continued to accu-mulate thiamine during growth for lOhr butthe concentration of this vitamin accumulated

18 Y. Deguchi, T. Morishita and M. Mutai

was higher in strain E-319 than in strain 5C-1(Figs. 6a and b), substantiating the results

presented in the previous section (see Fig. 1).Whenthiamine was exogenously added to themedium, the level of thiamine accumulatedduring growth was significantly reduced instrain 5C-1 (Fig. 6a). On the other hand, theaddition of the exogenous thiamine had vir-

tually no effect on the level of thiamine accu-mulated in strain E-319 (Fig. 6b). These resultsstrongly suggest that the thiamine biosyntheticpathway is controlled by an end-product, thia-mine, in lower-accumulator strain 5C-1 but

not in higher-accumulator strain E-319.

DISCUSSION

The data presented here indicate that manyof the strains of Bifidobactqriurn investigatedcan synthesize thiamine, nicotinic acid, folic

acid, pyridoxine and vitamin B12 when grownin the semi-synthetic medium. These results aregenerally in good agreement with the previousfindings that bifidobacteria possess the abilityto produce several vitamins.1~3'21~23> How-

ever, the concentrations of the vitmains

accumulated widely varied among the differentspecies and strains. As for thiamine, nicotinic

acid and folic acid, particularly, the levels weresignificantly higher in B. bifidum and B. in-fantis (higher-accumulators) than in B. breve,B. longum and B. adolescentis (lower-

accumulators). In the latter three species, somestrains did not accumulate measurable quan-tities of these vitamins (non-accumulators). B.adolescentis was regarded as a low-

accumulator for folic acid and in most cases asa non-accumulator for thiamine and nicotinicacid. The fact that the non-accumulator strainsof B. adolescentis as to thiamine and nicotinic

acid exhibited growth requirements for thesetwo vitamins means that the pathways leadingto the synthesis of these vitamins are blockedin these bacteria (non-synthesizers). In con-trast, the lack of the requirements for thia-

mine, nicotinic acid and folic acid in the non-accumulator strains of B. longum and B. brevesuggests that these vitamins are synthesized

but not accumulated in these organisms, pre-

sumably because the vitamins synthesized areexhaustively utilized for vitamin-mediated cel-lular metabolism.

The reduced level of thiamine synthesis inthe lower-accumulator strain of B. longumduring growth in the presence of exogenousvitamin (Fig. 6a) means that thiamine syn-thesis is subjected to feed-back repression orinhibition in this strain. Similar control ofthiamine synthesis has been reported inSalmonella typhimurium2Ar ~25) and Escherichiacoli.26'21) In contrast, the little or no re-duction of thiamine synthesis in the higher-

accumulator strain of B. bifidum in the pres-ence of exogenous vitamin (Fig. 6b) suggeststhat neither feed-back repression nor inhib-ition is concerned in the regulation of thia-mine biosynthesis and thus thiamine would besynthesized constitutively in this strain. Theseresults also imply that the sensitivities of thia-mine synthesis to end-product control are gen-erally different between higher- and lower-accumulator strains of bifidobacteria. Similarexperiments with various strains other than B.bifidum E 319 and B. longum 5C-1 are inprogress. The response of the syntheses ofnicotinic acid and folic acid to feed-backcontrol may also differ betwen higher- andlower-accumulator strains. As to the mech-anism underlying vitamin synthesis in bifi-dobacteria, our present knowledge is too lim-ited to permit more meaningful discussion.Together with that on thiamine biosynthesis,therefore, further work on the enzymatic andgenetic basis of biosyntheses of these vitaminsmay provide useful information on cellularmetabolism and its regulation in these strictlyanaerobic bifidobacteria.

From all the findings and the discussionabove, it may also be possible that the abili-ties of bifidobacteria to produce these vita-mins are demonstrated in the humanintesti-nal tract. The lower-accumulator strains, inwhich the biosynthesis of vitamins is sensitiveto feed-back regulation, would show little orno synthesis of the vitamins in their naturalhabitats that are rich in the vitamins and re-

Vitamin Synthesis in Bifidobacteria 19

lated compounds such as precursors. Incontrast, the higher-accumuator strains, in

which the biosynthesis of vitamins is insensi-tive to feed-back control, would continue tosynthesize the vitamins in their habitats, un-less another regulatory mechanismaffectingthe level of the vitamin synthesized is pres-ent in these organisms. Therefore, the high-er accumulators rather than the lower accu-mulators are expected to be good vitaminproducers in the human intestinal tract.Whether these points can be substantiatedfor the individual vitamins remains to bedetermined by future investigations. In con-

nection with this, it would be interesting toexamine the ability of the higher accumula-tors to produce the vitamins, e.g., thiamine,in the digestive tract after oral administra-tion of these bacteria to appropriate experi-mental animals.

Acknowledgments. We are deeply indebted to Mr. T.Terashima and Dr. T. Yura for the useful discussion andadvice and also Dr. T. Sakurai for his interest andencouragementthroughout this study.

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