Effects of calcium gluconate (GACa) and a new compound of calcium gluconate and calcium lactate (Medical Super Ca; MSCa) on the growth of Bifidobacterium were investigated in vivo and in vitro. In in vitro experiments, GACa and MSCa stimulated the growth of B. bifidum in synthetic VLM medium. However, these substances did not stimulate, rather inhibited in part, the growth of B. bifidum in the complex medium (GAM broth). When 2.2 mg per mouse per day of GACa or MSCa was administered to mice, and feces were assessed for anaerobic bacteria on VLM medium and for facultative anaerobic bacteria on BTB-L agar, there was no difference among the 3 groups of mice administered with or without GACa or MSCa. Colony forming units (CFU) of B. bifidum in feces of the mice drinking 2.2 mg of MSCa was 3.5 × 105 organisms per g of feces, a value intermediate between those drinking 2.2 mg GACa (6.8 × 104) and controls (3.3 × 106). These results suggested that GACa and MSCa stimulated the growth of B. bifidum in synthetic media, and that the effects of MSCa on the growth of bifidobacteria in vivo and in vitro were intermediate between those of GACa and control

Matsuba, Takashi

Tanaka, Yoshinori

Tomioka, Shinichiro

Zhang, Jien-Ling

Yonago Acta Medica

Tottori University Research Result Repository

137Yonago Acta medica 2001;44:137–142Effects of a New Compound of Calcium Gluconate and CalciumLactate on the in vivo and in vitro Growth of BifidobacteriumShinichiro Tomioka, Jien-Ling Zhang, Takashi Matsuba and YoshinoriTanakaDepartment of Bacteriology, Tottori University Faculty of Medicine,  Yonago 683-0826JapanEffects of calcium gluconate (GACa) and a new compound of calcium gluconate andcalcium lactate (Medical Super Ca; MSCa) on the growth of Bifidobacterium were in-vestigated in vivo and in vitro.  In in vitro experiments, GACa and MSCa stimulated thegrowth of B. bifidum in synthetic VLM medium.  However, these substances did notstimulate, rather inhibited in part, the growth of B. bifidum in the complex medium(GAM broth).  When 2.2 mg per mouse per day of GACa or MSCa was administered tomice, and feces were assessed for anaerobic bacteria on VLM medium and for facultativeanaerobic bacteria on BTB-L agar, there was no difference among the 3 groups of miceadministered with or without GACa or MSCa.  Colony forming units (CFU) of B. bifidumin feces of the mice drinking 2.2 mg of MSCa was 3.5 × 105 organisms per g of feces, avalue intermediate between those drinking 2.2 mg GACa (6.8 × 104) and controls (3.3 ×106).  These results suggested that GACa and MSCa stimulated the growth of B. bifidumin synthetic media, and that the effects of MSCa on the growth of bifidobacteria in vivoand in vitro were intermediate between those of GACa and control.Key words:  Bifidobacterium; calcium gluconate; calcium lactate; MPN medium; VLM mediumMany substances are well known as so-called“foods for health”.  These include fructo-oligo-saccharides (Gibson and Wang, 1994; Gibson etal., 1995; Sghir et al., 1998; Sharp et al., 2001),galacto-oligosaccharides (Sharp et al., 2001),gluconic acid (Asano et al., 1994), lactic acid,and lactosucrose (Ohkusa et al., 1995).  Fructo-oligosaccharide stimulates the growth of Bifido-bacterium (Gibson and Wang, 1994; Gibson etal., 1995; Sghir et al., 1998; Sharp et al., 2001)and administration of fructo-oligosaccharideincreases fecal bifidobacteria in healthy hu-mans (Bouhnik et al., 1999).Gluconic acid is widely distributed in natur-al products such as rice, honey, wine, vinegar,etc., and has been shown to stimulate thegrowth of Bifidobacterium in the human intes-tine and to have beneficial effects on the bacte-Abbreviations:  BTB-L agar, bromthymol blue lactose agar; GACa, calcium gluconate; GAM, Gifu anaerobicmedium; MSCa, a compound of calcium gluconate and calcium lactaterial flora in the human intestine (Asano et al.,1994).  Calcium ions are necessary for thestrengthening of bones and teeth.  However, theordinary Japanese diet is lacking in calcium.Calcium gluconate (GACa) is stable at roomtemperature (Rubio and Moldenhauer, 1995)and has been suggested to be useful as a health-promoting food additive.Calcium lactate dissolves readily and freelyin water and reduces intraoral demineralizationof enamel if ingested in the diet (Kashket andYaskell, 1992, 1997).In the present study we investigated theeffects of a compound of calcium gluconate andcalcium lactate (MSCa) on the growth of Bifi-dobacterium in vivo and in vitro.  MSCa dissolvesmore rapidly and freely in water than GACa, andis therefore expected to be a better food additive.S. Tomioka et al.138Materials and MethodsReagents and mediaCalcium gluconate (GACa) was purchased fromSigma (St. Louis, MO).  A compound of calciumgluconate and calcium lactate (MSCa), which isfreely soluble in water, was obtained fromNihon Medical (Tottori, Japan).  GAM for an-aerobic bacteria and bromthymol blue lactoseagar (BTB-L agar) were obtained commerciallyfrom Nissui Seiyaku (Tokyo, Japan).  VLM me-dium was used as a nonselective medium for an-aerobic bacteria (Tanaka et al., 1997).  Resazurinwas purchased from Wako Pure Chemicals(Osaka, Japan) and cysteine-HCl was obtainedfrom Sigma.  MPN medium was used as a selec-tive medium for Bifidobacterium (Tanaka andMutai, 1980).  Biotin, riboflavin, adenine, gua-nine, uracil and Tween 80 were purchased fromWako Pure Chemicals.  Xanthine and pyruvicacid were from Sigma, and pantothenic acid andnalidixic acid were from Nakalai Chemicals(Kyoto, Japan).  GACa and MSCa were dis-solved in GAM medium and VLM medium,and autoclaved before use.  The ingredients in100 mL of anaerobic dilution fluid were as fol-lows:  3.75 mL of 0.78% K2HPO4, 3.75 mL of asalt solution consisting of 0.47% KH2PO4, 1.18%NaCl, 1.2% (NH4)2SO4, 0.12% of CaCl2 and0.25% MgSO4  • H2O, 0.1 mL of 0.1% resazurin,0.1 mL of 5% L-cysteine  • HCl, 0.2 mL of 25% L-ascorbic acid, 5 mL of 8% Na2CO3 and distilledwater.Bacterial strainBifidobacterium bifidum strain Hino used inthis experiment was kindly supplied by Dr. M.Ueda, Tottori University College of Medical CareTechnology and stored in GAM broth at –30˚C.An aliquot of the stock solution of Bifido-bacterium was added to fresh GAM broth in atest tube with a tight-fitting cap, anaerobicallyincubated at 37˚C overnight and used for the ex-periments.Growth of Bifidobacterium in culturemediaAliquots of 10 µL of the culture were addedunder a stream of hydrogen peroxide gas to testtubes with GAM broth containing GACa at afinal concentration of 1% (GACa1) or 2%(GACa2) or MSCa at a final concentration of1% (MSCa1) or 2% (MSCa2).  These test tubeswere closed tightly with a cap, and anaerobi-cally incubated at 37˚C for 16 h.  After serialdilution with anaerobic dilution fluid under astream of hydrogen peroxide gas, aliquots of100 µL of the suspension were put onto VLMagar plates, incubated in an anaerobic jar at37˚C for about 1 week and the viable organismswere enumerated by the colony counting meth-od.  Similar experiments were performed usingVLM medium containing GACa1 or GACa2and MSCa1 or MSCa2, and put onto VLM agarplates for colony counting.Isolation of bacteria from feces of miceGACa and MSCa were suspended in water at aconcentration of 0.75% and sterilized by auto-claving.  Six-week-old BALB/c mice (4 to 6;female) were bred in cages, and were given freeaccess to solid food (CE-2; Clea Japan, Tokyo)and water containing GACa or MSCa.  Thedoses of GACa and MSCa consumed by mouseper day were calculated from the decrease inamount of water in the bottle.  In another ex-periment, granules of 20 mg of MSCa were ad-ministered directly into the animal’s mouthwith a small spoon.After at least 2 weeks under these condi-tions, the feces were collected directly in a vinylbag with 1 mL of anaerobic dilution fluid.  Imme-diately after closing the vinyl bag and weighing,the suspension of feces was serially diluted withanaerobic dilution fluid under a stream of car-bon dioxide gas.  Aliquots of 100 µL of each sus-pension were put onto VLM agar plates, MPNagar plates and BTB-L agar plates, and incubat-ed in an anaerobic jar at 37˚C for about 2 weeks,followed by colony counting.Biological effect of calcium gluconate139MSCa1 MSCa2 GACa1 GACa2Addition to VLM medium20 1010 Viable cells (ratio to control)Addition to GAM mediumMSCa1 MSCa2 GACa1 GACa2100806040200 (%)Viable cellsStatistical analysisStudent’s t-test was used for statistical analysisto test for differences between the groups.ResultsGrowth of B. bifidum in GAM broth andVLM mediumB. bifidum grew in GAM broth and reached 2.1× 108 cells per mL after 16 h in culture at 37˚C.The numbers of bacteria in GAM broth contain-ing GACa1 and GACa2 were 60% and 46%, re-spectively, of those in control GAM broth withno additive.  More bacteria grew in GAM brothcontaining MSCa1 or MSCa2 than in that con-taining GACa1 or GACa2 (Fig. 1).  Interesting-ly, the numbers of bacteria grown in all GAMbroth containing GACa or MSCa were less thanthat in control GAM broth.  Thus, GACa andMSCa did not have any positive effect on thegrowth of Bifidobacterium.  The numbers of vi-able organisms in GAM broth containing GACaand MSCa decreased in a dose-dependent man-ner.Fig. 2.  Growth of B. bifidum in VLM medium supplemented with MSCa andGACa.  The growth of B. bifidum in VLM medium was approximately 2.0 × 107organisms per mL of medium.  B. bifidum growth in VLM medium containingMSCa and GACa is shown as the relative ratio of the number of viable organismsto that in controls.  Symbols are the same as in Fig. 1.Fig. 1.  Growth of Bifidobacterium bifidum in GAMbroth supplemented with MSCa and GACa.  Thegrowth of B. bifidum in GAM broth was approxi-mately 2.1 × 108 organisms per mL of medium, andthis is shown as 100% viable organisms.  Other dataare shown as percentages relative to this value.MSCa1 ( ) and MSCa2 ( ) indicate GAM brothcontaining MSCa at final concentrations of 1% and2%, respectively.  GACa1 ( ) and GACa2 ( ) indi-cate GAM broth containing GACa at final concen-trations of 1% and 2%, respectively.  (–) indicatesGAM broth with no additive ( ).  Typical data from1 of 3 separate experiments are shown.B. bifidum grew in VLM medium and reach-ed 2.0 × 107 organisms per mL.  In contrast toGAM broth, B. bifidum grew well in VLM me-dium containing GACa1 and GACa2, andreached 2.6 × 108 and 4.5 × 108 organisms permL, respectively, which were 13-fold and 23-fold, respectively, greater than the control (Fig.2).  The bacteria also grew in VLM mediumcontaining MSCa1 and MSCa2, as shown inFig. 2.  The viable cell number in VLM mediumcontaining GACa and MSCa increased in adose-dependent manner.S. Tomioka et al.1401086420CFU/g of fecesMSCaAdministration into mouse(log10)Addition to waterMSCa GACa10864**CFU/g of feces(log10) DiscussionBifidobacterium is thought to exert health-promoting effects for humans (Sghir et al., 1998).Its effects include inhibition of abnormal fer-mentation, production of vitamin B complexes,acceleration of peristaltic movement, stimula-tion of the immune system and inhibition of theproduction of carcinogenic substances.  The invitro experiments indicated that the growth ofBifidobacterium was stimulated, while that ofClostridium perfringens was not stimulated,rather inhibited, by gluconic acid or oligofruc-tose (Asano et al., 1994; Catala et al., 1999).The growth of Bifidobacterium was inhibit-ed by adding GACa and MSCa to GAM brothand stimulated by adding GACa and MSCa toIsolation of bacteria from feces of miceThe number of bacteria isolated on VLM agarplates from the feces of mice fed approximately2.2 mg of GACa or MSCa per mouse per daywas about 1.3 × 109 organisms per g of feces(Fig. 3).  There was no difference among the 3groups of mice examined.  However, feces ofmice drinking 2.2 mg of GACa yielded only 6.8× 104 organisms per g of feces on MPN agarplates.  That value was significantly less thanthat (3.3 × 106 organisms) in the control group.The number of viable bacteria in feces of micedrinking 2.2 mg of MSCa, was 3.5 × 105organisms per g of feces, a value intermediatebetween the group drinking 2.2 mg of GACaand controls.To search for facultative anaerobic bacteriaand anaerobic bacteria in feces, we put the sam-ples from feces onto BTB-L agar plates andVLM agar plates.  BALB/c mice were orally ad-ministered 20 mg of MSCa every day and theirfeces were collected for isolation of bacteria.As shown in Fig. 4, there were no differences inthe number of viable organisms associated withuse of BTB-L agar plates and VLM agar plates.Fig. 3.  Isolation of anaerobic bacteria from feces ofmice given water containing MSCa or GACa.  Num-bers of colony forming units (CFU) of viable organ-isms in feces were assessed on VLM agar plates ( )and MPN agar plates ( ).  Each column shows anaverage ± SD of 3 to 5 experiments.  **P < 0.05relative to control.Fig. 4.  Numbers of colony forming units (CFU) of mouse intestinal bacteria.Bacterial suspensions were prepared from the feces of mice, fed 20 mg of MSCaevery day.  A fixed amount of the suspension was serially diluted with anaerobicdilution fluid, and viable organisms were enumerated on BTB-lactose agar plates( ) and VLM agar plates ( ).  (–) indicates CFU from feces of mice withoutMSCa.  Each column shows the average ± SD of 5 to 8 separate experiments.Biological effect of calcium gluconate141VLM medium (Figs. 1 and 2).  As GAM brothcontains peptone, soy peptone, proteose peptone,decomposed serum, yeast extract and liver ex-tract, it is a nutrient-rich medium.  Thus, GAMbroth may contain sufficient amounts of glucon-ic acid and calcium ions for bacterial growth.However, a large excess of calcium ions mightinhibit the growth of Bifidobacterium.  VLMmedium is less nutrient-rich, and therefore addi-tion of gluconic acid and calcium ions to thismedium might improve the growth of Bifido-bacterium.There are large numbers of anaerobic bacte-ria and facultative anaerobic bacteria in themouse intestine.  The total numbers of these an-aerobic bacteria and facultative aerobic bacteriain the feces of the mice drinking water supple-mented with 0.75% MSCa were similar to thosein the feces of controls (Fig. 4).  However, therewere fewer Bifidobacterium organisms inmouse feces as compared with the human fecesdescribed by Asano et al. (1994).  When 2.2 mgof GACa was given to mice in the drinkingwater, the number of Bifidobacterium was sig-nificantly decreased.  Administration of 2.2 mgof MSCa reduced the number of Bifidobacte-rium but this effect was not significant.  As thesolid food given to mice is nutrient-rich andmay contain sufficient amounts of substancessimilar to gluconic acid and calcium ions, itmight be unnecessary to add MSCa to the feed.The excessive administration of calcium ionsmight inhibit the growth of Bifidobacterium inthe mouse intestine.In these experiments, we found that thegrowth of B. bifidum in GAM broth supple-mented with MSCa was inhibited, but not to agreater extent than that in GAM broth withGACa.  Addition of MSCa to VLM mediumstimulated the growth of B. bifidum.  Further-more, by administration of MSCa in the drink-ing water, the number of Bifidobacterium orga-nisms isolated from the feces of the mice wasreduced relative to the controls, although thiseffect was not significant.  For application ofMSCa for human use, further studies on theisolation of Bifidobacterium from the feces ofsubjects taking MSCa are required.Acknowledgments:  The authors would like to thankDr. M. Ueda (Tottori University College of MedicalTechnology) for supplying the bacteria.This work was supported in part by a grant fromNihon Medical Co., Tottori, Japan.References 1 Asano T, Yuasa K, Kunugita K, Teraji T, MitsuokaT.  Effects of gluconic acid on human faecal bac-teria.  Microb Ecol Health Dis 1994;7:247–256. 2 Bouhnik Y, Vahedi K, Achour L, Attar A, Salfati J,Pochart P, et al.  Short-chain fructo-oligosaccharideadministration does-dependently increases fecalbifidobacteria in healthy humans.  J Nutr 1999;129:113–116. 3 Catala I, Butel MJ, Bensaada M, Popot F, TessedreAC, Rimbault A, et al.  Oligofructose contributesto the protective role of bifidobacteria in experi-mental necrotising enterocolitis in quails.  J MedMicrobiol 1999;48:89–94. 4 Gibson GR, Beatty ER, Wang X, Cummings JH.Selective stimulation of bifidobacteria in the hu-man colon by oligofructose and inulin.  Gastroen-terology 1995;108:975–982. 5 Gibson GR, Wang X.  Enrichment of bifido-bacteria from human gut contents by oligofruc-tose using continuous culture.  FEMS MicrobiolLett 1994;118:121–127. 6 Kashket S, Yaskell T.  Effect of timing of admin-istered calcium lactate on the sucrose-inducedintraoral demineralization of bovine enamel.Arch Oral Biol 1992;37:187–191. 7 Kashket S, Yaskell T.  Effectiveness of calciumlactate added to food in reducing intraoral demin-eralization of enamel.  Caries Res 1997;31:429–433. 8 Ohkusa T, Ozaki Y, Sato C, Mikuni K, Ikeda H.Long-term ingestion of lactosucrose increasesBifidobacterium sp. in human fecal flora.  Diges-tion 1995;56:415–420. 9 Perrin S, Grill JP, Schneider F.  Effects of fructo-oligosaccharides and their monomeric compo-nents on bile salt resistance in three species ofbifidobacteria.  J Appl Microbiol 2000;88:968–974.10 Rubio SL, Moldenhauer JE.  Effect of 24 hourroom temperature hold time on the heat resistanceof Bacillus coagulans spores suspended 10% cal-cium gluconate, USP.  PDA J Pharm Sci Technol1995;49:50–52.11 Sharp R, Fishbain S, Macfarlane GT.  Effect ofshort-chain carbohydrates on human intestinal bi-fidobacteria and Escherichia coli in vitro.  J MedMicrobiol 2001;50:152–160.S. Tomioka et al.14212 Sghir A, Chow JM, Mackie RI.  Continuous cul-ture selection of bifidobacteria and lactobacillifrom human faecal samples using fructooligosac-charide as selective substrate.  J Appl Microbiol1998;85:769–777.13 Tanaka R, Mutai M.  Improved medium for selec-tive isolation and enumeration of Bifidobacterium.Appl Environ Microbiol 1980;40:866–869.14 Tanaka Y, Tanioka S, Tanaka M, Tanigawa T,Kitamura Y, Minami S, et al.  Effects of chitin andchitosan particles on BALB/c mice by oral and par-enteral administration.  Biomaterials 1997;18:591–595.Received June 4, 2001; accepted July 6, 2001Corresponding author:  Dr. Yoshinori Tanaka

Effects of a New Compound of Calcium Gluconate and Calcium Lactate on the in vivo and in vitro Growth of Bifidobacterium

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Effects of a New Compound of Calcium Gluconate and Calcium Lactate on the in vivo and in vitro Growth of Bifidobacterium

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