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*Corresponding author.Tel.:#[phone];fax:#[phone].E-mail address:jorreinhqus.unl.edu.ar(J.A.Reinheimer)International Dairy Journal 9(1999)497505Culture media for the enumeration of Bixdobacterium bixdumand Lactobacillus acidophilus in the presence of yoghurt bacteriaC.G.Vinderola,J.A.Reinheimer*Programa de Lactologn&a Industrial,Facultad de Ingeniern&a Qun&mica(Universidad Nacional del Litoral),Santiago del Estero 2829,3000 Santa Fe,ArgentinaReceived 8 January 1999;accepted 14 May 1999AbstractA proposed methodology for the enumeration of probiotic bacteria(bidobacteria and Lactobacillus acidophilus)in the presence ofyoghurt bacteria is described.Commercial mixed cultures of yoghurt(four cultures),L.acidophilus(two cultures)and Bixdobacteriumbixdum(two cultures)were assayed using a total of 15 culture media from four agar categories:nonselective(Skim Milk(SM),MRSand M17);modied(Galactose MRS(G-MRS),Galactose M17(G-M17)and Trehalose MRS(T-MRS);di!erential(Lactic BacteriaDi!erential(LBD),Lactic-and L-S);and selective(Nalidixic Paromomycin Neomycin Lithium MRS(NPNL-MRS),NalidixicNeomycin Lithium MRS(NNL-MRS),Nalidixic Kanamycin Lithium MRS(NKB-MRS),Bile-MRS,Oxgall Gentamicin MRS(OG-MRS)and Lithium Propionate MRS(LP-MRS).Every culture used was tested for its growth on each medium under threedi!erent incubation conditions(aerobic,anaerobic with combustion and anaerobic in GasPak)at 373C for 72 h.SM and MRS agarwere taken as reference media for yoghurt and probiotic bacteria,respectively.For each organism,the cell recovery rate on eachmedium was compared statistically with that obtained on the reference media.The ability of every medium to di!erentiate or inhibitthe organisms was also tested.SM and LBD agar(aerobic)were useful to enumerate yoghurt bacteria and inhibit bidobacteria.T-MRS and Bile-MRS agar(aerobic)only allowed the growth of L.acidophilus cultures.For Bixdobacterium bixdum,a selective countcould be performed on LP-MRS agar(anaerobic,GasPak).This set of media was demonstrated to be e!ective for the enumeration ofS.thermophilus,Lactobacillus delbrueckii subsp.bulgaricus,L.acidophilus and Bixdobacterium bixdum in commercial dairy prod-ucts.(1999 Elsevier Science Ltd.All rights reserved.Keywords:Bixdobacterium bixdum;Lactobacillus acidophilus;Yoghurt1.IntroductionAllover the world,fermenteddairyproductshave beenconsumed for nutrition and maintenance of good healthfor a very long time.The overall popularity of theseproducts has expanded over the last decade.Yoghurtconsumption has increased signicantly,presumably be-cause of its perceived health benets(Wood,1992).Fur-thermore,almost 100 products containing Lactobacillusacidophilus and bidobacteria are available on the mar-kets world-wide(Dave&Shah,1996).The addition ofthese probiotic bacteria to milk or fermented dairy prod-ucts ensures their presence in the gut micro#ora.Thebenets of probiotic bacteria for consumers are widelyrecognised(Bottazzi,Zacconi&Sarra,1985;Nakazawa&Hosono,1992;Wood,1992;Roissart&Luquet,1994;Cogan&Accolas,1996;Bottazzi,1997;Mustapha,Jiang&Savaiano,1997;Kailasapathy&Rybka,1997).Althoughthereare suggestedlevels for theseprobioticsorganisms in fermented milk by di!erent authors(Ar-royo,Cotton&Martin,1994;Medina,Jopaz&Zor-dono,1995;Shah,Lankaputhra,Britz&Kyle,1995;Rybka&Kailasapathy,1995),there are few recommen-ded methodologies for an accurate enumeration of themicro#orain yoghurts containingthese.This causes di$-culties in quality control(Rybka&Kailasapathy,1996)and does not allow establishment of o$cial levels,noro$cial methodologies for the enumeration of thesemicroorganisms in fermented dairy products.Several media have been suggested for the enumer-ation of L.acidophilus(IDF,1995;Rybka&Kailasa-pathy,1996;Dave&Shah,1996)or Bixdobacterium sp.(Beerens,1990;Lapierre,Undeland&Cox,1992;Lim,0958-6946/99/$-see front matter(1999 Elsevier Science Ltd.All rights reserved.PII:S0 9 58-6 9 4 6(9 9)0 0 1 20-XHuh&Baek,1995;Arroyo et al.,1994;Rybka&Kailasapathy,1996;Pacher&Kneifel,1996;Dave&Shah,1996;Silvi,Rumney&Rowland,1996)in purecultures and commercial products.However,not all me-dia give good results when selective or di!erential enu-meration of L.acidophilus and B.bixdum is required inthe presence of S.thermophilus and Lactobacillus del-brueckii subsp.bulgaricus(Dave&Shah,1996),especiallywhen the media are applied to perform a colony count ofstrains that are di!erent from those employed to designthe methodology.It is important to bear in mind thatmedia proposed for a di!erential or selective enumer-ation of L.acidophilus and/or B.bixdum should not becomplex or require time-consuming procedures to pre-pare and should o!er a good cell recovery for the micro-organisms(Lim et al.,1995).Many of the assays toformulate a medium where the four microorganismscould be counted fail because of the lack of recovery ofone or more species of these bacteria(Lankaputhra&Shah,1996);lack of selectivity(Lim et al.,1995;Pacher&Kneifel,1996);or di!erentiation among colonies(Kneifel&Pacher,1993;Ghoddusi&Robinson,1996;Nighswonger,Brashears&Gilliland,1996).In variouscountries,there is great confusion within the dairy indus-try and regulatory organizations for selecting propermethods to control the levels of bidobacteria andL.acidophilus in fermented dairy products like yoghurt.The aim of this work was to select a set of culturemedia for the enumeration of L.acidophilus and B.bi-xdum in presence of yoghurt bacteria in fermented dairyproducts.2.Materials and methods2.1.CulturesPure lyophilised cultures of Bixdobacterium bixdum(strains BBI and BB12),Lactobacillus acidophilus(strainsLAI and LA5)and mixed cultures of S.thermophilus andLactobacillus delbrueckii subsp.bulgaricus(identied asY-SD,W1,W2 and JA)were supplied by local industries.These cultures are widely used in Argentinian fermenteddairy products(yoghurt and cultured milk).All the cul-tures were maintained according to manufacturers in-structions.2.2.Culture mediaFifteen media from four categories were selected forthe assays:non selective media*Skim Milk agar(SM)(Marshall,1992),MRS agar and M17 agar(Biokar,Be-auvais,France);modied media*Galactose MRS agar(G-MRS)(Sneath,1986),Galactose M17 agar(G-M17)and Trehalose MRS agar(T-MRS)(IDF,1995);di!eren-tial media*Lactic Bacteria Di!erential agar(LBD)(HIMedia Laboratories,Bombay,India),Lactic agar(Matalon&Sandine,1986)and L-S agar(Oxoid,Eng-land,1977);and selective media as shown in Table 1.Themodied media were prepared from individual ingredi-ents in our laboratory;all components of MRS and M17agar were included,but glucose was substituted with anequal amount of galactose(Galactose MRS-and M17agar)or trehalose(Trehalose MRS agar).All the sugarswere dissolved in distilled water,sterilized by ltration(lters Millipore,type HA,0.45 lm,Millipore Corpora-tion,Bedford,MA,USA)and aseptically added to theautoclavedbase mediumpreviously cooledto503C at theconcentration of 2%(w/v).The aim of the addition ofgalactose or trehalose was to allow the growth ofB.bixdum and L.acidophilus respectively,since strainsof S.thermophilus and L.delbrueckii subsp.bulgaricus arenormally not able to ferment these sugars(Sneath,1986).To formulate selective media,MRS agar was used asa basal medium to which inhibitory compounds wereadded.The selective compounds were selected from thebibliography available and the nal compositions ofthe media are shown in Table 1.2.3.Media performanceThe suitability of the media was tested by platingdecimal dilutions of the lyophilized mixed cultures ofyoghurt bacteria(S.thermophilus and L.delbrueckiisubsp.bulgaricus)and of pure cultures of B.bixdum andL.acidophilus.Thus,a sample of 1 g of each culture wasdecimally diluted in sterile peptone water(0.1%)and0.1 ml aliquot dilutionsplatedover thedi!erent media,intriplicate.Plates were incubated 72 h at 373C under threedi!erent conditions:aerobic,anaerobic produced bycombustion and anaerobic using the GasPak(GasPakSystem-Oxoid,Basingstoke,Hampshire,England).Theanaerobic incubation consisted in putting the plates in-side a 13 cm wide and 15 cm tall metal tin with a burningcandle inside and then hermetically closed.The candleburnt until the oxygen was depleted and a micro-aerophilic atmosphere was created.The colony formingunits(CFU)were counted and the characteristics of thecolonies were recorded for each di!erent medium.Toconrm the identity of the colonies,catalase testing(MacFaddin,1980)was carried out and cell morphology(phase contrast,1000 Microscope Jenamed 2 CARLZEISS)was registered.2.4.Statistical analysisExperiments were replicated at least three times fol-lowing a randomized block design.All data were ana-lyzed using the one-way ANOVA procedure of SPSS.The di!erences among means were detected by theDuncansmultiplerange test(Lizasoain&Joaristi,1995).498C.G.Vinderola,J.A.Reinheimer/International Dairy Journal 9(1999)497505Table 1Selective media used in this study for viable cell counts of Bixdobacterium bixdum and Lactobacillus acidophilusAgar mediumInhibitory agentFinal composition of selective compounds in the media(%w/v)NPNL-MRSNPNL solution(Martin&Chou,1992)Nalidixic acid0.075Paromomycin sulfate0.001Neomycin sulfate0.0005Lithium chloride0.015NNL-MRSNNL solution(Catalogue Nu-trish Cultures,1996)Nalidixic acid0.0015Neomycin sulfate0.01Lithium chloride0.3NKB-MRSNKB solution(Arany et al.,1995)Nalidixic acid0.003Kanamycin sulfate0.005Polimixin B100!Bile-MRSBile(Sigma)solution(IDF,1995)Bile(Oxgall)0.15OG-MRSOG solution(Lim et al.,1995)Bile(Oxgall)0.02Gentamicin0.003LP-MRSLP(Lapierre et al.,1992)Lithium chloride0.2Sodium propionate0.3!International units l1.NPNL:Nalidixic acid,Paromomycin sulfate,Neomycin sulfate and Lithium chloride.NNL:Nalidixic acid,Neomycin sulfate and Lithium chloride.NKB:Nalidixic acid,Kanamycin sulfate and Polimixin B.OG:Oxgall and Gentamicin.LP:Lithium chloride and Sodium propionate.2.5.Analysis of fermented dairy productsA total of seven products containing bibobacteriaand/or L.acidophilus as well as S.thermophilus and/orL.delbrueckii subsp.bulgaricus were purchased in SantaFe(Argentina)supermarkets and immediately examinedmicrobiologically.One of the products purchased wasa soft,rindless cheese with S.thermophilus used asa starter culture and containing L.acidophilus andbidobacteria;it had a moisture content of 58%,a fatcontent of 12%,a ripening time ranging of 20 days anda shelf life of 1 month.Samples(1.0 ml)of the products(except soft cheese)were decimally diluted in sterile pep-tone water(0.1%)and 0.1 ml aliquot dilutions werespread over the surfaces of plates of the di!erent media.Plates of LBD-and Bile-MRS agar were incubated aero-bically and plates of LP-MRS agar were incubated an-aerobically(GasPak,Oxoid)at 373C for 3 days.In thecase of soft cheese,samples used for microbiologicalassays were collected aseptically into sterile#asks.Por-tions of 20 g were homogenized with a 180 ml sterilealiquot sodium citrate solution(2%)for 3 min in a stom-acher.Decimal dilutions of the soft cheese homogenateswereproducedin peptone water(0.1%)and nally platedon the same media as before and incubated in the sameway.The experiments were replicated three times.3.Results and discussionTable 2 shows the plate counts of yoghurt bacteriafromlyophilisedculturesinthedi!erentculturemedia under all the incubation conditions tested,whileTables 3 and 4 show the results obtained from thelyophilized cultures of L.acidophilus and B.bixdum,respectively.For B.bixdum cultures the data refer only toan incubationin the GasPak System because the cultureswere unable to grow under aerobic and anaerobic(com-bustion)conditions.SM agar was chosen as a referencemedium for the simultaneous colony count of Lactobacil-lus delbrueckii subsp.bulgaricus and S.thermophilus andMRS agar was chosen as a reference medium for L.acidophilus and B.bixdum.Since the highest colonycounts were obtained in these media,they were used toevaluate the cell recovery in the others.3.1.Performance of media for diwerential/selectivecolony counts3.1.1.Non selective mediaPreliminary studies carried out at our laboratory havedemonstrated that SM agar was a very suitable mediumfor total counts of lactic acid bacteria in natural whey(Reinheimer,Sua H rez,Bailo&Zalazar,1995)and milkC.G.Vinderola,J.A.Reinheimer/International Dairy Journal 9(1999)497505499Table 2Enumeration(log CFU g1)of S.thermophilus(St)and L.delbrueckii subsp.bulgaricus(Lb)on di!erent media(72 h at 373C)from lyophilised yoghurtcultures.The values are the mean of three determinationsMediumIncubationunderYoghurt cultureY-SDW1W2JAStLbStLbStLbStLbSMAer.10.15!8.34!9.49!9.62!8.83!8.56!9.98!8.49!An.Comb.10.18!8.38!9.48!9.70!8.99!8.34!9.96!8.60!An.GasPak10.23!8.78!9.49!9.58!9.04!8.95!10.08!8.85!MRSAer.9.49,$8.32!9.208.008.728.34!An.Comb.9.45,$8.67!9.72!8.62!8.658.64!An.GasPak10.04!,8.30!8.709.65!8.238.67!8.768.52!M17Aer.9.91!,8.708.92!9.98!An.Comb.9.26,$8.778.70!9.90!An.GasPak9.48,$8.768.85!10.00!G-MRSAer.7.70#8.58!8.57#8.45!An.Comb.8.00#8.59!9.51!8.36!An.GasPak8.89$8.60!9.63!8.45!G-M17Aer.9.85!,An.Comb.9.87!,An.GasPak9.87!,T-MRSAer.An.Comb.An.GasPakLBDAer.10.26!8.50!9.04#8.76#8.70!8.46!10.15!8.34!An.Comb.10.38!8.65!9.52!9.208.91!8.62!10.15!7.95!An.GasPak9.96!,8.40!9.23$9.26!,8.88!8.58!10.18!8.85!LacticAer.9.95!,8.48!8.789.30!,7.48#8.56!9.49!An.Comb.9.90!,8.58!8.709.30!,8.118.84!9.36!7.00An.GasPak10.04!,8.76!8.90#9.41!,8.88!8.58!8.188.85!L-SAer.9.93$8.41!9.00#9.40!,9.04!9.15!9.78!7.30An.Comb.9.86$8.46!8.41%9.30!,9.15!8.96!9.62!7.30An.GasPak9.91$8.46!9.18$9.59!9.12!8.089.91!8.60!NPNL-MRSAer.9.30,$7.708.49%9.48!,7.00$9.48!8.28!An.Comb.8.99,$7.638.54%,9.30!,7.32#9.60!8.32!An.GasPak9.36,$7.788.53%,9.43!,7.48#9.61!8.30!NNL-MRSAer.An.Comb.An.GasPakNKB-MRSAer.9.22,$7.98!7.62&7.13$8.94!An.Comb.9.17,$8.13!7.89&7.27#8.85!An.GasPak9.59$8.18!7.85&7.62#9.08!Bile-MRSAer.An.Comb.An.GasPakOG-MRSAer.An.Comb.An.GasPakLP-MRSAer.An.Comb.An.GasPak!,#,$,%,&Means in row with a common superscript do not di!er(P0.05).Aer.:aerobiosis;An.Comb.:anaerobiosis by combustion;An.GasPak:anaerobios by GasPak System.:no growth.500C.G.Vinderola,J.A.Reinheimer/International Dairy Journal 9(1999)497505Table 3Enumeration(log CFU g1)of L.acidophilus on di!erent media(72 hat 373C)from lyophilised cultures.The values are the mean of threedeterminationsMediumIncubationunderCultureLAILA5SMAer.10.45!10.45!An.Comb.10.64!10.54!An.GasPak10.66!10.41!MRSAer.10.84!10.64!An.Comb.10.84!10.86!An.GasPak10.86!10.79!M17Aer.9.899.41An.Comb.9.5610.34!An.GasPak9.5110.58!G-MRSAer.10.68!10.34!An.Comb.10.77!10.66!An.GasPak10.78!10.34!G-M17Aer.10.68!10.58!An.Comb.10.64!10.72!An.GasPak10.81!10.52!T-MRSAer.10.74!10.62!An.Comb.10.60!10.71!An.GasPak10.82!10.75!LBDAer.10.53!10.58!An.Comb.10.53!10.86!An.GasPak10.81!10.81!LacticAer.10.72!10.45!An.Comb.10.71!10.58!An.GasPak10.83!10.74!L-SAer.10.63!10.43!An.Comb.10.70!10.48!An.GasPak10.80!10.57!NPNL-MRSAer.10.04!,10.08!An.Comb.10.11!,10.10!An.GasPak10.11!,10.18!NNL-MRSAer.9.60An.Comb.9.62An.GasPak9.66NKB-MRSAer