taxonomy and physiology of probiotic lactic acid bacteria

International Journal of Food Microbiology 41 (1998) 103–125

Taxonomy and physiology of probiotic lactic acid bacteria

1*¨Gunter Klein , Alexander Pack, Christine Bonaparte , Gerhard Reuter

¨Institute of Meat Hygiene and Technology, Veterinary Faculty, Free University of Berlin, Brummerstr. 10, D-14195 Berlin, Germany

Accepted 6 March 1998

Abstract

The current taxonomy of probiotic lactic acid bacteria is reviewed with special focus on the genera Lactobacillus,Bifidobacterium and Enterococcus. The physiology and taxonomic position of species and strains of these genera wereinvestigated by phenotypic and genomic methods. In total, 176 strains, including the type strains, have been included.Phenotypic methods applied were based on biochemical, enzymatical and physiological characteristics, including growthtemperatures, cell wall analysis and analysis of the total soluble cytoplasmatic proteins. Genomic methods used were pulsedfield gel electrophoresis (PFGE), randomly amplified polymorphic DNA-PCR (RAPD-PCR) and DNA–DNA hybridizationfor bifidobacteria. In the genus Lactobacillus the following species of importance as probiotics were investigated: L.acidophilus group, L. casei group and L. reuteri /L. fermentum group. Most strains referred to as L. acidophilus in probioticproducts could be identified either as L. gasseri or as L. johnsonii, both members of the L. acidophilus group. A similarsituation could be shown in the L. casei group, where most of the strains named L. casei belonged to L. paracasei subspp. Arecent proposal to reject the species L. paracasei and to include this species in the restored species L. casei with a neotypestrain was supported by protein analysis. Bifidobacterium spp. strains have been reported to be used for production offermented dairy and recently of probiotic products. According to phenotypic features and confirmed by DNA–DNAhybridization most of the bifidobacteria strains from dairy origin belonged to B. animalis, although they were often declaredas B. longum by the manufacturer. From the genus Enterococcus, probiotic Ec. faecium strains were investigated with regardto the vanA-mediated resistance against glycopeptides. These unwanted resistances could be ruled out by analysis of the39 kDa resistance protein. In conclusion, the taxonomy and physiology of probiotic lactic acid bacteria can only beunderstood by using polyphasic taxonomy combining morphological, biochemical and physiological characteristics withmolecular-based phenotypic and genomic techniques. 1998 Elsevier Science B.V.

Keywords: Lactic acid bacteria; Molecular typing; Physiology; Probiotics; Taxonomy

1. Introduction

*Corresponding author. Tel.: 1 49 30 838 2793; fax: 1 49 30Lactic acid bacteria (LAB) comprise a wide range838 2792.

1 of genera including a considerable number of¨Present address: Institut fur Milchforschung und Bakteriologie,species. It is generally accepted (Stiles and Holzap-¨ ¨Universitat fur Bodenkultur, Gregor-Mendel-Str. 33, A-1180

Wien, Austria. fel, 1997) that LAB are Gram-positive and usually

0168-1605/98/$19.00 1998 Elsevier Science B.V. All rights reserved.PI I : S0168-1605( 98 )00049-X

104 G. Klein et al. / International Journal of Food Microbiology 41 (1998) 103 –125

catalase-negative bacteria which grow under mi- a major end product. Some physiological characteris-croaerophilic to strictly anaerobic conditions, and are tics are of interest for their function as probiotics, anon-spore forming. Endospore-forming lactic acid pre-condition of which is survival in the gastro-producing bacteria that are aerobic to facultatively intestinal tract. This is based on their resistance toaerobic are found in the genera Bacillus and low pH and/or bile and their temperature growthSporolactobacillus. Traditionally, Bacillus spp. are ranges (Fuller, 1989). These abilities are, however,not considered as LAB because of their physiological of minor taxonomic interest. Important physiologicaland biochemical characteristics. features for taxonomic considerations are carbohy-

The most important genera of LAB are Lac- drate fermentation patterns, resistance to differenttobacillus, Lactococcus, Enterococcus, Streptococ- NaCl concentrations, growth on different nutrientcus, Pediococcus, Leuconostoc, Weissella, Carnobac- media, growth at defined temperatures and resistanceterium, Tetragenococcus, and Bifidobacterium. against antibiotics. The taxonomy of lactobacilli hasPhylogenetically, Gram-positive bacteria are divided been based on these phenotypic properties for de-into two major branches. With the exception of the cades. They were divided into three subgenera,bifidobacteria, all the above-mentioned genera of ‘Thermobacterium’, ‘Streptobacterium’, andLAB belong to the Gram-positive phylum with a low ‘Betabacterium’ (Orla-Jensen, 1919, 1924; Sharpe etG 1 C (guanine plus cytosine) content ( , 50%) al., 1966; Reuter, 1973; Kandler and Weiss, 1986),(Schleifer and Ludwig, 1995). Nevertheless, the according to their growth temperatures and theirlatter are also considered as LAB, because of similar hexose fermentation pathway (homofermentative orphysiological and biochemical properties and the heterofermentative). Modern molecular methodssharing of some common ecological niches such as showed that these subgroups are inconsistent withthe gastro-intestinal tract (GIT). the phylogenetical relationship of the species

Species of these genera can be found in the GIT of (Schleifer, 1987). The new molecular based group-man and animal and also in fermented food. Strains ing has been reviewed by Hammes and Vogel (1995)used as probiotics usually belong to species of the and is therefore only summarized in Table 1 togethergenera Lactobacillus, Enterococcus, and Bifidobac- with the classical system of the subgenera. Hammesterium. These LAB are typically chemoorgano- and Vogel (1995) based their subdivision of thetrophic and ferment carbohydrates with lactic acid as lactobacilli on the peptidoglycan type of the cell wall

Table 1Division of the genus Lactobacillus into subgenera (no longer valid) and into molecular based subgroups

Group of lactobacilli Fermentation pathway Growth temperature Representatives used as probioticsaSubgenera

b‘Thermobacterium’ Homofermentative 158C negative L. acidophilus group208C negative458C positive

b‘Streptobacterium’ Homofermentative 158C positive L. casei groupc458C negative L. sake /curvatus

b‘Betabacterium’ Heterofermentative No general rule L. reuteri /L. fermentumdMolecular based subgroups

Group A Obligatory homofermentative, Not applicable L. acidophilus groupno fermentation of pentoses

Group B Facultatively heterofermentative Not applicable L. casei group, L. sake /curvatus(gas from pentoses)

Group C Obligatory heterofermentative Not applicable L. reuteri /L. fermentum(gas from glucose and pentoses)

aAccording to Sharpe et al. (1966).bGas from glucose.cUsed as protection culture.dSummarized by Hammes and Vogel (1995).

G. Klein et al. / International Journal of Food Microbiology 41 (1998) 103 –125 105

and the fermentation pathway of pentoses and hex- relevant for probiotics, namely L. gasseri and L.oses. johnsonii, were described quite recently in the years

Modern taxonomic methods applied to LAB in- from 1980 to 1992. This may be the reason why theclude both phenotypic characterization and genotypic identification of strains as L. acidophilus was quiteanalysis. Phenotypic methods used are the analysis common and not justified in most cases. The speciesof the cell wall composition (especially for cannot easily be differentiated by classical methods.bifidobacteria), protein fingerprinting by analysis of Most of the descriptions in the literature are relatedthe total soluble cytoplasmatic proteins and the to L. acidophilus, although one of the other twoelectrophoretic mobility of certain enzymes. How- species is intended. Using DNA–DNA homologyever, fatty acid methyl ester (FAME) analysis does analysis two major groups could be defined by twonot seem to be reliable for LAB. The identification authors (Table 2) and identification is easily pos-of LAB is based on the molecular level with DNA– sible. We tried to confirm these results by proteinDNA homology analysis as reference method. Nu- fingerprinting.cleic acid probes have been developed for several Only L. gasseri, L. johnsonii and, in part, L.Lactobacillus species, often in combination with acidophilus and L. crispatus are used as probiotics.priming methods (Drake et al., 1996; Tilsala-Timis-¨jarvi and Alatossava, 1997). Classification is also 1.1.2. L. casei group

possible with genotypic methods if RAPD-PCR, This group comprises the recently revived L. zeaeribotyping or similar techniques are applied. and L. casei, L. paracasei and L. rhamnosus. The

The following groups of bacteria are of special latter three are used as probiotics in man and animal.interest with regard to their application as probiotics Historically, the L. casei group comprised only oneand their taxonomy. We applied two or more of the species, L. casei, which was divided into five sub-above-mentioned methods to classify or identify species: the L. casei subspecies casei, alactosus,strains of these species. pseudoplantarum, tolerans and rhamnosus. Collins

et al. (1989) proposed a reclassification of the L.1.1. Groups within the genus Lactobacillus casei group. They described two new species: L.

paracasei and L. rhamnosus. The former subspecies1.1.1. L. acidophilus group L. casei casei was transferred to the species L. casei

In addition to L. acidophilus sensu stricto five without any subspecies. On the other hand, L.other species form part of this group. If one consid- paracasei comprised two subspecies: the subspeciesers the chronological order of the descriptions of paracasei including the former L. casei subspeciesthese six species (Table 2) it is obvious that only L. alactosus and pseudoplantarum and the subspeciesacidophilus sensu stricto has been known for a long tolerans from the L. casei subspecies with the sametime. This species was described by Moro (1900) as name.‘Bacillus acidophilus’ and renewed by Hansen and The new species L. rhamnosus consisted only ofMoquot (1970). L. crispatus was described in 1953, the strains of the former subspecies rhamnosus.and the other four species, including the species However, the description of the new species was

Table 2Taxonomic description and DNA-homology groups of the species of the L. acidophilus group

Species First description DNA-homology group

Johnson et al. (1980) Lauer et al. (1980)

L. acidophilus sensu stricto Moro, 1900; Hansen and Moquot, 1970 A1 IaL. crispatus Brygoo and Aladame, 1953; Cato et al., 1983 A2 IbL. amylovorus Nakamura, 1981 A3 IcL. gallinarum Fujisawa et al., 1992 A4 Id

L. gasseri Lauer and Kandler, 1980 B1 IIaL. johnsonii Fujisawa et al., 1992 B2 IIb


based only on a limited number of DNA–DNA strain of L. fermentum biotype IIb (Hansen, 1968).homology experiments and the phenotypical descrip- The strain was deposited by Reuter in 1964 at thetion of the strains was very poor. Only L. rhamnosus American Type Culture Collection (ATCC 23272)could be easily identified. The cell wall of L. and was later transferred to the Deutsche Sammlungrhamnosus contains rhamnose and it is one of the von Mikroorganismen (DSM 20016). Kandler et al.few species that is able to ferment rhamnose. L. casei (1980) described this biotype as L. reuteri, a newand L. paracasei could not easily be differentiated species of heterofermentative lactobacilli, accordingbiochemically. Therefore, the taxonomic position of to DNA–DNA homology.L. paracasei was unclear. L. reuteri is of great interest as a bacteriocin

These historical developments are important for producing probiotic and is used in animal nutrition asthe understanding of the recent discussions. Dicks et well as in yoghurt type products and pharmaceutical. (1996) proposed a revision of the L. casei group preparations (Casas and Dobrogosz, 1996; Dob-after a first ‘Request for an Opinion’ in 1991, which rogosz and Casas, 1996). The species L. reuteri andwas not successful (Dellaglio et al., 1991). Only the L. fermentum are phenotypically closely related. It isspecies L. rhamnosus was not subject to alterations obvious that a differentiation based on biochemicalfor the reasons mentioned above. But, according to features is difficult, but is possible if growth tem-Dicks et al. (1996), L. paracasei should be rejected peratures are tested under controlled conditionsand all strains should be included in the species L. (Reuter, 1997b). Moreover, a molecular phenotypiccasei. Furthermore, the type strain of L. casei, ATCC characteristic is a useful tool for differentiating these393, should be transferred to the revived species L. two species. The peptidoglycan type differs sig-zeae. The current status of this proposal and the nificantly between L. reuteri and L. fermentumposition of the type strains and important reference (Table 4).strains are shown in Table 3. As the species L. caseiconsists at the moment only of the type strain and a 1.2. The genus Enterococcusfew additional strains, this alteration is very im-portant for the naming of production strains. All Strains of enterococci are mainly used in pig andprobiotic strains, even those which are named L. poultry nutrition. However, there are pharmaceuticalcasei, taxonomically belong either to L. paracasei as products which contain enterococci as probioticdescribed by Collins et al. (1989) or to the changed cultures for human clinical therapy. The genusand enlarged species L. casei as proposed by Dicks Enterococcus harbours many different species (De-et al. (1996). vriese and Pot, 1995), but only two of them are of

importance as probiotics: Ec. faecium is mainly used1.1.3. L. reuteri /fermentum group as an animal probiotic but also for human applica-

A specific biotype of Lactobacillus fermentum tion, whilst Ec. faecalis is primarily used as a human(biotype IIb) was first isolated by Lerche and Reuter probiotic. These two species can easily be differen-(1962). Strain F 275 was designated as the reference tiated from each other by a fermentation reaction

Table 3Alterations in the L. casei group as proposed by Dicks et al. (1996)

Species Status Alteration Type strain Reference strainsa T TL. zeae Revived name ATCC 15820 (former Inclusion of ATCC 393 (current type strain of L. casei)

L. rhamnosus strain)b TL. casei Inclusion of Neotype ATCC 334 Inclusion of DSM 5622 (current type strain of L. paracasei paracasei)

L. paracaseibL. paracasei Rejection Type strain should be Inclusion of all strains in L. casei

subspp. included in L. caseia TL. rhamnosus No changes ATCC 7469 Except for transfer of ATCC 15820 to L. zeae no changes

aIn force.bStill under discussion in the International Committee on Systematic Bacteriology (ICSB).


Table 4Discriminating features of L. fermentum and L. reuteri (modified according to Holzapfel et al., 1996) and own examinations

aSpecies Strain Cell wall Gas Ara Cell Man Treh Xyl Nit Mot 158C 208C 458C

Discriminating featuresL. reuteri Orn-D Asp na 1 2 2 2 2 na na na 2 naL. fermentum Lys-D Asp na v v ( 1 ) v v na na na 1 na

Own examinationsTL. reuteri DSM 20016 nd 1 1 1 2 2 2 nd 2 1 2 2 1

L. reuteri F 275 nd 1 1 1 2 2 2 nd 2 2 2 2 1

L. reuteri PE 3 nd 1 1 1 2 2 2 nd 2 2 2 2

TL. fermentum ATCC 14931 nd 1 2 2 2 2 nd 1 2 2 1 1

aType of peptidoglycan; Gas, gas from glucose; Ara, L( 1 )-arabinose; Cell, cellobiose; Man, mannitol; Treh, trehalose; Xyl, xylose; Nit,nitrate reduction; Mot, motility; 158C, 208C and 4560.18C, growth (waterbath); v, variable; nd, not done; na, not identified as adiscrimination feature.

(arabinose, sorbitol) and different growth tempera-tures (508C, for instance) (Facklam and Collins,1989; Nusser, 1991; Devriese and Collins, 1993).Strains of these two species are often isolated fromclinical material. They cause nosocomial infectionsin the immunocompromised and hospitalized host. Insome cases these enterococci are resistant against theantibiotics of first choice (i.e. ampicillin), and theycan acquire an additional resistance, the resistanceagainst glycopeptide antibiotics (e.g., vancomycinand teicoplanin, both of which can induce transfer-able resistance) (Leclercq and Courvalin, 1996), usedin the therapy of multiresistant enterococci. If thesestrains become glycopeptide-resistant the infection is

Fig. 1. Conventional identification key for the B. longum /animalisnot curable. Therefore, glycopeptide-resistant en-group according to Bonaparte and Reuter (1996).terococci should not be spread via the food chain as

probiotic cultures to hospital kitchens and to im-munocompromised hosts (Bates et al., 1994). The differentiation methods have been described, includ-spread of resistant bacteria into the gut or the ing protein electrophoresis of special enzymes (Royenvironment and the acquisition of special antibiotic et al., 1994), PFGE and PCR (Roy et al., 1996). Theresistances are unwanted side-effects in the applica- identification is only possible with molecular pheno-tion of probiotics. typic (cell wall analysis, Fig. 2a,b) or genotypic

(DNA–DNA homology) techniques. Additionally,1.3. Groups within the genus Bifidobacterium classification methods have been described using

numerical analysis of enzyme and fermentation1.3.1. B. longum /animalis group patterns (Gavini et al., 1991).

The most important species with regard to their Recently, Stiles and Holzapfel (1997) reviewedapplication as probiotic cultures are B. longum and the current taxonomy of lactic acid bacteria related toB. animalis. It seemed to be possible to differentiate food and feed. Another review by Charteris et al.these species based on their biochemical reactions, (1997) focused on rapid detection and selectionnamely melezitose fermentation (Fig. 1). But it was methods of probiotic lactobacilli and bifidobacteriashown (Bonaparte and Reuter, 1996) that this key including 16S and 23S rRNA-targeted hybridizationreaction is not applicable in every case, and especial- probes for the identification of species. This studyly not in strains isolated from dairy products. Several focuses on lactic acid bacteria present in probiotic


tion of the Institute of Meat Hygiene and Technologyof the Free University of Berlin and were subcul-tured prior to the investigation on MRS-Agar (DeMan et al., 1960; Reuter, 1985) under mi-croaerophilic conditions at 378C in the case oflactobacilli and enterococci. Bifidobacteria weresubcultured strictly anaerobically (Anaerocult A,Merck) on Biogarde agar (Reuter, 1990). The typestrains, important reference strains and probioticstrains from dairy or pharmaceutical products orstrains from the gastrointestinal or urogenital tractare listed in Tables 5 and 6.

2.2. Biochemical, physiological and enzymaticexaminations

Biochemical properties, such as fermentation ofcarbohydrates, gas production from glucose andgrowth at defined temperatures, were recorded usingclassical macrotube tests described by Lerche andReuter (1962) and Klein et al. (1995b) for all strains.

Fig. 2. Peptidoglycan type of important probiotic bifidobacteria Twenty-one carbon substrates were tested by acidifi-species. (a) Peptidoglycan type of B. longum, B. infantis and B.

cation reactions in a semi-solid medium (Reuter,suis. (b) Peptidoglycan type of B. animalis, B. choerinum, B.1964) after six days. Growth at a defined temperaturecuniculi and B. ruminantium. L-Ala, L-alanine; L-Orn, L-or-

nithine; MurNAc, N-acetylmuramic acid; D-Ala, D-alanine; L- was recorded after three days at 1560.18C, after twoThr, L-threonine; GlcNAc, N-acetylglucosamine; L-Lys, L-lysine; days at 2060.18C, and after one day at 45 orL-Ser, L-serine; D-Glu, D-glucosamic acid. 5060.18C, respectively.

Carbohydrates and their derivatives tested were:L( 1 )-arabinose (Merck 1492), D( 1 )-glucose(Merck 8342), lactose (Merck 7657), D( 1 )-sucrose

products and on their physiological characteristics (Merck 7651), D( 1 )-maltose (Merck 5910), D( 1 )-and their systematics, with special emphasis on trehalose (Merck 8353), D( 1 )-melibiose (Merckmolecular-based typing methods. Therefore, strains 12240), D( 1 )-cellobiose (Merck 2352), D( 1 )-raf-of the most important genera and species have been finose (melitose) (Merck 7549), D(2)-mannitolinvestigated by both classical phenotypic methods (Merck 5982), D( 1 )-salicin [2-o-(-D-such as biochemical fermentation patterns and mo- glucopyranosido)-benzylalcohol] (Merck 7665),lecular phenotypic or genotypic methods. L( 1 )-rhamnose (Merck 4736), D( 1 )-xylose

(Merck 8692), D( 1 )-mannose (Merck 5984),D( 1 )-melezitose (Serva 28550), myo-inositol(Merck 4728), D(2)-sorbitol (Merck 7758), D( 1 )-

2. Material and methods inulin (Merck 4733), dextrin (Merck 3006), D( 1 )-galactose (Merck 4062), D(2)-fructose (Merck

2.1. Bacterial strains 5323), D(2)-ribose (Merck 7605), L-arginine(Merck 1542), amygdalin (Fluka 10050) and Na-

Strains investigated in this study included the type gluconate (Merck 822058).strains of all relevant species of probiotic LAB. For bifidobacteria special miniaturized biochemi-Lyophilised cultures were kept in the culture collec- cal test kits (API, Biomerieux, France) were used.


Table 5aType strains of bacterial species investigated in this study

Species Strain designation (type strain) SourceTL. acidophilus DSM 20079 Human pharynxTL. crispatus DSM 20584 Human eye

TL. gallinarum LMG 9435 Crop, chickenTL. amylovorus DSM 20531 Intestine, cattleTL. gasseri DSM 20243 Human intestine

TL. johnsonii LMG 9436 Human bloodTL. casei ATCC 393 CheeseTL. paracasei paracasei DSM 5622 Unknown

TL. rhamnosus ATCC 7469 Pharmaceutical preparationTL. reuteri DSM 20016 Human intestine

TL. fermentum ATCC 14931 Fermented beetsTEc. faecium DSM 20477 UnknownTEc. faecalis DSM 20478 Unknown

TB adolescentis var. a CCUG 18363 Adult intestineTB. angulatum DSM 20098 Human faeces

TB. animalis ATCC 27527 Rat intestineTB. asteroides DSM 20089 Hindgut honeybeeTB. bifidum DSM 20456 Infant faecesTB. boum DSM 20432 Bovine rumenTB. breve var. a DSM 20213 Infant intestine

TB. catenulatum CCUG 18366 Human faecesTB. choerinum DSM 20434 Piglet faecesTB. coryneforme DSM 20216 Hindgut honeybeeTB. cuniculi DSM 20435 Rabbit faeces

TB. dentium CCUG 18367 Dental cariesTB. gallicum DSM 20093 Adult intestine

TB. gallinarum ATCC 33777 Chicken cecumTB. globosum ATCC 25865 Bovine rumen

TB. indicum DSM 20214 Hindgut honeybeeTB. infantis DSM 20088 Infant intestineTB. longum var. a DSM 20219 Adult intestineTB. magnum DSM 20222 Rabbit faecesTB. minimum DSM 20102 SewageTB. pseudocatenulatum DSM 20438 Infant faecesTB. pseudolongum DSM 20099 Pig faecesTB. pullorum DSM 20433 Chicken faecesTB. subtile DSM 20096 Sewage

TB. suis CUETM 89-42 Pig faecesTB. thermophilum DSM 20210 Pig faeces

aType strains not listed in the text or in the tables and figures are included as follows: Enterococcus type strains have been used as referencestrains for the identification of Enterococcus strains; Bifidobacterium type strains are all included in the dendrogram of Fig. 13 and cannotbe shown in full detail. They were used for the construction of a meaningful dendrogram.DSM, Deutsche Sammlung von Mikroorganismen, Braunschweig; LMG, Laboratorium voor Microbiologie, Gent; ATCC, American Type

¨ ´Culture Collection, Rockeville, MD; CCUG, Culture Collection University of Goteborg; CUETM, Collection Unite Ecotoxicologie,Villeneuve d’Ascq.

The test included 49 carbon substrates (acidification was calculated using the Dice index and the un-reactions, API 50 CH) and 89 enzymatic tests (LRA weighted pair group average linkage method.ZYM strips). The test was performed and interpreted For the delimitation of the species Ec. gallinarumaccording to Gavini et al. (1991) and a dendrogram and Ec. casseliflavus from the Ec. faecium group and


Table 6Probiotic strains from dairy or pharmaceutical products or strains from the gastrointestinal or urogenital tract investigated in this study

Genus /species Source No. of strains

L. acidophilus Pharmaceutical preparations 9Dairy products 10

L. crispatus Gastrointestinal tract 1Dairy products 1

L. gasseri Gastrointestinal tract 23Vaginal tract 2Pharmaceutical preparations 2

L. johnsonii Pharmaceutical preparations 2Dairy products 7Gastrointestinal tract 1

L. paracasei subspp. Pharmaceutical preparations 2Dairy products 1

L. rhamnosus Pharmaceutical preparations 1Dairy products 6Intestinal tract 1Urogenital tract 6

L. reuteri Human faeces 1Pigeon crop 1

Ec. faecium Feed probiotics 5Bifidobacterium spp. Intestinal tract / faeces 17

Dairy products 30Unknown 8

from Ec. faecalis the acidification of methyl-D- ized. Normalization of densitometric traces withglucopyranoside (Sigma, St. Louis, MO) was tested background subtraction (rolling disk) and conversionfor all isolates additionally (Devriese et al., 1996) were carried out with GelCompar version 3.1 (Ap-

plied Maths, Kortrijk, Belgium). Clustering was2.3. Numerical analysis of the total soluble performed by the unweighted average pair groupcytoplasmatic protein patterns with colloidal method with ‘none alignment’.Coomassie-blue

2.4. DNA extractionCulture of strains, protein purification and de-

termination of protein concentration were performed DNA from strains of the Lactobacillus acidophilusas described by Klein et al. (1995b) with the group was extracted according to Klaenhammerfollowing modifications (Klein et al., 1996; Pack, (1984) with the utilization of mutanolysine

21 211997): cells were harvested by rinsing of two agar (1 mg ml ) and lysozyme (1 mg ml ) for pulsedplates with 5 ml of 0.9% NaCl each. The protein field gel electrophoresis (PFGE) and the proceduressamples were diluted to a standard concentration of of Ausubel et al. (1991) for the randomly amplified

211.0 mg ml . Electrophoresis was carried out with polymorphic DNA-PCR (RAPD-PCR) with CTAB/Tris-Glycine-gradient gels (8–16%, 15 traces, NaCl. The concentration of purified DNA wasNovex) in a Novex Xcell apparatus, three gels per determined by a DNA-Fluorometer TKO (Hoefer).strain, with markers (Mark12, Novex) in positions 1,4, 8, 12, 15. Proteins were stained with Colloidal 2.5. Pulsed field gel electrophoresis (PFGE)Coomassie blue (Novex) and gels were dried with aspecial drying solution (Anarapid, Anamed, Ger- PFGE was performed according to Goering andmany) between two sheets of cellophane. The stained Duensing (1990) and Miranda et al. (1991) withprotein patterns were scanned at 1200 dpi (High- some modifications. Briefly, cells were embedded inscreen Flatbed colour IIs, Laserscanner) and digit- low-melting-point agarose prior to DNA extraction.


Several restriction endonucleases were tested. Diges- OPV-14 59-AGA TCC CGC C-39 (70% G 1 Ction with SmaI was most effective and the lac- content) and for OPV-19 59-GGG TGT GCA G-39

tobacilli strains were digested with this enzyme. The (70% G 1 C content). The gels were stained andrestriction fragments were separated by PFGE over visualized as described for PFGE.28 h. The initial pulse time (the time the current field The methods described in this section were ap-remains in one orientation before changing) of 5 s plied where appropriate, i.e. not every species orwas prolonged continually over the first 15 h to a strain was examined by all the methods described.pulse time of 60 s, and from hour 15 to hour 28 the Biochemical reactions were performed for all speciespulse time changed continually from 60 to 90 s. The and genera, protein fingerprinting was applied for allincluded angle (change in the orientation of the field) genera except the bifidobacteria, the API system waswas 1208. Gels were stained with ethidium bromide applied only for the bifidobacteria and DNA exami-for 15 min and examined by UV transillumination. nation was mainly carried out for strains from the L.

acidophilus group.2.6. Randomly amplified polymorphic DNA-PCR(RAPD-PCR)

3. ResultsRAPD-PCR was performed according to the

protocol described by Piehl (1995). For the amplifi- The results are presented separately for each genuscation a TRIO-Thermoblock (Biometra) was used and its taxonomic groups.with the following thermocycler program: 948C for2 min, 398C for 2 min, 728C for 2 min (two cycles) 3.1. Lactobacillus acidophilus groupand 948C for 15 s, 398C for 15 s, 728C for 1 min (35cycles). Several primers were tested and the follow- Classical phenotypic characterization of the L.ing primers were used because of their discriminat- acidophilus group resulted in different biotypes asory power: KAY3 (TIB MOLBIOL, Berlin) and already described by Reuter (1964). It was notOPV-06, -08, -14 and -19 (all from OPERON possible to define a specific biotype for one speciesTechnologies Inc., Alameda). The sequences were or to connect one species with one biotype (Table 7).for KAY3 59-CTG GCG ACT G-39 (70% guanine 1 For example, one species included different biotypescytosine [G 1 C] content), for OPV-06 59-ACG CCC (L. acidophilus) or one biotype was present inAGG T-39 (70% G 1 C content), for OPV-08 59- different species (biotype III).GGA CGG CGT T-39 (70% G 1 C content), for The analysis of the total soluble cytoplasmatic

Table 7Fermentation patterns and physiological characteristics within the L. acidophilus group (modified according to Mitsuoka, 1992, and Stilesand Holzapfel, 1997) and ‘key’ reactions of fermentation patterns

aSpecies Trehalose Melibiose Raffinose Lactose Growth in NaCl Biotype

3.5% 4.5% 5.0%

L. acidophilus v v 1 1 1 2 2 2 II, III, IVL. crispatus 1 1 1 1 1 1 v 1 2 2 IIIL. amylovorus 1 1 2 2 1 2 2 2 IL. gallinarum 2 1 1 1 1 v 1 1 2 ?L. gasseri 1 1 2 2 /v 1 1 1 v IL. johnsonii 1 1 1 1 1 1 v 1 1 1 III

aBiotypeI 1 1 2 2

II 1 1 2 1 1

III 1 1 1 1 1 1

IV 2 2 1 1

aBiotype according to Reuter (1964). v, variable.


protein patterns of strains from the L. acidophilus a relationship of r 5 68.2%. The strains of proteingroup resulted in two major protein groups, group A group A clustered at r 5 75.9%, and those of proteinand B (Fig. 3). Protein group A consisted of strains group B at r 5 72.9%. These groups are consideredof L. acidophilus sensu stricto, L. crispatus, L. as major protein groups. Minor groups could beamylovorus and L. gallinarum, including the type distinguished within these major groups. Withinstrains of these species. Group B contained the protein group A, four subgroups could be differen-species L. gasseri and L. johnsonii. Both groups had tiated, A1 to A4 (Fig. 4). Subgroup A1 contained all

Fig. 3. Dendrogram derived from the protein pattern analysis by Colloidal Coomassie blue staining of the Lactobacillus acidophilus group. rindicates the percentage of similarity. Analysis of the whole spectrum of protein patterns.


Fig. 4. Dendrogram derived from the protein pattern analysis by Colloidal Coomassie blue staining of the Lactobacillus acidophilushomology group A1 to A4 (Johnson et al., 1980); whole spectrum.

strains of the species L. acidophilus, including the strain of L. gasseri and included most of the isolatestype strain and probiotic strains. L. gallinarum was from the gastrointestinal tract. Group B2 was dividedincluded in this cluster, but the type strain of this into two clusters, from which one had a relationshipspecies could be differentiated as subgroup A4 from of r 5 44.5% to group B1 and the other r 5 61.5%.all other strains of subgroup A1 by partial analysis of The second cluster was more closely related to groupthe protein profiles (data not shown). The subgroups B1 than to the other B2 cluster, but these strainsA1 and A4 had a relationship to subgroups A2 and could be identified as L. johnsonii strains by RAPD-A3 of r 5 75.9%. Subgroup A2 included the type PCR (data not shown). Thus, group B2 consisted ofstrain of L. amylovorus and A3 the type strain and L. johnsonii strains including the type strain andreference strains of L. crispatus. most of the strains from dairy probiotic cultures.

Protein group B could be separated into several The partial analysis was necessary to differentiatesubgroups (Fig. 5) and a species differentiation some species within both major protein groups andbetween L. gasseri and L. johnsonii was not possible allowed a species-specific classification of strains.with the analysis of the whole protein spectrum. Furthermore, intra-specific differentiation was pos-After partial analysis of the most discriminating sible and strains of the same origin could be iden-bands two subgroups could be identified: subgroups tified.B1 and B2 (Fig. 6). Group B1 consisted of the type Pulsed field gel electrophoresis (PFGE) with the


Fig. 5. Dendrogram derived from the protein pattern analysis by Colloidal Coomassie blue staining of the Lactobacillus acidophilushomology group B1 and B2 (Johnson et al., 1980); whole spectrum.

restriction enzyme SmaI of all species of the L. primers are presented in this study. Fig. 8 shows theacidophilus group resulted in the typical DNA RAPD-PCR patterns of six different L. acidophiluspattern for each species (Fig. 7). The patterns were strains analyzed with four different primers. Eachreproducible and consisted of 11 to more than 15 primer resulted in a specific profile for these L.bands each. acidophilus strains, for instance primer OPV-04

RAPD-PCR was performed with more than 20 produced only one major band visible by UVdifferent primers. Only some of them were applic- illumination. In every case, strain 6 was differentable in the typing of Lactobacillus strains. Five from the other L. acidophilus strains but possessed


Fig. 6. Dendrogram derived from the protein pattern analysis by Colloidal Coomassie blue staining of the Lactobacillus acidophilus*homology group B1 and B2 (Johnson et al., 1980); partial analysis of the discriminating protein bands; B2 : confirmed as L. johnsonii by

RAPD-PCR analysis.

its own specific DNA pattern, which was characteris- 3.2. L. casei grouptic for the respective primer. Twenty-nine strains ofthe L. acidophilus group, including strains from all Biochemical and physiological characteristicssix species and one L. rhamnosus strain, are shown were not able to differentiate between the species ofin Fig. 9. They were analyzed by one single primer, the L. casei group with the exception of L. rham-primer KAY3. Each species has its specific band nosus (rhamnose fermentation).pattern with this primer and can be identified when Protein fingerprinting resulted in different clustersthe pattern for one specific primer is known. of the species of the L. casei group. Three clusters


fingerprinting technique was not useful because ofthe limited number of strains.

3.4. Enteroccocus

The five probiotic Enterococcus species were onlyinvestigated below the species level. Phenotypiccharacterization showed them belonging to Ec.faecium by biochemical and physiological key re-actions (data not shown). An intra-specific differen-tiation was made within these strains by proteinfingerprinting before and after a conjugation experi-ment with a vanA-positive strain (Fig. 11). It couldbe shown that the strains without the vanA genediffered significantly from the same strains afterincorporation of the vanA gene by conjugation (r 5

Fig. 7. DNA patterns as revealed by pulsed field gel electro- 87.8%). This was due to a resistance proteinphoresis of five type strains of the L. acidophilus group and the (39 kDa) which can be used as a phenotypic markertype strain of L. rhamnosus with the restriction enzyme SmaI. MI,

for enterococci harbouring the vanA gene.Marker (245–2000 kbp); MII, Marker (0.1–200 kbp); RH, typestrain of L. rhamnosus; GL, type strain of L. gallinarum; AM,type strain of L. amylovorus; GS, type strain of L. gasseri; JO, 3.5. Bifidobacteriumtype strain of L. johnsonii.

Twenty-six species of the genus Bifidobacteriumcould be defined according to the unweighted aver- with their type strains and 55 wild strains wereage pair group method (Fig. 10). Cluster R included examined by their biochemical, physiological andthe type strain of L. rhamnosus and 14 different L. enzymatic characters. Special focus was placed onrhamnosus strains isolated from the gastrointestinal the B. longum /animalis group, because of theiror urogenital tract. The relationship to cluster Z with occurrence in probiotic dairy products. No specificone single strain, the current type strain of L. casei key reactions applicable for all strains investigatedATCC 393, and to cluster C, with the type strain of could be detected. By numerical analysis of 49L. paracasei paracasei, was 89.5 and 87.5%, respec- fermentation reactions and 89 enzymatic reactions atively. Cluster C included another L. paracasei strain dendrogram showing different taxonomic groups wasand probiotic strains from a pharmaceutical and a created (Fig. 12). Seven clusters can be defined at adairy product. No probiotic strain could be clustered level of similarity of 52%. B. animalis strainsin cluster Z together with the L. casei type strain. (cluster III) and B. longum strains (cluster VII)

The PFGE patterns of the type strain of L. showed a relationship of r 5 37%. The B. longumrhamnosus differed significantly from those of the and B. infantis strains were grouped together in thetype strains from the L. acidophilus group (Fig. 7). same cluster. The other taxonomic groups were B.The same could be observed for the RAPD-PCR breve, B. boum /B. thermophilum, B. pseudolongum /pattern of the L. rhamnosus type strain (Fig. 9, strain B. globosum, B. bifidum and B. dentium /B. suis.30).

4. Discussion3.3. L. reuteri /fermentum group

4.1. Lactobacillus acidophilus groupPhenotypical examinations showed some differen-

tiation criteria between L. reuteri and L. fermentum Identification of species of the L. acidophilussuch as arabinose fermentation, nitrate reduction and group using biochemical and physiological charac-growth at 208C (Table 4). Application of the protein teristics only is unreliable, although some physiolog-


Fig. 8. RAPD-PCR of six L. acidophilus strains (lanes 1–6) with four different primers: I, OPV-06; II, OPV-08; III, OPV-14; IV, OPV-19. Mand L, marker.

ical characteristics have been used for the differentia- B with L. gasseri and L. johnsonii representing thetion (Mitsuoka, 1992; Stiles and Holzapfel, 1997). homology groups B1 and B2. Protein groups B1 andTwo important probiotic species, L. gasseri and L. B2 also consists of L. gasseri and L. johnsonii, in thejohnsonii, cannot be differentiated (Table 7). same order. These findings confirm earlier results

However, a molecular-based typing method, the obtained by protein electrophoresis and DNA probeanalysis of protein patterns, was able to differentiate hybridisations (Pot et al., 1993).between the two major DNA homology groups, PFGE is a strain-specific DNA typing methodaccording to Johnson et al. (1980) and Lauer et al. often used as an epidemiological tool (Tenover et al.,(1980). The major protein groups A and B confirm 1995). For taxonomic purposes the discriminatingexactly at the phenotypic level homology groups A power is generally too high to be used on the speciesand B of Johnson et al. (1980) or I and II of Lauer et level. However, it was possible to show typicalal. (1980) (Table 2, Fig. 3). Furthermore, after profiles for the type strains of all species of the L.application of partial analysis of the protein patterns acidophilus group (data for L. acidophilus sensuthe distribution of the protein groups is identical to stricto not shown), which were reproducible for thethe homology groups of the six species of the L. restriction enzyme SmaI (Fig. 7). This method isacidophilus group. L. acidophilus, L. crispatus, L. very reliable for quality assurance. It allows aamylovorus and L. gallinarum represent the DNA comparison of the identity of the original manufac-homology groups A1 to A4, respectively, and are turers stock-culture with the culture isolated from thesubdivided into the protein groups A1 to A4. The probiotic dairy (or other) products. A disadvantage issame scheme is applicable for DNA homology group the time-consuming procedure (4–5 days).


Fig. 9. RAPD-PCR of 12 L. acidophilus strains (lanes 1–8, 12–14, 29), nine L. gasseri strains (lanes 9–11, 16–21), four L. johnsonii strains(lanes 15, 22–24), one L. amylovorus strain (lane 25), one L. gallinarum strain (lane 26), two L. crispatus strains (lanes 27, 28) and one L.rhamnosus strain (lane 30) with one single primer (KAY3); M, marker.

Fig. 10. Dendrogram derived from the protein pattern analysis by Colloidal Coomassie blue staining of the Lactobacillus casei group. rindicates the percentage similarity. C, Z and R represent the new species groups according to Dicks et al. (1996) with C, L. casei; Z, L. zeae;and R, L. rhamnosus. P, strain from pharmaceutical product; M, strain from probiotic dairy product (yoghurt type).


primers is essential for good results. Several differentprimers have to be tried before a well-identifiedspecies can be recognised. Fig. 9 shows speciesidentification within the L. acidophilus group. Majorbands of amplicates (Piehl, 1995) are characteristicfor each species. The primer KAY3 seems to be veryuseful for the differentiation of the strains investi-gated in this study. Similar methods (PFGE andPCR) have been applied successfully for bifidobac-teria (Roy et al., 1996).

Fig. 11. Protein analysis of probiotic Ec. faecium strains beforeand after the transfer of the vanA gene (conjugation).

4.2. L. casei groupRAPD-PCR is a molecular-typing method which

is very suitable for identification of strains of the L. Our results strongly confirm the proposal of Dicksacidophilus group (Piehl, 1995; Pack, 1997). How- et al. (1996) for a revision of the L. casei group.ever, it is less time consuming than PFGE, but the Evidence on the phenotypical molecular level usingconstruction of a database is nearly impossible. If an protein analysis shows that there is no close relation-unidentified strain has to be investigated a set of ship between strains that have been declared as L.known strains has to be included in the same RAPD- casei by the producer of a probiotic product and thePCR procedure (Piehl, 1995). As can be seen (Figs. type strain of L. casei (Fig. 10). These strains, as8 and 9) the selection of one or more optimal well as other probiotic strains, cluster together with

Fig. 12. Dendrogram derived from 138 phenotypic characters (carbon substrates fermentation and enzymatic activities) based on unweighted*pair group average linkage. The bifidobacteria type strains of Table 5, which are not named in this dendrogram, remained unclustered. B.

ps., B. pseudolongum; B. glob., B. globosum; B. therm., B. thermophilum.


the L. paracasei paracasei type strain in cluster C. be valid for strain-specific characterization. As dem-The L. rhamnosus cluster R is well defined and onstrated by Holzapfel et al. (1996), some featuresseparated from both other clusters C and Z. It is may be variable (arabinose fermentation) and cannotevident that only three groups are present in the L. be used as key reactions. In the case of L. reuteri andcasei group. This is in agreement with the results of L. fermentum cell wall analysis is the method ofother investigations, where strains from the L. casei choice (Table 4). Other molecular-based techniquesgroup from other ecological niches have been in- such as gene probes or PFGE may also producevestigated (Hertel et al., 1993; Patarata et al., 1994). reliable results (McCartney et al., 1996). An addi-Therefore, the proposal of Dicks et al. (1996) to tional criterion may be the origin of the strain: aninclude all L. paracasei strains in the renewed isolate from the intestine usually belongs to thespecies L. casei and to include the L. casei type species L. reuteri rather than to L. fermentum.strain in the species L. zeae is strongly supported byour data. Another technique also supports this opin- 4.4. Enteroccocusion. Mori et al. (1997) showed by phylogeneticalanalysis of the genes encoding for 16S rRNA that L. The exclusion of the vanA gene is crucial for thezeae and L. casei, represented by their current type approvement of enterococci as probiotics. As shownstrains, are closely related and can be transferred to earlier (Klare et al., 1995) the 39 kDa protein isone species, L. zeae (Fig. 13). Furthermore, they expressed if the vanA gene is present in the strain.supported the rejection of L. paracasei and the Ec. faecium and Ec. faecalis strains, which arerevision of the species L. casei as proposed by Dicks vancomycin susceptible, can receive this gene byet al. (1996) and by this study. Protein fingerprinting conjugation, but probiotic strains differ significantlyconfirmed the proposals of Dicks et al. (1996). in the rate of transfer (Klein, 1996). They receive thePrevious studies showed that even an intra-species gene with a lower transfer rate than control ordifferentiation within the L. casei group was possible clinical strains. It is impossible to exclude reception(Klein et al., 1995b). of the vanA gene by conjugation, but one can

exclude the presence of the active vanA gene in a4.3. L. reuteri /fermentum group probiotic strain by phenotypic screening methods

such as protein analysis. Additionally, genetic tech-Phenotypical differentiation criteria between L. niques are applicable like the detection of the vanA

reuteri and L. fermentum can be shown. Because of gene by PCR (Klein et al., 1995a; Klare et al., 1995).the limited number of strains these criteria can only With RAPD-PCR and PFGE, further intra-specific

differentiation between probiotic and clinical strainsis possible (Piehl et al., 1995; Klein and Reuter,1997). Different screening methods can be appliedfor intra-specific differentiation of probiotic strainsfrom clinical strains.

4.5. Bifidobacterium

The phenotypic key reactions for the differentia-tion of the species B. longum and B. animalis (Fig.1) examined by Reuter (1963) and Mitsuoka (1969)for strains of human and animal origin cannot beapplied in general for strains of dairy origin(Bonaparte and Reuter, 1996). The application of the

Fig. 13. Unrooted tree showing the phylogenetic relationships of numerical analysis of 138 characters in total accord-the L. casei group as revealed by K values for 16S rDNAnuc ing to the method of Gavini et al. (1991) permittedaccording to the data of Mori et al. (1997). The evolutionary

the classification of B. longum and B. animalisdistances (K values) are the sum of the lengths of the horizontalnuc

lines between two species (Mori et al., 1997). strains in different clusters and hence to their dif-


ferentiation. However, the method did not allow the protein fingerprinting and numerical analysis ofdifferentiation of B. longum from B. infantis. This biochemical reactions have been applied as pheno-close relation between both species has already been typic methods, and PFGE and restriction enzymeshown by several authors using different methods analysis (REA) as genomic methods. Most of these(Scardovi et al., 1979; Lauer and Kandler, 1983; techniques were applied in this study. They must beBahaka et al., 1993). applied in combination, because only the so-called

DNA–DNA homology studies were performed polyphasic taxonomy (Vandamme et al., 1996) is(data not shown) in order to confirm the phenotypic able to differentiate species which are phenotypicallyclassification. They showed that most of the strains very similar but genotypically quite different.used in dairy products do not belong to the species Another main conclusion of this study is that mostB. longum, but to B. animalis (Bonaparte and Reuter, of the probiotic cultures used in probiotic products1996). Meile et al. (1997) recently described a new do not have the appropriate species designation. Thisspecies, B. lactis, which consisted exclusively of is the case for the genera Lactobacillus andoxygen-tolerant bifidobacteria isolated from fer- Bifidobacterium as shown for the L. acidophilusmented milk. Therefore, it would be interesting to group, the L. casei group and the B. longum /B.hybridize our strains with the B. lactis type strain. animalis group. Most of the bacteria declared as L.The DNA homologies should be about 30%, since acidophilus belonged either to L. johnsonii or to L.Meile et al. (1997) reported a DNA homology of ca. gasseri (Holzapfel et al., 1996; Pack, 1997; Pack et35% between the type strain of B. animalis and B. al., 1997). A similar situation could be shown in thelactis. However, according to Gavini (1997) the type L. casei group where nearly all ‘L. casei’ strainsstrain of B. animalis and B. lactis, both of which belonged to L. paracasei. Additionally, strainswere received from the DSM, highly hybridized named as ‘B. longum’ are often B. animalis strains in(85%). The latter hybridization was performed with dairy products. The reasons for the misidentificationthe optical renaturation technique, whereas Meile et are given above.al. (1997) used a newly developed method. The As a consequence, taxonomy (including identifica-taxonomic position of B. lactis must be reconsidered. tion and classification) is essential for quality assur-It should be ruled out that B. lactis strains are in fact ance as required by governmental law and by theB. animalis strains which are adapted to milk. consumer. It is obvious that knowledge of the genus

or the so-called species group of probiotic strains isnot sufficient. The species or subspecies must be

5. Conclusions identified reliably. Furthermore, for safety aspects(e.g., within L. rhamnosus or enterococci) intra-

The taxonomy and nomenclature of probiotic LAB species differentiation is advisable.is still changing. Since the description of the genus Molecular techniques should be able to solve theseLactobacillus by Kandler and Weiss (1986) and the problems and provide the basis for quality assurance.genus Bifidobacterium by Scardovi (1986) most of To underline the importance of the genera andthe taxonomic groups have undergone dramatic species examined in this study, Table 8 gives anchanges (Schleifer, 1987; Hammes et al., 1991; overview of probiotic dairy products and some otherKlein et al., 1994; Vandamme et al., 1996; Stiles and products on the European market (Reuter, 1997a).Holzapfel, 1997). The taxonomy is no longer based The species name and strain designation is givenmainly on physiological criteria such as growth according to the manufacturers’ designation. Thesetemperatures or fermentation key reactions. It is descriptions are often not in congruence with thebased on phenotypic and genomic characteristics current taxonomy of LAB, as could be shown inrevealed on the molecular level. Techniques which several cases (Hamilton-Miller et al., 1996; Holzap-have been applied with success are cell wall analysis fel et al., 1996; Pack et al., 1997).and protein analysis as phenotypic identification It is evident that probiotics should have no nega-methods and DNA–DNA hybridization, gene probes tive effect on the host or via the environment onand RAPD-PCR as genomic identification methods. public health. Therefore, careful selection of bacteri-For classification of strains at the species level al strains is important. An intra-specific differentia-


Table 8New probiotic products in Germany and Europe

aProduct code Producer and/or distributor Probiotic LAB (declaration)b(a) Yoghurt-type products

´A, June 1995 Nestle, D L. acidophilus La1B, Oct. 1995 Toni Lait AG, CH L. reuteri

L. acidophilus La5L. casei 01Bifidobacterium

C Campina Melkunie, NL L. acidophilus GillilandD Danone, F L. casei

¨E Sobbecke, D L. acidophilusBifidobacteriumL. casei

¨F Muller, D Bifidus longum 536G Bauer, D L. acidophilus

Bifidob. bifidumH, Aug. 1996 Tuffi, D L. acidophilus LA-H3

Bifidob. bifidum LB-H1L. casei LC-H2

b(b) Yoghurt drinksA Emmi, CH L. casei LGG

L. acidophilusSc. thermophilusBifidob. bifidum

¨B Sudmilch, D L. casei LGGC Campina Melkunie, NL L. acidophilus GillilandD Danone, B/D L. casei immunitassE Yakult, D/NL/UK/B L. casei ShirotaF Zott, D BactoLab cultures

¨G Schoppingen, D BB 536¨H Muller, D B. longum 536

I, Aug. 1996 Alpenmilch Salzburg, A L. caseiL. acidophilusSc. thermophilus‘L. bifidus’

¨J, Aug. 1996 Haska Upahl & Malmo, S L. plantarum 299K, Aug. 1996 Tuffi, D L. acidophilus LA-H3


(c) Fresh cheese and hard cheese (type Gouda and Maasdamer) and raw sausageA (cheese), Aug. 1996 Tuffi, D L. acidophilus LA-H3


B (raw sausage), May 1997 Reinert, D L. acidophilusL. caseiBifidobacterium

aDeclaration by the manufacturer.bAccording to Holzapfel et al. (1996) and own examinations.

tion is necessary to separate probiotic from potential analysis of enterococci. This intra-specific differen-pathogenic strains. This is possible with phenotypic tiation is not necessary for pure taxonomic purposes,fingerprinting techniques as has been shown in the but may serve safe biotechnology.


al., 1989). Request for an opinion. Int. J. Syst. Bacteriol. 41,Acknowledgements340–342.

De Man, J.C., Rogosa, M., Sharpe, M.E., 1960. A medium for the¨We thank Lilo Brautigam and Dorothea Jaeger forcultivation of lactobacilli. J. Appl. Bacteriol. 23, 130–135.

excellent technical assistance and Mr. Leinen (Free Devriese, L.A., Collins, M.D., 1993. Phenotypic identification ofUniversity of Berlin) for his photo documentation. the genus Enterococcus and differentiation of phylogenetically

distinct enterococcal species and species groups. J. Appl.¨We thank I. Piehl (Nurnberg) for the collaborationBacteriol. 75, 399–408.concerning the RAPD-PCR method. We thank E.

Devriese, L.A., Pot, B., 1995. The genus Enterococcus. In: Wood,¨ ¨Falsen (University of Goteborg, Goteborg, Sweden)B.J.B., Holzapfel, W.H. (Eds.), The Lactic Acid Bacteria, Vol.

for kindly providing strains. 2. Blackie Academic, London, pp. 327–367.Devriese, L.A., Pot, B., Kersters, K., Lauwers, S., Haesebrouck,

F., 1996. Acidification of methy-D-glucopyranoside: A usefultest to differentiate Enterococcus casseliflavus and Enterococ-

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