Detection of Antifungal Properties in Lactobacillus paracasei subsp. paracasei SM20, SM29, and SM63 and Molecular Typing of the Strains

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1 Journal of Food Protection, Vol. 68, No., 2005, Pages 9 Copyright, International Association for Food Protection Detection of Antifungal Properties in Lactobacillus paracasei subsp. paracasei SM20, SM29, and SM63 and Molecular Typing of the Strains SUSANNE MIESCHER SCHWENNINGER, * UELI VON AH, BRIGITTE NIEDERER, MICHAEL TEUBER, 2 A LEO MEILE Laboratory of Food Biotechnology and 2 Laboratory of Food Microbiology, Institute of Food Science and Nutrition, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland MS 03-50: Received 6 November 2003/Accepted 0 April 2004 ABSTRACT Lactobacilli isolated from different food and feed samples such as raw milk, cheese, yoghurt, olives, sour dough, as well as corn and grass silage, were screened for their antifungal activities. Out of,424 isolates tested, 82 were shown to be inhibitory to different yeasts (Candida spp. and Zygosaccharomyces bailii) and a Penicillium sp., which were previously isolated from spoiled yoghurt and fruits. Carbohydrate fermentation patterns suggested that a substantial portion, 25%, belonged to the Lactobacillus casei group, including L. casei, L. paracasei, and L. rhamnosus. The isolates SM20 (DSM454), SM29 (DSM455), and SM63 (DSM456) were classified by PCR using species-specific primers to target the corresponding type strains (L. casei, L. paracasei, and L. rhamnosus) as controls. Further molecular typing methods such as randomly amplified polymorphic DNA, pulsed-field gel electrophoresis, and sequencing analysis of the 6S rrna gene allowed classifying strains SM20, SM29, and SM63 as L. paracasei subsp. paracasei in accordance with the new reclassification of the L. casei group proposed by Collins et al. (Int. J. Syst. Bacteriol. 39:05 08). Fungal growth is a frequent problem in a variety of food and feed, causing spoilage associated with considerable economic losses. Moreover, the accumulation of mycotoxins in food products is an important public health problem. To inhibit undesirable fungal growth and to extend the shelf-life of susceptible foods and feed, different additives are used that then create the problem of indigestibilities or allergies. As a result, consumer concerns regarding these chemical preservatives have been increasing over the last few years, accompanied by a growing interest in biopreservation, i.e., the use of particular microorganisms and/or their metabolites to prevent spoilage (4). Due to their antimicrobial properties and natural occurrence in many fermented foods and feed, lactobacilli are an important group in this field. Along with the production of organic acids like lactic and acetic acid, lactobacilli are well represented in the class of bacteriocin producers and are also known to excrete ethanol, hydrogen peroxide, carbon dioxide, diacetyl, acetaldehyde, and other fermentation compounds (7). Although many studies have assessed the antibacterial effects of Lactobacillus spp. and their bacteriocins, as reviewed by Klaenhammer et al. (5), only a few research groups have studied antifungal compounds and their potential to increase food safety (3,, 7, 8, 9, 2). The aim of the present study was to obtain suitable Lactobacillus strains for biopreservation of food: first to select strains that inhibit spoilage fungi and then to clearly * Author for correspondence. Tel: ; Fax: ; susanne.miescher@ilw.agrl.ethz.ch. identify them at the molecular level. In the last few years, different reclassifications have taken place within the genus Lactobacillus (2, 5, 6, 8). Molecular typing such as sequencing analysis of the 6S rrna gene, species-specific PCR approaches, randomly amplified polymorphic DNA (RAPD), and pulsed-field gel electrophoresis (PFGE) allowed us to put suitable strains in an updated and modern classification system, such that their status as food-grade or generally recognized as safe (GRAS) could be verified. MATERIALS A METHODS Bacterial and fungal strains and growth conditions. Lactobacilli used throughout this study were propagated at 37 C in deman Rogosa Sharpe (MRS) broth containing 0.% Tween 80 (Biolife, Milan, Italy) (Table ). Indicator fungi originating from spoiled yoghurt and fruits were cultivated in yeast mold (YM) medium (Difco, Becton Dickinson, Sparks, Md.) at 25 C (20). Bacterial cultures were maintained as frozen stocks at 80 C in broth containing 30% (vol/vol) glycerol; fungi were maintained at 4 C on solid medium. Isolation of lactobacilli. A 0-g food or feed sample was treated in a stomacher for 2 min with 90 ml of diluent (0.85% NaCl containing 0.% peptone), serially diluted, and plated on MRS agar containing 0.% Tween 80. Liquid samples were directly diluted and plated. Agar plates were incubated under anaerobic conditions at 37 C for 3 days. From each sample, a variety of morphologically different colonies was selected to screen for antifungal activities. Screening method for antifungal activities. A preliminary screening for antifungal activities was done using a modified agar spot assay according to Grinstead and Barefoot (3). The test cul-

2 2 SCHWENNINGER ET AL. J. Food Prot., Vol. 68, No. TABLE. Lactobacilli used for molecular typing in this study Strain a Identification by API 50 CHL (identification comment) Source/reference Lactobacillus casei ATCC 393 T ATCC 334 Lactobacillus paracasei subsp. paracasei ATCC T SM5 SM20 (DSM454 b ) SM28 SM29 (DSM455 b ) SM30 SM63 (DSM456 b ) SM65 SM66 SM70 Lactobacillus rhamnosus ATCC 7469 T LMG8030 Lactobacillus zeae ATCC 5820 T 97.0% (doubtful) 99.4% (very good) 94.8% (doubtful) 94.8% (doubtful) 94.8% (doubtful) c 99.9% (excellent) 99.2% (very good) 98.8% (good) Salami isolate, this study Salami isolate, this study Salami isolate, this study Salami isolate, this study Universiteit Gent, Laboratorium voor Mikrobiologie, Gent, Belgium a Superscript T indicates type strain. b Deposited at German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany. c Identified as Lactobacillus curvatus (64.7%) using API 50 CHL and growth behavior at 5 C. tures were spot inoculated onto agar plates and incubated anaerobically at 37 C for 3 days. Each plate was then overlaid with tempered (50 C) YM soft agar (0.6%, 6 ml) containing about 0 4 cells per ml of one of the following indicator fungi: Candida sp. -50/5, C. krusei 3-69/2, C. magnoliae -35/, C. parapsilosis 4-5/, C. pulcherrima -50/3, C. reukaufii 4-73/4, C. silvicola 4-42/, C. valida -48/8, Zygosaccharomyces bailii -48/, and Penicillium sp. 2-2/7. The inhibition areas around the test colonies were recorded first after 3 days at 25 C and then daily. Preliminary characterization of bacterial isolates. Bacterial isolates grown on MRS agar were Gram stained and examined microscopically for cellular morphology. Furthermore, catalase activity was tested by spotting colonies with 30% hydrogen peroxide and spore staining was done with the putative lactobacilli using malachite green. Preliminary strain identification of MRS isolates was achieved using the API 50 CHL identification system as described by the supplier (biomérieux, Lyon, France). Additionally, selected strains were further characterized by their proteolysis activity in MRS agar containing either 5 or 0% skim milk and by their production of organic acids in liquid media, which was analyzed enzymatically using kits of Boehringer Mannheim (Roche Diagnostics GmbH, Mannheim, Germany). Growth conditions were determined in MRS broth at different temperatures (5 to 45 C) and with salinities of 5 or 0% at 32 C for 72 h. Identification of selected strains by partial sequencing of the 6S rrna gene. Amplification of the 6S rrna gene was carried out using a rapid method without a DNA extraction step. Briefly, a single colony of the corresponding strain was obtained from an agar plate and used for PCR. The colony was added to the PCR mix (50 l), containing 50 mm KCl,.5 mm MgCl 2, 0 mm Tris-HCl (ph 9.0), 200 M of each dntp, M of each universal oligonucleotides bak4 and bakw (Table 2), and 2.5 U Taq DNA polymerase (Amersham Biosciences, Uppsala, Sweden). The reaction was started with an initial heating step at 95 C for 3 min, followed by 40 cycles of the following sequence: 95 C for 5 s, 56 C for 30 s, and 72 C for 2 min; and a final annealing at 72 C for 7 min. Amplified DNA was purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences). Nucleotide sequencing of both strands was performed using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit and the ABI PRISM ABI 30 Genetic Analyzer apparatus (Perkin Elmer, Wellesley, Mass.), as recommended by the supplier. DNA sequence analysis, sequence alignments, and sequence database searching were conducted with programs covered by the GCG Software Package (version 0.0, Accelrys, San Diego, Calif.) using the GenEMBL database copy. Sequencing primers targeting conserved regions of the 6S rrna gene are listed in Table 2. Differentiation of L. casei, L. paracasei, and L. rhamnosus by PCR. Amplification of the 6S rrna gene for Lactobacillus strains was carried out as described by Ward and Timmins (27) with some modifications. A 290-bp fragment of the 6S rrna gene was amplified using Lactobacillus species-specific primers (casei, para, or rham) in combination with primer Y2 corresponding to a conserved region of the bacterial 6S rrna gene (Table 2). PCR mixtures were prepared as described above and the temperature program was set as recommended by Young et al. (29): 93 C for 5 min, 35 cycles of the following: 93 C for 45 s, 62 C for 45 s, and 72 C for 2 min; and a final annealing at 72 C for 5 min. PCR amplification products were separated by electrophoresis on a 2% agarose gel, and puc9-dna/mspi(hpaii) (MBI

3 J. Food Prot., Vol. 68, No. MOLECULAR TYPING OF ANTIFUNGAL LACTOBACILLI 3 TABLE 2. Oligonucleotides used for identification purposes a Name Sequence Comment/property Reference OPL-0 OPL-05 Y2 casei para rham bak4 bakw eub338 uni55 uni785 uni088 uni GGCATGACCT-3 5 -ACGCAGGCAC-3 5 -TGGCTCAGAACGAACGCTAGGCCCG-3 5 -TGCACTGAGATTCGACTTAA-3 5 -CACCGAGATTCAACATGG-3 5 -TGCATCTTGATTTAATTTTG-3 5 -AGGAGGTGATCCARCCGCA-3 5 -AGTTTGATCMTGGCTCAG-3 5 -ACTCCTACGGGAGGCAGC-3 5 -ACCGCGGCTGCTGGCAC-3 5 -GGMTTAGATACCCTGGTAGTCC-3 5 -GGTTAAGTCCCGCAACGAGC-3 5 -GTACACACCGCCCGTCA-3 Primer for RAPD (Lactobacillus) Primer for RAPD (Lactobacillus) Specific for Lactobacillus casei 6S rrna gene Specific for Lactobacillus paracasei 6S rrna gene Specific for Lactobacillus rhamnosus 6S rrna gene a Synthesized by Mycrosynth, Balgach, Switzerland. Fermentas, Hanover, Md.) was used as a molecular weight standard. Randomly amplified polymorphic DNA genotyping of Lactobacillus strains. DNA for RAPD genotyping was prepared according to the method of Tynkkynen et al. (25), with some modifications. Single colonies were selected from MRS agar plates and each transferred to a.5-ml reaction tube containing 00 l TE buffer (0 mm Tris-HCl, mm ETDA [ph 8.0]). Cells were well mixed on a vortex, supplemented with 50 l of 0% SDS, homogenized, and finally incubated for 30 min at 65 C. Bacterial suspensions were centrifuged (2,200 g, 4 C, 5 min) and the supernatants discarded. The tubes containing the cell sediments were heated in a microwave oven for 5 min at 650 W. Each pellet was then dissolved in 500 l TE buffer and centrifuged (2,200 g, 4 C, 5 min). DNA concentrations of supernatants were determined in an Eppendorf biophotometer as described by the supplier. RAPD analysis was performed according to the method of Du Plessis and Dicks (9) with some modifications. PCR was done with 5 ng of DNA in a volume of 50 l containing 50 mm KCl, 2 mm MgCl 2, 0 mm Tris-HCl (ph 9.0), 200 M of each dntp, 0.4 M 0-base primer OPL-0 or OPL- 05 (Table 2), and 2.5 U Taq DNA Polymerase (Amersham Biosciences). DNA amplification was performed in a Biometra T- gradient thermoblock (Biometra GmbH, Goettingen, Germany). The PCR cycling program used was 94 C for 3 min followed by 45 cycles of 94 C for min, 36 C for min, and 72 C for min. Final hybridization and synthesis was performed at 36 C for 3 min and 72 C for 5 min. PCR amplification products were analyzed by electrophoresis in 0.8% agarose gels with -kb DNA ladder (New England Biolabs Inc., Beverly, Mass.) as a molecular weight standard. Pulsed field gel electrophoresis of Lactobacillus strains. The preparation of genomic Lactobacillus DNA in situ in agarose blocks and the following restriction with SfiI was performed according to the method of Tynkkynen et al. (25). Briefly, the digests were electrophoretically separated on % agarose gels in 0.5 TBE buffer ( TBE: 0. M Tris-HCl, 0. M boric acid, 0.2 mm EDTA [ph 8.0]) on a CHEF-DR II system (BioRad, Hercules, Calif.) for 22 h at a voltage of 90 V and 4 C. The pulse time was 5 to 30 s and the lambda ladder PFGE marker (New England Biolabs Inc.) was used as a size standard. Agarose gels were stained with ethidium bromide (5 g/ml) and the DNA was visualized under UV light. Characterization of antifungal activities. To characterize antifungal compounds, extracts from MRS agar plates with fully grown lactobacilli cultures (72 h at 37 C) were prepared by scraping the bacterial lawn and cutting the remaining agar in pieces of 2 by 2 mm. After extraction in 50 ml of water for 4 days at 5 C, the aqueous phase was filtered (0.2 m) and evaporated to 0- fold (crude extract). Effects of protease treatment. A sample of ml of crude extract was dissolved in 9 ml of YM broth supplemented with proteinase K or protease (each mg/ml, Sigma, St. Louis, Mo.) and inoculated with 0.2 ml of a 3-day culture of C. pulcherrima -50/3. During incubation for 4 days at 25 C, the optical densitiy was measured every 2 h. A control sample was prepared without enzyme treatment. Effects of heat treatment. A sample of ml of crude extract was heat treated for 0 min at 00 C and then mixed with 9 ml of YM broth inoculated with 0.2 ml of a 3-day culture of C. pulcherrima -50/3. A control sample was prepared without heat treatment and growth of indicator yeasts was observed as described above. Nucleotide sequence accession number. The nucleotide sequences reported here have been submitted to the EMBL Nucleotide Database under accession numbers AJ4405 (strain SM20 [DSM454]), AJ4406 (strain SM29 [DSM455]), and AJ4407 (strain SM63 [DSM456]). RESULTS Successful screening for antifungal activities in Lactobacillus spp. A preliminary screening for antifungal activities was performed with,424 isolates originating from raw milk, cheese, yoghurt, black olives, sour dough, salami, as well as corn and grass silage on MRS agar. A total of 82 strains showed antifungal activities in an agar-spot assay against a set of 0 fungi representing predominant contaminations of spoiled yoghurt and fruit (20). These isolates were further characterized by Gram-staining, catalase activity, microscopy, and forming of spores. The species belonging to the L. casei group showed predominant antifungal activities, accounting for 25% of all isolates studied (Table 3). Identification analysis by API 50 CHL allowed classifying them as L. casei, L. paracasei subsp. paracasei,

4 4 SCHWENNINGER ET AL. J. Food Prot., Vol. 68, No. TABLE 3. Composition of 82 isolates on MRS agar originating from raw milk, cheese, yoghurt, black olives, salami, as well as corn and grass silage showing antifungal activities in an agar spot assay Identification (API 50 CHL) L. paracasei subsp. paracasei L. rhamnosus L. casei L. curvatus a L. plantarum L. delbrueckii subsp. lactis L. delbrueckii subsp. bulgaricus L. fermentum L. brevis L. fructivorans L. buchneri L. lindneri L. acidophilus Unidentified Cocci Proportion (%) L. casei group % a Identified as Lactobacillus paracasei/l. casei analyzing the 6S rrna gene. and L. rhamnosus. Furthermore, the species L. plantarum (4%) showed high inhibitory activities toward the tested fungi. The analysis of acidification profiles of API 50 CHL strips showed that a total of 6% of the isolates producing antifungal compounds were cocci and 8% remained unidentified. Molecular typing and characterization of three high antifungal lactobacilli: DNA sequencing analysis. Three isolates, SM20 (DSM454), SM29 (DSM455), and SM63 (DSM456), with particularly high antifungal activities were further studied using sequencing analysis of the 6S rrna gene. Based on their carbohydrate metabolisms, SM20 and SM29 were previously identified as L. paracasei subsp. paracasei and SM63 as L. curvatus (Table ). The species attributed to SM20 and SM29 were confirmed by 6S rrna gene analysis. Strain SM20 showed an identity of 00% in,59 nucleotides of a total of,520 nucleotides identified that overlapped with the partial sequence of the 6S rrna gene of L. paracasei JCM830 (D7922) and with the partial 6S rrna gene sequence of L. casei (D8657). A number of,528 nucleotides sequenced from strain SM29 showed an identity of 99.9% in,520 nucleotides overlapping with the partial sequence of the 6S rrna gene of L. casei (D8657) and of L. paracasei JCM830 (D7922). Contrary to the API classifications, strain SM63 was also identified as member of the casei group because,55 nucleotides identified from the 6S rrna gene of strain SM63 showed an identity of 99.9% in,493 nucleotides that overlapped with the corresponding sequence of L. casei (D8657) and with L. paracasei JCM830 (D7922). Therefore, we classified SM63 in the species L. paracasei and L. casei. Specific PCR for the L. casei group. Because members of the L. casei group form a very homogeneous group concerning phenotypic criteria as well as sequence data of the 6S rrna gene, Ward and Timmins (27) developed a simple PCR approach to differentiate the species L. casei, L. paracasei, and L. rhamnosus based on the reclassification of Collins et al. (2). Figure shows a specific product of 290 bp for strains SM20, SM29, and SM63 amplified in a PCR with the L. paracasei-specific primer used in combination with primer Y2, both targeting the 6S rrna gene (lanes 2, 5, and 8). As a control, the type strains of L. casei (ATCC 393 T ) and L. paracasei subsp. paracasei (ATCC T ) also revealed a 290-bp band with the corresponding primers (lanes 0 and 4). As expected, L. rhamnosus type strain (ATCC 7469 T ) did not amplify a specific fragment with the specific primers casei or para (lanes 6 and 7), but likewise no signal was present with oligonucleotide rham under the experimental conditions (lane 8). RAPD genotyping. For further classification, strains SM20, SM29, and SM63 were analyzed in parallel with 2 control strains of L. casei, L. paracasei subsp. paracasei, L. rhamnosus, and L. zeae (Table ) by the RAPD genotyping technique. Using primer OPL-0, two distinct RAPD genotypes, R and R2, were detected among the 5 tested strains (Fig. 2, Table 4). Genotype R was represented by two strains of L. rhamnosus, LMG8030 (lane ) and ATCC 7469 T (lane 2), which produced two strong ampli- FIGURE. Two percent agarose gel of the amplification of a 290-bp PCR product obtained after using the casei group-specific oligonucleotides in combination with primer Y2 (Table 2). (A) L. paracasei subsp. paracasei SM20; (B) L. paracasei subsp. paracasei SM29; (C) L. paracasei subsp. paracasei SM63; (D) L. casei ATCC393 T ; (E) L. paracasei subsp. paracasei ATCC T, and (F) L. rhamnosus ATCC 7469 T. The primer combinations were casei/y2 in lanes, 4, 7, 0, 3, 6; para/y2 in lanes 2, 5, 8,, 4, 7; and rham/y2 in lanes 3, 6, 9, 2, 5, 8; 9, negative control; bp, puc9-dna/mspi(hpaii) marker.

5 J. Food Prot., Vol. 68, No. MOLECULAR TYPING OF ANTIFUNGAL LACTOBACILLI 5 FIGURE 2. RAPD patterns of Lactobacillus strains obtained with primer OPL-0 (A) and OPL-05 (B). Lane, L. rhamnosus LMG 8030; 2, L. rhamnosus ATCC 7469 T ;3,L. zeae ATCC 5820 T ;4,L. casei ATCC 393 T ;5,L. paracasei subsp. paracasei SM63; 6, L. casei ATCC 334; lanes 7 to 5, L. paracasei subsp. paracasei: 7, ATCC T ; 8, SM70; 9, SM66; 0, SM65;, SM20; 2, SM30; 3, SM28; 4, SM5; 5, SM29; 6, -kb DNA ladder. TABLE 4. Differentiation of strains of the Lactobacillus casei group and L. zeae ATCC 5820 T by RAPD (using primer OPL- 0 or OPL-05) and PFGE analysis Strain L. rhamnosus LMG8030 ATCC 7469 T RAPD a OPL-0 OPL-05 PFGE b R R R R L. zeae ATCC 5820 T Unique R2 B P L. casei ATCC 393 T ATCC 334 L. paracasei subsp. paracasei SM63 ATCC T SM70 SM66 SM65 SM20 SM30 SM28 SM5 SM29 R2 A R2 A R2 A3 R2 A3 R2 A3 R2 A3 R2 B Unique P a R and R2, RAPD genotypes and 2; A, A2, and A3, clusters, 2, and 3 within the RAPD genotype R; B, B2, B3, clusters, 2, and 3 within the RAPD genotype R2. b P and, PFGE genotypes and 2., not determined. fication products of 0.5 kb and approximately 0.4 kb, respectively. Genotype R2, represented by strains of L. casei, L. paracasei subsp. paracasei, and L. zeae also showed the 0.4-kb fragment but, in addition, a -kb,.4-kb, or 2-kb fragment was amplified. Consequently, these strains were classified in three different clusters, A, A2, and A3. A, represented by L. casei ATCC 393 T (lane 4) and L. paracasei subsp. paracasei SM63 (lane 5), produced the -kb fragment; cluster A2, represented by L. casei ATCC 334 (lane 6), L. paracasei subsp. paracasei strains ATCC T (lane 7), SM70 (lane 8), SM66 (lane 9), SM65 (lane 0), and SM20 (lane ) produced the.4-kb and 2.0-kb fragments; and cluster A3, represented by L. paracasei subsp. paracasei strains SM30 (lane 2), SM28 (lane 3), SM5 (lane 4), and SM29 (lane 5) lacked the.4-kb fragment. L. zeae ATCC 5820 T (lane 3) showed a unique profile, producing only the 0.4-kb fragment. RAPD genotyping with primer OPL-05 gave a similar discrimination into genotype R and R2. Genotype R, represented by L. rhamnosus strains LMG8030 (lane ) and ATCC 7469 T (lane 2), showed a profile with two fragments just beneath 0.5 kb. Genotype R2 could likewise be divided into three different clusters, B, B2, and B3. In the B cluster, L. zeae ATCC 5820 T (lane 3) and L. casei ATCC 393 T (lane 4) had two close fragments just above 0.5 kb (B). In B2, L. casei ATCC 334 (lane 6), L. paracasei subsp. paracasei strains ATCC T (lane 7), SM70 (lane 8), SM66 (lane 9), and SM65 (lane 0) had three typical fragments of approximately 0.5 kb. The B3 cluster, represented by L. paracasei subsp. paracasei strains SM20 (lane ), SM30 (lane 2), SM28 (lane 3), SM5 (lane 4), and SM29 (lane 5), gave three to four weak bands around the -kb frag-

6 6 SCHWENNINGER ET AL. J. Food Prot., Vol. 68, No. RAPD patterns was verified in the laboratory by four repetitions carried out by two different experimentors. FIGURE 3. PFGE profiles of Lactobacillus strains determined with restriction enzyme SfiI. Lane, L. zeae ATCC 5820 T ;2,L. casei ATCC 393 T ;3,L. casei ATCC 334; lanes 4 to 7, L. paracasei subsp. paracasei: 4, ATCC T ; 5, SM63; 6, SM29; 7, SM20; 8, PFGE marker. ment. L. paracasei subsp. paracasei SM63 (lane 5) showed a unique profile. L. rhamnosus was clearly distinguished from other members of the casei group, such as L. casei and L. paracasei, by RAPD analysis with primers OPL-0 and OPL-05 therefore confirming the results from the specific PCR analysis (Fig. ). The reproducibility of the PFGE. PFGE was performed to discriminate between the closely related strains classified in RAPD genotype R2, L. casei, L. paracasei, and L. zeae (Fig. 2, Table 4). Strains SM20, SM29, and SM63 were compared with L. casei ATCC 393 T, L. casei ATCC 334, L. paracasei subsp. paracasei ATCC T, and L. zeae ATCC 5820 T using PFGE. Genomic DNA digested with SfiI finally allowed the differentiation of two PFGE genotypes in the L. casei paracasei zeae subgroup even though the strains all had unique profiles (Fig. 3 and Table 4). Genotype P, represented by L. zeae ATCC 5820 T (lane ) and L. casei ATCC 393 T (lane 2), gave a double or triple band of approximately 240 kb and only a few weak bands at smaller sizes. Genotype, represented by L. casei ATCC 334 (lane 3), L. paracasei subsp. paracasei strains ATCC T (lane 4), SM63 (lane 5), SM29 (lane 6), and SM20 (lane 7), gave distinct profiles with fragments ranging from sizes smaller than 48 kb to approximately 240 kb. Metabolic properties. Strains SM20, SM29, and SM63 were further characterized based on biochemical properties and growth behavior (Table 5). Different profiles were detected by API 50 CHL: strain SM29 converted more carbohydrates than strains SM20 and SM63, but all produced acid from ribose, galactose, D-glucose, D-fructose, D- mannose, mannitol, sorbitol, N-acetyl-glucosamine, maltose, lactose, trehalose, melezitose, and D-tagatose. Furthermore, no proteolytic activities were detected in any of the three strains. NaCl concentrations between 5 and 0% were tolerated by all three strains. SM20 exhibited a considerable TABLE 5. Phenotypic differences within Lactobacillus paracasei subsp. paracasei strains SM20, SM29 and SM63 Strain Acid produced from a : Fermentation products (g/liter) b Growth conditions NaCl tolerance (%) Proteolytic activity SM20 Ribose, galactose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, N-acetyl-glucosamine, amygdaline, arbutin, aesculine, salicine, cellobiose, maltose, lactose, saccharose, trehalose, inulin, melezitose, -gentiobiose, D-turanose, D-tagatose L-Lactate: 7. D-Lactate:.0 Acetate: C (opt.: C) 0 c Negative SM29 Ribose, adonitol, galactose, D-glucose, D-fructose, D-mannose, rhamnose, dulcitol, mannitol, sorbitol, -methyl-d-mannoside, -methyl-d-glucoside, N-acetyl-glucosamine, amygdaline, arbutin, aesculine, salicine, cellobiose, maltose, lactose, saccharose, trehalose, inulin, melezitose, -gentiobiose, D-turanose, D-tagatose L-Lactate: 7.5 D-Lactate: 0.9 Acetate: C (opt.: C) 5 d Negative SM63 Ribose, galactose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, N-acetyl-glucosamine, maltose, lactose, trehalose, melezitose, D-tagatose L-Lactate: 9.8 D-Lactate: 0.8 Acetate: C (opt.: C) 5 e Negative a Determined using API 50 CHL. b Enzymatically determined in cell free MRS culture supernatant (Boehringer Mannheim; Roche Diagnostics GmbH, Mannheim, Germany). c Good growth at a salinity of 5% and fairly good growth at 0%. d Good growth at a salinity of 5% NaCl and very weak growth at 0%. e Good growth at a salinity of 5% and weak growth at 0%.

7 J. Food Prot., Vol. 68, No. MOLECULAR TYPING OF ANTIFUNGAL LACTOBACILLI 7 salt tolerance because it grew in the presence of 0% NaCl (Table 5). L. paracasei subsp. paracasei SM20, SM29, and SM63 produced in MRS broth 7. to 9.8 g/liter L-lactate, approximately 0.8 to.0 g/liter D-lactate, and only traces of acetic acid, 0.0 to 0. g/liter (Table 5). Clearly, these strains (SM20, SM29, and SM63) produced more total lactate in MRS broth compared with the noninhibitory L. delbrueckii subsp. bulgaricus DSM2008 T, which produced only 0. g/liter L-lactate and 7.9 g/liter D-lactate. Acetic acid contents of strain DSM2008 T were in the same range as in antifungal strains (0.2 g/liter; data not shown). Preliminary characterization of antifungal compounds. Crude extracts of strains SM20, SM29, and SM63 did not lose their antifungal activity after treatment with proteinase K or protease, which suggests that the antifungal compounds are not proteinaceous. Furthermore, heat treatment of the crude extract at 00 C for 0 min did not affect antifungal activity for these strains (data not shown). DISCUSSION In this study, 82 out of,424 isolates grown on MRS agar showed inhibitory properties toward the tested fungi. A proportion of 25% of the active isolates belonged to the L. casei group, including L. casei, L. paracasei, and L. rhamnosus. Antimicrobial properties of members of this group have been described in different studies (4, 22, 24, 26, 28). Furthermore, we characterized a second cluster comprising 4% of high antifungal isolates and belonging to the species L. plantarum, which has already been associated with inhibitory properties (7, 9, 2). Finally, this study concentrated on the three high antifungal strains SM20, SM29, and SM63. They were clearly identified and characterized on a molecular level using different genotyping methods such as RAPD, PFGE, a specific PCR approach, and sequencing analysis of the 6S rrna gene. Although the strains showed different characteristics of acid production, they all belonged to the species L. paracasei subsp. paracasei according to the classification of Collins et al. (2). Over the last few years, the status of members of the L. casei group has been changed frequently and reclassifications have occurred. In 980, on the basis of phenotypic criteria, five subspecies of L. casei were recognized, i.e., L. casei subsp. alactosus, L. casei subsp. casei, L. casei subsp. pseudoplantarum, L. casei subsp. rhamnosus, and L. casei subsp. tolerans (23). Based on DNA- DNA hybridization experiments, Collins et al. (2) proposed that members of L. casei subsp. alactosus, L. casei subsp. pseudoplantarum, L. casei subsp. tolerans, and the majority of L. casei subsp. casei strains be given a separate species status, namely, L. paracasei sp. nov., i.e., L. paracasei subsp. paracasei ATCC T and L. paracasei subsp. tolerans ATCC T. Moreover, L. casei subsp. rhamnosus was elevated to species status as L. rhamnosus sp. nov. (ATCC 7469). L. casei is still represented by its type strain ATCC 393. In this study, sequencing analysis of the 6S rrna gene of strains SM20, SM29, and SM63 revealed 99.9 to 00% identity in approximately,500 nucleotides that overlapped with the partial sequence of the 6S rrna gene of L. paracasei JCM830 (D7922) and with the partial sequence of the 6S rrna gene of L. casei (D8657). These findings showed the close relationship between the two species L. casei and L. paracasei. As given in Table, SM20 was identified with a reliability of 99.4% as L. paracasei subsp. paracasei by carbohydrate fermentation patterns from API 50 CHL tests. In contrast, the identification of SM29 was doubtful with an accuracy of 94.8%. Interestingly, SM63 was identified as L. curvatus (64.7%) by API 50 CHL and by its ability to grow at 5 C. A species-specific PCR approach, developed to differentiate the L. casei group (27), revealed that the three strains SM20, SM29, and SM63 belong to the species L. paracasei by comparison with the corresponding type strains. On the basis of acid production described by Collins et al. (2), SM20, SM29, and SM63 were divided within the subspecies paracasei. Surprisingly, L. rhamnosus (ATCC 7469 T ) did not show the specific band after PCR with the primer pair Y2/rham (Fig. ). Furthermore, no PCR products were observed when this primer combination (Y2/rham) was used with the strains to be identified confirming the distinction of L. rhamnosus and L. casei/l. paracasei. To verify the identification of strains SM20, SM29, and SM63 as L. paracasei subsp. paracasei, further genotyping methods such as RAPD and PFGE were compared with the API 50 CHL test, the sequencing analysis of the 6S rrna gene, and the species-specific PCR approach according to Ward and Timmins (27). RAPD analysis with primers OPL- 0 and OPL-05 yielded two distinct genotypes, R and R2, which allowed clearly discriminating L. rhamnosus and L. casei/l. paracasei subsp. paracasei (Table 4) and supporting the classification of L. rhamnosus as a separate species according to Collins et al. (2). Likewise, Tynkkynen et al. (25) could distinguish all non L. rhamnosus strains from L. rhamnosus strains by RAPD analysis using another random sequence primer. Recently, Dellaglio et al. (6) concluded that the species L. casei is not correctly represented by its type strain ATCC 393. They proposed that this strain be reclassified as L. zeae and that L. casei ATCC 334 and L. paracasei strains become members of the same taxon, under the name L. casei. This would imply that ATCC 334 be the neotype strain and that the name L. paracasei be rejected. The findings of Dellaglio et al. (6) support our observations of the close relationship between L. casei and L. paracasei as shown by sequencing analysis of the 6S rrna gene. Furthermore, RAPD analysis with primer OPL-05 revealed that L. zeae ATCC 5820 T and L. casei ATCC 393 T were in the same cluster, R2-B (Table 4). Using OPL-0, L. casei ATCC 393 T and strain SM63 formed a cluster (R2-A) whereas L. zeae ATCC 5820 T showed a unique profile. Because strain SM63 also showed a unique profile using primer OPL-05, this strain seems to be closely related to L. zeae ATCC 5820 T and L. casei ATCC 393 T. Likewise, strains of L. paracasei subsp. paracasei, including L. casei ATCC 334, formed two further clusters with OPL-0 (R2- A2/R2-A3) or OPL-05 (R2-B2/R2-B3) that seemed to be closely related to each other (Fig. 2 and Table 4). Strain

8 8 SCHWENNINGER ET AL. J. Food Prot., Vol. 68, No. typing with PFGE revealed also a division between L. zeae ATCC 5820 T /L. casei ATCC 393 T and L. paracasei subsp. paracasei strains/l. casei ATCC 334 (P/; Table 4 and Fig. 3), which supports the findings of Dellaglio et al. (6). The reproducibility of RAPD patterns is often a problem and the technique is very sensitive to experimental conditions. Our data are based on four reproducible repetitions done by two different experimentors, indicating a stable RAPD system. Due to their antifungal features, lactobacilli and particularly the L. casei group have great potential for biopreservation of food and feed systems. However, future industrial use requires not only the identification of active strains but also the development of effective methods to monitor cultures during production and use. For safety reasons, it is crucial to differentiate industrial strains and to verify their genetic stability. Genotyping methods such as RAPD, PFGE, or DNA sequencing analysis have the potential to give species-specific or even strain-specific information. Finally, the use of clearly identified and characterized strains with strong antimicrobial properties will contribute to improved food safety and perhaps reveal other novel applications. Further research will focus on determining the nature of antifungal compounds produced by novel strains as well as evaluating their technological suitability. This study showed that antifungal substances produced by L. paracasei subsp. paracasei strains SM20, SM29, and SM63 were heat resistant but not proteinaceous. In addition, these strains produced markedly higher amounts of total lactate (8. to 20.6 g/liter) compared with the noninhibitory L. delbrueckii subsp. bulgaricus DSM2008 T (8.0 g/liter). However, data also suggest that these concentrations were not solely responsible for antifungal activities. Further studies of antifungal compounds produced by the strains described in this study are currently under investigation. ACKNOWLEDGMENTS We thank Janice Sych for critical reading of the manuscript and for helpful discussions. This work was supported by a grant from the Swiss National Science Foundation SPP Biotechnology Parts of it are protected by a European patent application. REFERENCES. Amann, R. I., W. Ludwig, and K.-H. Schleifer Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59: Collins, M. D., B. A. Phillips, and P. Zanoni Deoxyribonucleic acid homology studies of Lactobacillus casei, Lactobacillus paracasei sp. nov., subsp. paracasei and subsp. tolerans and Lactobacillus rhamnosus sp. nov., comb. nov. Int. J. Syst. Bacteriol. 39: Corsetti, A., M. Gobbetti, J. Rossi, and P. 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