System. Appl. Microbiol. 22, 87-96 (1999) SVSTEIVL4TIC AND © Urban & Fischer Verlag _h_tt~p:_V_w_ww __ .u_r_ba_n_fi_s_ch_e_r._d_e/~jo_u_rn_a_ls_1_sa_m ___________________________________ J4~ED ~~~"

A Polyphasic Study on the Taxonomic Position of Industrial Sour Dough Yeasts

V. H. MANTYNEN\ M. KORHOLA2, H. GUDMUNDSSON3, H. TURAKAINEN2, G. A. ALFREDSSON3, H. SALOVAARA\ and K. LINDSTROM 1

1 University of Helsinki, Dept. App!. Chem. and Microbiology, Viikki Biocenter, Helsinki, Finland 2 Alkomohr Biotech Ltd., Helsinki, Finland 3 University of Iceland, Microbiology laboratory, Institute of Biology, Reykjavik, Iceland 4 University of Helsinki, Dept. Food Technology, Helsinki, Finland

Received December 22, 1998

Summary

The sour dough bread making process is extensively used to produce wholesome palatable rye bread. The process is traditionally done using a back-slopping procedure. Traditional sour doughs in Finland comprise of lactic acid bacteria and yeasts. The yeasts present in these doughs have been enriched in the doughs due to their metabolic activities, e.g. acid tolerance. We characterized the yeasts in five major sour bread bakeries in Finland. We found that most of the commercial sour doughs contained yeasts which were similar to Candida milleri on the basis of 18S rDNA and EF-3 PCR-RFLP patterns and metabolic activities. Some of the bakery yeasts exhibited extensive karyotype polymorphism. The mini­mum growth temperature was 8 °C for C. milleri and also for most of sour dough yeasts.

Key words: Candida milleri - sour dough - PCR-RFLP - karyotype polymorphism - minimum growth temperature - Biolog

Introduction

The sour dough bread making process is extensively used to produce wholesome palatable rye bread in Northern Europe. Technologically the sour dough bread making process is essential for baking from whole meal rye, this process improves dough machinability, bread crumb properties, keeping properties and flavour (SALO­VAARA 1998). The yeasts occuring in the Finnish rye sour doughs have earlier been identified as Torulopsis holmii, Saccharomyces cerevisiae, and Torulopsis stellata, and as sporadic contaminants Endomycopsis fibuliger and Hansenula anomala (SALOVAARA and SAVOLAINEN 1984). Yeast species found in traditional Portuguese bread doughs include S. cerevisiae, Issatchenkia orientalis, Pichia membranaefaciens, Torulaspora delbrueckii, P. anomala, S. kluyveri, Kluyveromyces marxianus and I. occidentalis (ALMEIDA and PAIS 1996). Some strains pre­viously identified as Torulopsis holmii have subsequently been assigned to the new species Candida milleri sp. nov. (YARROW 1978), which is associated with sour ferments (OTIOGALLI et al. 1996, VOGEL 1997).

The traditional systematics and identification of yeasts have been based on biochemical tests, and morphological

and physiological criteria (BARNETI et al. 1990). Al­though traditional yeast taxonomy has long accepted the premise that mating is synonymous to conspecificity, in­dication of species affinity by this technique should be considered with caution (VAUGHAN-MARTINI and MAR­TINI 1987). Furthermore, imperfect fungi (e.g. heteroge­neous genus Candida; BARNS et al. 1991) cannot be stud­ied using traditional genetics (BOEKHOUT et al. 1993) and moreover the physiological features of industrial yeasts have been shown to alter when changes occur in growth conditions (ROHM and LECHNER 1990, BOTHA and KOCK 1993, CODON and BENITEZ 1995).

Molecular characteristics have defined and changed the taxonomy of yeasts (KREGER-VAN RIJ 1980, KURTZ­MAN 1993, KURTZMAN 1994, KURTZMAN and BLANZ 1998). Karyotyping has a high potential when classifying different yeasts (DE ]ONGE et al. 1986). Together with DNA reassociation, pulsed-field gel electrophoresis has been used to study the taxonomic positioning of yeasts (MILLS and MCCLUSKEY 1990, NAUMOV et al. 1992), but it has been found that even species with high DNA reas­sociation rate exhibit electrophoretic karyotype poly-

0723-2020199/22/01-087 $ 12.0010

88 V. H. MANTYNEN et al.

morhisms (VAUGHAN-MARTINI et al. 1993). Comparative 18S rRNA sequence analysis using the 18S rDNA-RFLP method or 18S rDNA sequences data have been used to study the taxonomic positions of the yeast species (MAGEE et al. 1987, BARNS et al. 1991, BRUNS et al. 1991, BOWMAN et al. 1992, MOLINA et al. 1992, WILMOTIE et al. 1993, CAl et al. 1996, MASNEUF et al. 1996, JAMES et al. 1997, SMOLE-MoZINA et al. 1997). 18S rDNA studies have shown that some yeast genera are phylogenetically heterogeneous ie. Candida, Kluyveromyces, and Saccha­romyces and the members of these genera have been found to be intermixed with each other (BARNS et al. 1991, CAl et al. 1996, JAMES et al. 1997). Elongation fac­tor 3 (EF-3) is a specific protein in fungi which is needed in translation (COLTHURST et al. 1992, BELFIELD and TUITE 1993) and its expression correlates with growth rate (SWOBODA et al. 1994). EF-3 is a single copy gene and its DNA sequence has 79% identity between S. cere­visiae and C. albicans (MYERS et al. 1992). Therefore, the DNA sequences of EF-3 can be used to study the phylo­genetic relationships of fungi (COLTHURST et al. 1991). The molecular tools are thus powerful for resolving phy­logenetic relationships between yeasts.

Some yeasts are psychrophilic and the growth of these yeasts can occur at temperatures as low as 0 °C (STOKES 1971). Cold storage is a common practise to preserve the sour doughs in the production environment between pro­duction days. Yeast strains in sour doughs should be able to survive the cold storage step. The Biolog (Biolog, USA) system, which is a new method to test the ability of microorganisms to utilize or oxidize a preselected panel of different carbon sources. Many sour dough yeasts can­not utilize maltose, which is major sugar component of rye (SAVOLA et al. 1982, SALOVAARA and SAVOLAINEN 1984, HANSEN and HANSEN 1994, VOGEL 1997). Biolog system analyses the physiological activities of the yeast isolates thus providing a metabolic fingerprint, which can be used to characterize the food borne yeast isolates, although with the current data base, it identifies food as­sociated yeasts only poorly (68%) (PRAPHAlLONG et al. 1997)

We applied a polyphasic approach for studying the taxonomic relationships of yeasts isolated from sour doughs used in five major Finnish industrial bakeries and compared the results with the reference strains using 18S rDNA PCR-RFLP, EF-3 PCR-RFLP, karyotyping, Biolog system, and minimum growth temperature determina­tion methods.

Materials and Methods

Yeast strains were collected from five industrial rye bread­making processes applying a daily back-slopping procedure for the maintenance of the sour dough. Samples were taken from mature, fully fermented (>12 h, 27-30 °C, pH 3.8) sour sponges used for dough preparation. The samples were diluted and plated for isolated colony counts (107-108 CFU/g). The most prevalent colony type was picked as a representative for the sour dough yeast. From two samples also a minority colony type (strains B11 and C11) appeared and they were analyzed in

the present study. In addition 36 reference strains originating from various culture collections were used (Table 1). Some of these strains were earlier isolated from Finnish rye sour doughs corresponding to the processes and isolation procedures described above. From the bakeries D and E the sampling was done twice.

DNA extraction and 18S rDNA amplification: Yeast strains were grown in 10 ml of YPD broth (Sigma Chemical Co., ST. Louis, Mo.) overnight at room temperature or longer for slow growing strains. The cells were collected and washed twice with 1 ml of TE buffer (10 mM Tris-HCI, 1 mM EDTA, pH 8.0; Sigma) by centrifuging for 5 min at 6000 rpm. Pellet was resus­pended in 565 pi of TE buffer, 6 pi of 1 % SDS, 6 pi of 10 M mercapto ethanol, 20 pi of Iyticase (10 mg/ml, Sigma) and 3 pi of proteinase K (20 mg/ml, Promega, Madison, Wi,) was added. The suspension was incubated for 1 h at 65°C. Then 300 pi 10% CTAB/0.7 N NaCI (E. Merck AG, Darmstadt, Germany), 420 pi of 5 M NaCI (Merck) and the volume was adjusted to 3000 pi with TE buffer followed by incubation for 10 min at 65°C. The DNA was purified twice with phenol (Merck) and chloroform (Merck) extractions, precipitated with isopropanol (Merck) and resuspended into 100 pi of TE buffer.

For 18S rDNA analysis specific primers were designed using the GCG program package (Genetics Computer Group, Madi­son, WI, USA) and 18S rDNA sequences of seven different yeasts in Genbank and EMBL sequence databases, namely J01353, X58052, X58053, X58054, X58055, X58056, X58057, so that a 958 bp long DNA fragment of the 185 rDNA sequence could be amplified from S. cerevisiae and from other yeasts. The primer sequences were yfor2 5'-GCAGTTAAAAA GCTCGTAGTTGAAC-3' and yrev2 5'-CTTACTAGGAAT TC­CTCGTTGAAGA-3'

Ca. 10 pg of DNA from the strains was used as template in the PCR. The PCR conditions were optimized for annealing temperature and MgCI2 concentration. The final reaction mix­ture (50 pi) contained 10 mM Tris-HCI (pH 8.8, at 25°C), 50 mM KCI, 0.1 % Triton X-I00, 2.5 mM MgCI2, 200 pM con­centrations of each dNTP, and 50 pmol of primers yfor2 and yrev2. The reactions were subjected to a pre-denaturation step at 94°C for 5 min. Then the Dynazyme DNA polymerase en­zyme (Finnzymes Oy, Espoo, Finland) was added using "hot start" at 80°C (D'AQUILA et al. 1991). PCR amplifications were performed in a MJ PTC-200 thermocycler with a heated lid. The thermal cycles were as follows: 35 cycles at 94°C for 45 s, at 65°C for 45 s, and at 72 °C for 60 s, and after PCR cycles at 72 °C for 4 min. The resulting PCR products were analyzed in 1 % agarose gels (0.5 x TAE buffer; 0.02 M Tris-acetate, 0.001 M EDTA, Sigma) containing ethidium bromide (0.25 mg/ml, Sigma), and electrophoresis was carried out for 1 hat 200 V.

For EF-3 analysis specific primers were designed using EF-3 sequences of S. cerevisiae (J05583) and C. albicans (Z11484). The primer sequences were for ef3for: 5'-ATTGCTGTCATTG­GTCCAAATGGTG-3' and for ef3rev: 5'-CAATCTTGTTACC­CATAGCATCGAA-3'. The expected amplification product would be 929 bp with J05583 sequence. The final reaction mix­ture (50 pi) for EF-3 amplification reactions contained 10 mM Tris-HCI (pH 8.8, at 25°C), 50 mM KCI, 0.1 % Triton X-IOO, 3 mM MgCI2, 200 pM concentrations of each dNTP, and 50 pmol of primers ef3for and ef3rev. The pre-amplification steps were the same as in 185 rDNA amplification. The thermal cycles were as follows: 35 cycles at 94°C for 30 s, at 65°C for 60 s, and at 72 °C for 60 s, and after PCR cycles at 72 °C for 4 min. The analyses of the amplifications were done as above.

18S rDNA and EF-3 -PCR -FLP analysis: After PCR reac­tions, the 18S rDNA amplification products were digested with four restriction enzymes, namely AluI, HinfI, Mboll (all from Prom ega, Madison, WI, USA), and MvaI (Mbi Fermentas, Vil­nius, Lithuania). The digested peR products were separated by

A Polyphasic Study on the Taxonomic Position of Industrial Sour Dough Yeasts 89

Table 1. Yeast strains used in this study.

Species Strain code Biolog identication Biolog Minimum Origin of the strain ID growth tem-score perature °C

Candida albicans ATCC 10231 Candida albicans 0.985 7.3 man with bronchomycosis Candida albicans ATCC 18804T Candida albicans 0.995 7.3 human skin lesion of erosio

interdigitalis, Uruquay Candida albicans ATCC 28516 Candida albicans 0.865 7.3 human, Belgium Candida geochares ATCC 36852T Candida geochares 0.806 3.4 grassland soil, South Africa Candida glabrata ATCC 2001T Pichia trehalophila 0.675 4.4 feces Candida milleri ATCC 62655 Kluyveromyces lodderae 0.937 8.6 sour dough, USA Candida milleri ATCC 60592 Kluyveromyces lodderae 0.997 ND sour dough, USA Candida milleri CBS 2664 Kluyveromyces lodderae 0.997 8.2 alphecin, Spain Candida milleri CBS 6897T Kluyveromyces lodderae 0.994 8.8 sour dough, USA Candida milleri CBS 7541 Kluyveromyces lodderae 0.997 8.2 bread, Morocco Candida milleri VTT C-87175 Kluyveromyces lodderae 0.859 8.5 sour dough, Finland Candida milleri VTT C-95232 Kluyveromyces lodderae 0.792 8.6 sour dough, Finland Candida milleri VTT C-96250 Kluyveromyces lodderae 0.823 8.2 sour dough, Finland Candida tropicalis ATCC 750T Candida albicans 0.656 8.2 bronchomycosis Cryptococcus laurentii ATCC 18803T Cryptococcus laurentii 0.917 8.3 Palm wine, Congo Kluyveromyces lactis CBS 2359 Kluyveromyces lactis 0.812 2.0 creamery, USA Pichia fermentans VTT C-74031 lssatchenkia scutulata 0.818 2.2 buttermilk, Netherlands Rhodosporidium toruloides CBS 14 Rhodosporidium sphaericus 0.561 2.9 wood pulp from Coniferea,

Sweden Saccharomyces castellii CBS 4309' Saccharomyces dairensis 0.918 1.4 soil, Finland Saccharomyces cerevisiae ALKO 743 Saccharomyces cerevisiae 0.940 4.8 commercial baker's yeast,

Finland Saccharomyces cerevisiae VTT C-81117 Kluyveromyces lactis 0.633 8.7 sour dough, Finland Saccharomyces cerevisiae VTT C-95236 Z ygosaccharomyces fermentati 0.545 0.7 sour dough, Finland Saccharomyces cerevisiae YNN295 Schizosaccharomyces japonicus 0.754 1.3 unknown Saccharomyces dairensis CBS 42fI Saccharomyces dairensis 0.898 1.8 dry fruit of Diospyros sp.

(persimmon) Saccharomyces exiguus CBS 135 Kluyveromyces lodderae 0.914 5.8 buttermilk, Netherlands Saccharomyces exiguus CBS 379T Kluyveromyces lodderae 0.997 7.8 unknown Saccharomyces exiguus CBS 7901 Kluyveromyces lodderae 0.888 9.2 sour dough, Italy Saccharomyces exiguus VTTC-81115 Kluyveromyces lodderae 0.930 8.7 sour dough, Finland Saccharomyces kluyveri CBS 3082T Cryptococcusluteolus 0.851 3.5 Drosophila pinicola (fruit fly) Saccharomyces pastorianus NCYC 396 Z ygosaccharomyces florentinus 0.969 1.1 Carlsberg laboratory,

Denmark Saccharomyces servazzii CBS 4311' Saccharomyces dairensis 0.764 ND soil, Finland Saccharomyces unisporus CBS 398T Saccharomyces dairensis 0.756 4.6 Carlsberg laboratory,

Denmark Schizosaccharomyces pombe VTT C 82133 Schizo saccharomyces pombe 0.999 9.1 sulphited grape juice Torulaspora delbrueckii CBS 1146 Saccharomyces dairensis 0.561 1.4 unknown Torulaspora delbrueckii TS207 Schizosaccharomyces pombe 0.919 2.8 sour dough, Finland Zygosaccharomyces rouxii CBS 732 Zygosaccharomyces rouxii 0.619 3.9 concentrated black-grape

must, Italy Baker's isolate All Kluyveromyces lodderae 0.997 8.3 sour dough, Finland Baker's isolate A12 Kluyveromyces lodderae 0.997 8.3 sour dough, Finland Baker's isolate Bl1 Trigonopsis triangularis var 0.862 8.7 sour dough, Finland

variabilis Baker's isolate B12 Kluyveromyces lodderae 0.997 8.5 sour dough, Finland Baker's isolate Cll Candida spandovensis 0.815 ND sour dough, Finland Baker's isolate C12 Kluyveromyces lodderae 0.997 8.3 sour dough, Finland Baker's isolate Dll Kluyveromyces lodderae 0.997 8.5 sour dough, Finland Baker's isolate D12 Kluyveromyces lodderae 0.997 8.6 sour dough, Finland Baker's isolate D21 Saccharomyces boulardii 0.646 8.3 sour dough, Finland Baker's isolate D22 Kluyveromyces lodderae 0.997 8.3 sour dough, Finland Baker's isolate Ell Kluyveromyces lodderae 0.997 8.5 sour dough, Finland Baker's isolate E12 Kluyveromyces lodderae 0.997 8.4 sour dough, Finland Baker's isolate E21 Saccharomyces boulardii 0.654 8.5 sour dough, Finland Baker's isolate E22 Saccharomyces boulardii 0.893 8.5 sour dough, Finland

ND= not determined

90 V. H. MANTYNEN et al.

electrophoresis at 200 V for 4.0 h in 5% agarose gels (0.5 x TAE buffer) containing 0.25 mg/ml ethidium bromide by using a water-cooled apparatus (Hoefer Scientific Instruments, San Francisco, USA). Further analysis of the gel results was done with GelCompar program package (GeICompar, comparative analysis of electrophoresis patterns version 4.1; Applied Maths, Kortrijk, Belgium). A dendrogram was created from these re­striction patterns with a computer program in GeiCompar using Dice correlation coefficient and the UPGMA clustering method. MboII, MspI, RsaI, and TaqI (all from Promega) were used for EF-3 amplification products and further analysis was done as for 185 rDNA-RFLP.

time ramping from 240 s to 300 s for 24 h. S. cerevisiae YNN 295 was used as a standard for electrophoresis.

Karyotyping: The samples were prepared according to Nau­mov et al. (1991). The pulsed field gel electrophoresis (CHEF­DR(II apparatus, BioRad Laboratories, Richmond, CA, USA) was carried out at 12 °C and 150 V with a switching time ramping from 110s to 150 s for 24 h and followed by switching

Minimum growth temperature: Yeasts were grown in YPD broth overnight at room temperature (180 rpm). The growth suspension (1 ml) was transferred into YPD agar cuvettes (88 mm by 15 mm) and were left on the bench for 30 s and the excess fluid was removed by aspiration. The analysis was per­formed according to the manufacturer's instruction. Shortly, the cuvettes were placed in the gradient temperature apparatus Gradiplate® WI0 (Biodata Oy, Finland). The temperature gra­dient in these compartments were achieved with two cold water baths containing 15% ethylenglycol. The one end of the sample compartment with a lower temperature (average of 2.7 0c) and another with higher temperature (average 8.1 0c). The temper­ature gradient varied between 5.1-5.7 °C (average 5.4 0c) in different growth experiments. The incubation was done in a cold room (average 6.8 0c) and the formula used to calculate

50 60 70 80 90 100 1"·1., •• "".1."" •• ,.1." •••• ,.1.",.,.,.1 •• ,.,,.,.1

-

'"-

r--

..---

.--

I .--

,.-

I

----1 I

r--

I

Saccharomyces pastorianus NCVC 396 Candida glabrata ATCC 2001 Torulaspora delbrueckii CBS 1146 Torulaspora delbrueckii TS 207 Saccharomyces cerevisiae vn C-81117 Saccharomyces cerevisiae vn C-95236 Saccharomyces cerevisiae ALKO 743 Candida milleri vn C-87175 Candida milleri vn C-95232 Saccharomyces exiguus vn C-81115 Saccharomyces exiguus CBS 135 Candida milleri CBS 7541 Saccharomyces castellii CBS 4309 Saccharomyces cereviaiae VNN 295 Candida milleri ATCC 62655 Candida milleri ATCC 60592 Candida milleri vn C-96244

I

Candida milleri vn C-96250 Saccharomyces dairensis CBS 421 Saccharomyces exiguus CBS 379 Saccharomyces exiguus CBS 7901

I I

Baker's isolate All Baker's isolate A 12 Baker's isolate B 11 Baker'S isolate C12 Baker's isolate 011 Baker's isolate 012 Baker's isolate 021 Baker's isolate 022 Baker'S isolate Ell Baker's isolate E12 Baker's isolate E21 Baker's isolate E22 Baker's isolate B12 Candida milleri CBS 2664 Saccharomyces unisporus CBS 398 Saccharomyces servazzii CBS 4311 Zygosaccharomyces rouxii CBS 732 Saccharomyces kluyveri CBS 3082 Candida albicans ATCC 18804 Candida tropical is ATCC 750 Candida albicans ATCC 10231 Rhodosporidium toruloides CBS 14 Schizosaccharomyce pombe vn C-82133 Cryptococcus laurentii ATCC 18803 Kluyveromyces lactis CBS 2359 Baker's isolate Cll Pichia fermentans vn C-74031

Fig. 1. A dendrogram of the strains based on the 185 rDNA PCR-RFLP assay. In construction of the dendro­gram GelCompar program package was used with Dice's band matching coefficient and UPGMA clustering method.

A Polyphasic Study on the Taxonomic Position of Industrial Sour Dough Yeasts 91

the minimum temperatures corrects the variations occuring out­side the apparatus. The samples were grown for three weeks, the edge of growth was measured using a stereo microscope and the results were transformed from the growth distances into temperatures.

BIOLOG identification: Yeast strains were analyzed with the Biolog system (Biolog Inc. Hayward, Ca) according to the man­ufacturer's instructions. Yeasts were grown in BUY media (Bio­log) at 26°C for 24 h or additional 24 h was needed for some strains to obtain enough growth. The isolate to be identified was suspended in prewarmed sterile water, within the density of range specified [44% to 51 % transmittance] using the Biolog turbidimeter. Then the suspension was inoculated into the YT Microplate™, and incubated for either 24, 48, or 72 hours to allow the pattern to form. The metabolic pattern was then in­terpreted by Biolog's MicroLog 3 computer program (Yeast database release 3.50), which automatically assesses the colour and turbidity changes in the wells and cross-references the pat­tern to a library of species. If an adequate match is found, the system identifies the isolate.

40 50 60

Results

RFLP analysis

• 18S rDNA PCR-RFLP analysis: The analysis com­prised altogether 33 different fragment sizes in the 18S rDNA PCR-RFLP. Thirteen of the baker's isolates were clustered at 80% confidence limit with C. milleri and S. exiguus strains. They were intermixed with strains of species C. glabrata, C. laurentii, S. castellii, S. cerevisiae, S. dairensis, S. pastorianus, and T. delbrueckii (Figure 1). The baker's isolates, except Cll, were almost identical in 18S rDNA PCR-RFLP analysis. Two Saccharomyces sensu lato strains, S. servazzii and S. unisporus, were grouped together and close to Z. rouxii, but distinct from the baker's isolates. Two C. albicans strains were grouped together with C. tropicalis and S. kluyveri and close to R. toruloides strain. The strains of species K. lac-

70 80 90 100 I I , I , , I I , I ,J , I I I , , , I " , I , , , , , , ,J , , , " ' , lei, , , , I I , I , I I , I , , , " ' I

Fig. 2. A dendrogram of the strains based on the EF-3 PCR-RFLP assay. In construc­tion of the dendrogram GeI­Compar program package was used with Dice's coeffi­cient band matching and UPGMA clustering method.

Candida milleri vn C-87175 Candida milleri vn C-96244

'----Candida milleri vn C-96250 '-------Saccharomyces exiguus vn C-81115

'----------Candida milleri vn C-95232 Baker's isolate A 11 Baker's isolate A 12 Baker's isolate B 11 Baker'S isolate C 12 Baker's isolate D11 Baker's isolate D12 Baker's isolate E 11 Baker's isolate E12 Baker's isolate D22

'------Baker's isolate B12

L __ --1C===Candida milleri ATCC 62655 Candida milleri A TCC 60592 Candida milleri CBS 7541 Candida milleri CBS 2664 Saccharomyces exiguus CBS 7901

'--------------------Candida tropical is ATCC 750 Torulaspora delbrueckii CBS 1146 Torulaspora delbrueckii TS 207

'--------Saccharomyces kluyveri CBS 3082 '-------------Zygosaccharomyces rouxii CBS 732

,..--------Kluyveromyces lactis CBS 2359 '---------Saccharomyces servazzii CBS 4311

'-----------Saccharomyces pastorianus NCYC 396 Saccharomyces cerevisiae YNN 295 Baker's isolate D21 Baker'S isolate E21 Baker'S isolate E22 Saccharomyces cerevisiae vn C-81117

'---------1 Saccharomyces cerevisiae vn C-95236 Saccharomyces cerevisiae ALKO 743

L _____ C==========Candida glabrata ATCC 2001 Baker's isolate C11

r __ --{======Candida albicans ATCC 10231 Candida albicans ATCC 18804 Saccharomyces unisporus CBS 398 Saccharomyces castellii CBS 4309 Saccharomyces dairensis CBS 421

L ____ -[========Saccharomyces exiguus CBS 379 Saccharomyces exiguus CBS 135

92 V. H. MANTYNEN et al.

tis, P. {ermentans and S. pombe, together with baker's isolate Cll, were clustered further apart from the other strains used in this study.

• EF-3 PCR-RFLP analysis: The EF-3 PCR-RFLP analysis was based on the distribution of 79 different sized fragments. The EF-3 DNA sequence could not be amplified from strain.s of C. geochares, C. lauren:ii~ P. fermentans, R. toruloldes, and S. pombe. The baker s ISO­

lates formed two distinct groups. Ten of the Baker's iso­lates (All, A12, Bll, B12, C12, Dll, D12, D22, Ell, E12) were grouped close to C. milleri strains ATCC 60592, ATCC 62655, CBS 2664, CBS 7541, and S. ex­iguus strain CBS 7901 (Figure 2). Other C. milleri strains (CBS 6897, VTT C-87175, VTT C-95232, VTT C-96250) and a S. exiguus strain VTT C-81115 formed an­other subgroup. Three of the baker's isolates (D21, E21, E22), which later were identified as S. boulardii in ~iolog assay were grouped closely together with the strams of Saccharomyces cerevisiae. Two S. exiguus strains CBS 135 and CBS 379 were clustered closer to the other Sac­charomyces sensu lato yeasts S. castellii, S. dairensis, and S. unisporus. Also C. albicans strains were rather close to the Saccharomyces sensu lato species. However, the S. kluyveri strain remained together with T. delbrueckii group. The S. pastorianus strain was grouped together with the K. lactis strain in EF-3 analysis. The baker's iso­late C11 was only distantly grouped together with C. glabrata and S. servazzii. Thus, the overall results with EF-3 PCR-RFLP did not totally agree with the results of 18S rDNA PCR-RFLP assay.

Karyotyping

The bakery isolates fell into two distinct groups in pulsed field gel electrophoresis karyotyping patterns. The isolates which were similar in PCR-RFLP assays were similar also in their karyotypes (Fig. 3). Ten isolates which grouped together with C. milleri strains (Fig. 3. lanes 2-7) and one S. exiguus strain (lane 13) in EF-3 PCR-RFLP assay all had a karyotype characteristic for C. milleri (lane 9) except for isolates from bakery A (lane 8), which had only 8 bands ranging from about 370 kb up to 1600 kb and had therefore similar, though distinct, karyotype with C. milleri CBS 2664 (lane 5). The charac­teristic pattern for C. milleri was 14 bands ranging from about 430 kb up to 2200 kb. The C. milleri strains had distinct karyotypes from the S. exiguus strains (lanes 10-12) The isolate Cll, which was different in all the other assays was also different from the others in PFGE analysis (lane 15) and had large size chromosomes simi­lar to C. albicans (lane 14). The isolates which were grouped closely with S. cerevisiae strains in the EF-3 PCR-RFLP assay and which were later identified as S. boulardii in the Biolog system each had similar karyo­type (lane 20) with S. cerevisiae strains (lanes ~, 17-19). The T. delbrueckii sour dough yeast had a umque kary­otype (lane 16).

Minimum growth temperature

The minimum growth temperatures of the yeast strains are presented in Table 1. The minimum growth

1 2 34 56 7 8 9 1011121314151617181920 I\b

2200

1600

112S

1020

'us

IISO HOO 710 700 G 0 :'1110 ~60

J70 290 Wi

Fig. 3. Ethidium bromide stained 1 % agarose gel after CHEF gel elec­trophoresis. In lane 1. S. cerevisiae YNN 295, 2. C. milleri VTT C-87175,3. C. milleri CBS 6897, 4. C. milleri VTT C-96250, 5. C. milleri CBS 2664, 6. C. milleri CBS 7541, 7. C. milleri ATCC 60592, 8. Bak­er's isolate AIl, 9. Baker's isolate B12, 10. S. exiguus VTT C-81115, 11. S. exiguus CBS 135, 12. S. exi­guus CBS 379, 13. S. exiguus CBS 7901, 14. C. albicans ATCC 18804, 15. Baker's isolate Cll, 16. T. del­brueckii TS207, 17. S. cerevisiae VTT C-81117, 18. S. cerevisiae VTT C-95236, 19. S. cerevisiae ALKO 743, and 20. Baker's isolate E22.

A Polyphasic Study on the Taxonomic Position of Industrial Sour Dough Yeasts 93

temperature ranged between 8.3-8.7 °C (average 8.4 °C, n = 13) of the Baker's isolates. The C. milleri reference strains had similar minimum temperature limitations average of 8.4 °C (n = 7). The minimum temperatures of S. cerevisiae (range 1-8.7 °C, average 3.9 °C, n = 4) and S. exiguus (range 5.8-9.2 °C, average 7.9 °C, n = 4) strains varied more. All three C. albicans strains had the same temperature limit of 7.3 °C, n = 3. One strain of S. cerevisiae could grow even at 0.7 °C and the reference strains could generally grow at lower temperatures than the baker's isolates.

Biolog identification

The identification of the strains is presented in Table 1. The Biolog system identified only eleven of 39 refer­ence strains correctly, and five additional strains were identified to the genus level. The Biolog system identified nine of the baker's isolates as Kluyveromyces lodderae and three as Saccharomyces boulardii which is a syno­mym for S. cerevisiae. There were single isolates of the species Candida spandovensis (Cll) and Trigonopsis tri­angularis var variabilis (Bll). However, all eight C. mil­leri and four S. exiguus reference strains were also identi­fied as Kluyveromyces lodderae. This indicated that nine yeasts isolated from sour doughs were similar to C. mil­leri and S. exiguus strains in their physiological activities. In fact nine of the sour dough yeasts (All, A 12, B12, Cll, Dll, D12, D22, Ell, E12) actually used the same substrates as the C. milleri reference strains ATCC 60592, CBS 2664, CBS 6897, and CBS 7541. These in­cluded a-D-glucose, D-galactose, raffinose, stachyose, sucrose, and D-trehalose. The remaining three sour dough isolates (D21, E21, E22) used maltose and these strains were identified as S. boulardii. Other strains of C. milleri that have been previously isolated from sour doughs, namely VTT C-87175, VTT C-95232, and VTT C-96250 had lower score for identification as K. lodder­ae. The species S. castellii, S. dairensis, S. servazzii, and S. unisporus were all identified as S. dairensis, probably because it was the only Saccharomyces sensu lato yeast in the Biolog database.

Discussion

The genetic basis of phenotypic characters is poorly known, therefore current taxonomy is unlikely to predict accurately evolutionary relationships (KURTZMAN 1994). Candida spp. are members of Deuteromycetes (fungi im­perfect) because "they have either lost the ability to un­dergo sexual development or have not chosen this path when mycologists were around to notice" (NELSON 1996). Ribosomal RNA molecules have also served as an evolutionary marker for yeasts (MAGEE et al. 1987, BARNS et al. 1991, BRUNS et al. 1991, BOWMAN et al. 1992, MOLINA et al. 1992, WIlMOTTE et al. 1993, MAS­NEUF et al. 1996, JAMES et al. 1997, SMOLE-MoZINA et al. 1997). HENDRIKS et al. (1989) proposed that C. albicans is a member of ascomycetous yeasts rather than fungi im-

perfect based on DNA nucleotide sequence coding for small ribosomal subunit RNA. Polyphasic studies using several methods to classify and characterize microbial strains approach a phenetic species concept and could serve as an approach to study yeast strains. In this study we used two different target DNA sequences (18S rDNA and EF-3), electrophoretic karyotyping and a metabolic fingerprinting method (Biolog) together with minimum growth temperature studies to characterize the yeast strains isolated from Finnish sour doughs.

The suitable criteria for grouping of strains depend on samples, methods and analyses used (SNEATH and SOKAl 1973). We used here Dice correlation coefficient and UPGMA clustering method to analyse the PCR-RFLP based results. The taxonomic positioning of the reference strains was used to evaluate the accuracy of the used as­says.

According to 18S rDNA PCR-RFLP analysis thirteen baker's isolates formed their own unique cluster close to strains of C. milleri and S. exiguus. The "sour dough yeast" clusters were intermixed with strains of C. glabra­ta, C. laurentii, S. cerevisiae, S. castellii, S. dairensis, S. exiguus, S. pastorianus, and T. delbrueckii. The physio­logical and immunological methods used to distinguish species S. exiguus, C. milleri, and S. cerevisiae have been found unsatisfactory (BARNETT 1992, MIDDElHOVEN and NOTERMANS 1993), and the results of physiological tests of the C. milleri and S. exiguus strains have been found contradictory (VAUGHAN-MARTINI 1995). The members of the genera Kluyveromyces, Saccharomyces, Torula­spora, and Zygosaccharomyces have also been found to intermix according to molecular methods (CAl et al. 1996, JAMES et al. 1997). Even though S. exiguus and S. cerevisiae have distinct 18S-28S rRNA spacer regions (ODA et al. 1997), the 18S rRNA DNA sequence analysis has shown that S. cerevisiae, S. exiguus, and T. del­brueckii are intermixed with each other and well separat­ed from two Saccharomyces sensu lato species, ie. S. unisporus and S. servazzii; C. albicans forms a coherent group with C. maltosa, C. parapsilosis, C. tropicalis, and C. viswanathii (KURTZMAN 1992, CAl et al. 1996, JAMES et al. 1997, ODA et al. 1997). Similar results were ob­tained also in this study using 18S rDNA PCR-RFLP assay.

The relatively large group of C. milleri and S. exiguus strains, together with the majority of baker's isolates, were clustered in their own branch according to the EF-3 assay. Special attention should be given to S. exiguus CBS 7901, which has been isolated from sour dough. In both 18S rDNA and EF-3 PCR-RFLP assays this strain was more closely related to the C. milleri strains and baker's isolates than to the other two S. exiguus strains, CBS 135 and CBS 379. The high DNA reassociation found by VAUGHAN-MARTINI (1995) between the S. exiguus strains CBS 135 and CBS 379 was supported by both 18S rDNA PCR-RFLP and EF-3 PCR-RFLP analyses, and further­more by the results obtained using the Biolog system. However, the electrophoretic karyotypes of CBS 135 and CBS 379 have been shown to be significantly different (NAUMOVA et al. 1996). S. unisporus CBS 398 and S. ser-

94 V. H. MANTYNEN et al.

vazzii CBS 4311 were closely grouped together in 18S rDNA assay, but separated in EF-3 assay supporting the results of VAUGHAN-MARTINI et al. (1993). Both 185 rDNA and EF-3 PCR-RFLP analyses gave similar overall classification on the baker's isolates, although the dis­tances between the strains were greater in EF-3 assay than in 185 rDNA assay and the strains identified as S. boulardii in Biolog system were nicely separated in EF-3 assay together with the S. cerevisiae strains. The EF-3 DNA sequence could not be amplified from strains of C. geochares, C. laurentii, P. fermentans, R. toruloides, and Schz. pombe. This together with the other results ob­tained from this assay confirmed that EF-3 gene is rather heterogeneous among yeasts.

VAUGHAN-MARTINI et al. (1993) found that S. baya­nus, S. castellii, S. cerevisiae, S. kluyveri, S. paradoxus, and S. pastorianus show uniform electrophoretic karyo­types, but that S. dairensis, S. exiguus, S. servazzii, and S. unisporus comprise heterogenous taxa. The latter strains were found to intermix with each other in all DNA­based comparisons used in the present study. Our 185 rDNA PCR-RFLP analysis of the C. milleri strains CBS 2664 and CBS 6897 agreed well with the high DNA reas­sociation found by VAUGHAN-MARTINI (1995). C. milleri reference strains were found to exhibit extensive chro­mosome length polymorphism. The sour dough yeasts which were grouped closely in 185 rDNA and EF-3 PCR­RFLP assays were also similar in their karyotypes and similar with some C. milleri reference strains isolated from bread environments e.g. C. milleri CBS 7541 and C. milleri ATCC 60592 (in figure 3). The Finnish sour dough yeasts had similar karyotypic characters with most C. milleri reference strains, except C. milleri CBS 2664, which had similar, though distinct karyotype with baker's isolate A 11. They all were found to be distinct from S. cerevisiae and T. delbrueckii strains, which have been considered as bread leavening yeasts (ALMEIDA and PAIS 1996). The karyotypes of the reference strains of S. cerevisiae and T. delbrueckii included in this study agreed with earlier studies (e.g. NAUMOV et al. 1992, ODA and TONOMURA 1995). The karyotype of isolate Cl1 (lane 15 in figure 3), which was identified as C. spandovensis ac­cording to Biolog system differed from the karyotype of C. albicans.

The minimum growth temperatures of sour dough starter yeasts were rather high with average of 8,4 0c. This basic growth requirement can be used as additional criteria when classifying yeasts. Sour dough yeasts in­cluding C. milleri reference strains exhibited similar min­imum growth temperature requirements.

The reference strains of S. cerevisiae, C. milleri, S. exi­guus,and C. albicans formed their own groups in both of the PCR-RFLP assays and their taxonomic relationships correlated quite well with earlier studies (CAl et al. 1996, JAMES et al. 1997, ODA et al. 1997). In all assays the C. milleri and S. exiguus strains were distributed to several groups indicating a large amount of heterogeneity among these species. The result that baker's isolates are only dis­tantly related with pathogenic C. albicans should also be noted.

Nine strains of the Finnish bakery starters were identi­fied by metabolic pattern as Kluyveromyces lodderae, which is closely related to S. exiguus and its anamorph C. holmii according to 185 rRNA DNA sequence studies (JAMES et al. 1997). The strains also formed their own cluster in EF-3 analysis. The species C. milleri is not in­cluded in current data base version of Biolog system. Therefore, if the identification of C. milleri and S. exi­guus strains as K. lodderae and furthermore if the identi­fication of strains S. castellii, S. dairensis, S. servazzii, and S. unisporus as S. dairensis was considered as a cor­rect identification according to current data base, then the identification percent would be 75%. This result is in fairly good agreement with the results obtained earlier with the Biolog system (PRAPHAlLONG et al. 1997). How­ever, the Biolog system at present identifies poorly differ­ent yeast strains found in nature, though broadening the current Biolog data base may improve the identification percent.

A polyphasic approach to phylogeny will give confi­dence to the taxonomic estimates obtained with different methods. We have shown that DNA-based methods pro­vide more detailed information, and that the PCR-RFLP analysis can be used to classify the bakery strains. Mini­mum growth temperature studies can provide informa­tion about the physiological factors influencing the starters storage and provides additional information for classification of yeasts.

Acknowledgements: The authors wish to thank Prof. SEPPO I. NIEMELA and TErpo

RAUTAVESI for guidance on determination of minimum growth temperatures, and ANNE MAKINEN and MARJO TOIVO for excel­lent technical support. The work is part of a Nordic Industry Foundation NORDFOOD project Rapid Control Methods (P93155) funded by TEKES (Technology Development Centre, Finland), Finnzymes Oy, Finland, and Ingman Foods Oy Ab, Fin­land. The project was also related to a local sour dough project (Tehoraski projekti) financed by TEKES and five industrial bak­eries (Fazer bakeries Ltd., Vaasa bakeries Ltd., Linkosuo Ltd., Primula Ltd. and Uotilan leipomo Ltd). Additional funding was provided by The Academy of Finland (ABS Graduate School).

References

ALMEIDA, M. j., and PAIS, C. Leavening ability and freeze toler­ance of yeasts isolated from traditional corn and rye bread doughs. Appl. Environ. Microbiol. 62,4401-4404 (1996).

BARNETT, J. A.: The taxonomy of the genus Saccharomyces Meyen ex Reess: a short review for non-taxonomists. Yeast. 8,1-23 (1992).

BARNETT, J. A., PAYNE, R. W., and YARROW, D.: Yeasts: charac­teristics and identification 2nd ed. Cambridge, Cambridge University Press (1990).

BARNS, S. M., LANE, D. j., SOGIN, M. L., BIBEAU, c., and WEIS­BURG, W. G.: Evolutionary relationships among pathogenic Candida species and relatives. J. Bacteriol. 173, 2250-2255 (1991).

BELFIELD, G. P. and TUITE, M. E: Translation elongation factor 3:a fungus-specific translation factor? Mol. Microbiol. 9, 411-418 (1993).

A Polyphasic Study on the Taxonomic Position of Industrial Sour Dough Yeasts 95

BOEKHOUT, T., RENTING, M. SCHEFFERS, W. A., and BOSBOOM, R.: The use of karyotyping in the systematics of yeasts. An­tonie van Leeuwenhoek. 63, 157-163 (1993).

BOTHA, A. and KOCK, J. L. E: Application of fatty acid profiles in the identification of yeasts. Int. J. Food Microbiol. 19, 39-51 (1993).

BOWMAN, B. H., TAYLOR, J. W., and WHITE, T J.: Molecular evolution of the fungi:Human pathogens. Mol. BioI. Evol. 9, 893-904 (1992).

BRUNS, T D., WHITE, T. J., and TAYLOR, J. W.: Fungal molecular systematics. Annu. Rev. Ecol. Syst. 22, 525-564 (1991).

CAl, J., ROBERTS, I. N., and COLLINS, M. D.: Phylogenetic rela­tionships among members of the ascomycetous yeast genera Brettanomyces, Debaryomyces, Dekkera, and Kluyveromy­ces deduced by small-subunit rRNA gene sequences. Int. J. Syst. Bacteriol. 46, 542-549 (1996).

CODON, A. c., and BENiTEZ, T: Variability of the physiological features and of the nuclear and mitochondrial genomes of the Bakers' yeasts. System. Appl. Microbiol. 1S, 343-352 (1995).

COLTHURST, D. R., SANTOS, M., GRANT, C. M., and TUITE, M. E: Candida albicans and three other Candida species contain an elongation factor structurally and functionally analogous to elongation factor 3. FEMS Microbiol. Lett. SO, 45-50 (1991).

COLTHURST, D. R., SCHAUDER, B. S., HAYES, M. Y., and TUITE, M. E: Elongation factor 3 (EF-3) from Candida albicans shows both structural and functional similarity to EF-3 from Saccharomyces cerevisiae. Mol. Microbiol. 6, 1025-1033 (1992).

D'AQUILA, R. T, BECHTEL, L. J., VIDELER, J. A., ERON, J. J., GORCZYCA, P., and KAPLAN, J. c.: Maximizing sensitivity and specificity of PCR by pre-amplification heating. Nucl. Acids Res. 19,3749 (1991).

DE JONGE, P., DE JONGH, E C. M., MEIJERS, R., STEENSMA, H. Y., and SCHEFFERS, W. A.: Orthogonal-field-alternation gel elec­trophoresis banding patterns of DNA from yeasts. Yeast. 2, 193-204 (1986).

HANSEN, B. and HANSEN, A.: Volatile compounds in wheat sourdoughs produced by lactic acid bacteria and sourdough yeasts. Zeitschrift fiir Lebensmittel Untersuchung und Forschung. 198,202-209 (1994).

HENDRIKS, L., GORIS, A., NEEFS, J.-M., VAN DE PEER, Y., HEN­NEBERG, G., and DE WACHTER, R.: The nucleotide sequence of the small ribosomal subunit RNA of the yeast Candida albi­cans and the evolutionary position of the fungi among the Eukaryotes. System. Appl. Microbiol. 12,223-229 (1989).

JAMES, S. A., CAl, J. ROBERTS, I. N., and COLLINS, M. D.: A phy­logenetic analysis of the genus Saccharomyces based on 18S rRNA gene sequences: Description of Saccharomyces ku­nashirensis sp. nov. and Saccharomyces martinae sp. nov. Int. J. Syst. Bacteriol. 47, 453-460 (1997).

KREGER-VAN-RIj, N. J. W.: Generic differentiation in yeasts. pp. 29-49. In: The Society for Applied Bacteriology symposium series no 9. Biology and Activities of yeasts. (E A. SKINNER, S. M. PASSMORE, and R. R. DAVENPORT, eds.). San Francisco, Academic Press (19S0).

KURTZMAN, C. P.: rRNA sequence comparisons for assessing phylogenetic relationships among yeasts. Int. J. Syst. Bacte­riol. 42,1-6 (1992).

KURTZMAN, C. P.: Systematics of the ascomycetous yeasts as­sessed from ribosomal RNA sequence divergence. Antonie van Leeuwenhoek 63,165-174 (1993).

KURTZMAN, C. P.: Molecular taxonomy of the yeasts. Yeast. 10, 1727-1740 (1994).

KURTZMAN, c. P. and BLANZ, P.A.: Ribosomal RNNDNA se­quence comparisons for assessing phylogenetic relationships.

pp. 69-74. In: The Yeasts:A taxonomic study. (c. P. KURTZ­MAN and J. W. FELL, eds.) 4th ed., Amsterdam, Elsevier Science B.Y. (1998).

MAGEE, B. B., D'SOUZA, T. M., and MAGEE, P. T.: Strain and species identification by restriction fragment length polymor­phisms in the ribosomal DNA repeat of Candida species. J. Bacteriol. 169, 1639-1643 (1987).

MASNEUF, I., AIGLE, M., and DUBOURDIEU, D.: Development of a polymerase chain reaction/restriction fragment length poly­morphism method for Saccharomyces cerevisiae and Saccha­romyces bayanus identification in enology. FEMS Microbio!. Lett. 138,239-244 (1996).

MIDDELHOVEN, W. J. and NOTERMANS, S.: Immuno-assay tech­niques for detecting yeasts in foods. Int. J. Food Microbiol. 19,53-62 (1993).

MILLS, D. and MCCLUSKEY, K.: Electrophoretic karyotypes of fungi: the new cytology. Mol. Plant. Microbe Interaction. 3, 351-357 (1990).

MOLINA, EI., INOUE, T., and JONG, S.-c.: Ribosomal DNA re­striction analysis reveals genetic heterogeneity in Saccha­romyces cerevisiae Meyen ex Hansen. Int. J. Syst. Bacteriol. 42,499-502 (1992).

MYERS, K. K., FONZI, W. A., and SYPHERD, P.S.: Isolation and se­quence analysis of the gene for translation elongation factor 3 from Candida albicans. Nucl. Acids Res. 20, 1705-1710 (1992).

NAUMOV, G., NAUMOVA, E., TURAKAINEN, H., SUOMINEN, P., and KORHOLA, M.: Polymeric genes MELS, MEL9 and MELlO -new members of a-galactosidase gene family in Saccharo­myces cerevisiae. Curro Genet. 20, 269-276 (1991).

NAUMOV, G. I., NAUMOVA, E. S., LANTTO, R. A., LOUIS, E. J., and KORHOLA, M.: Genetic homology between Saccharomyces cerevisiae and its sibling species S. paradoxus and S. bayanus: electrophoretic karyotypes. Yeast. S, 599-612 (1992).

NAUMOVA, E. S., NAUMOV, G. I., and KORHOLA, M.: Use of molecular karyotyping for differentiation of species in the heterogeneous taxon Saccharomyces exiguus. J. Gen. App!. Microbiol. 42, 307-314 (1996).

NELSON, M. A.: Mating systems in ascomycetes: a romp in the sac. Trends in Genetics. 12,69-74 (1996).

ODA, Y. and TONOMURA, K.: Electrophoretic karyotyping of the yeast genus Torulaspora. Lett. App!. Microbio!. 21,190-193 (1995).

ODA, Y., YABUKI, M., TONOMURA, K., and FUKUNAGA, M.: A phy­logenetic analysis of Saccharomyces species by the sequence of 18S-28S rRNA spacer regions. Yeast. 13, 1243-1250 (1997).

OTTO GALLI, G., GALLI, A., and FOSCHINO, R.: Italian bakery products obtained with sour dough: Characterization of the typical microflora. Adv. Food Sci. 18, 131-144 (1996).

PRAPHAILONG, W., VAN GESTEL, M., FLEET, G. H., and HEARD, G. M.: Evaluation of the Biolog system for the identification of food and beverage yeasts. Lett. Appl. Microbio!. 24, 455-459 (1997).

ROHM, H. and LECHNER, E Evaluation and reliability of a sim­plified method for identification of food-borne yeasts. App!. Environ. Microbio!. 56, 1290-1295 (1990).

SALOVAARA, H., and SAVOLAINEN, J.: Yeast type isolated from Finnish sour rye dough starters. Acta Alimentaria Polonica 10,241-245 (1984).

SALOVAARA, H.: Lactic acid bacteria in cereal products. pp. 115-13S. In Lactic acid bacteria - Technology and Health ef­fects 2nd ed. (S. SALMINEN and A. VON WRIGHT, eds.). Marcel Dekker (1998).

SAVOLA, P., SALOVAARA, H., and ENQVIST, J.: Concentrations of carbohydrate fractions in the sour dough rye bread process. pp. 465-469, Proc. 7th World cereal and bread congress, Prague (1982).

96 V. H. MANTYNEN et al.

SMOLE-MoZINA, S., DLAUCHY, D., DEAK, T., and RASPOR, P.: Identification of Saccharomyces sensu stricto and Torulaspo­ra yeasts by PCR ribotyping. Lett. Appl. Microbiol. 24, 311-315 (1997).

SNEATH, P. H. A., and SOKAL, R. R.: Numerical taxonomy: the principles and practise of numerical classification. pp. 573. San Francisco, W.H. Freeman and Company (1973).

STOKES, J. L.: Influence of temperature on the growth and metabolism of yeasts. The Yeasts. Physiology and biochem­istry of yeasts. (A. H. ROSE and J. S. HARRISON eds.). New York, Academic Press vol 2, 119-134 (1971).

SWOBODA, R. K., BERTRAM, G., COLTHURST, D. R., TUITE, M . F., Gow, N. A. R., GOODAY, G. W., and BROWN, A. J. P.: Regula­tion of the gene encoding translation elongation factor 3 dur­ing growth and morphogenesis in Candida albicans. Micro­biology 140, 2611-2616 (1994).

VAUGHAN-MARTINI, A.: Saccharomyces barnettii and Saccha­romyces spencerorum: two new species of Saccharomyces sensu lato (van der Walt). Antonie van Leeuwenhoek 68, 111-118 (1995).

VAUGHAN-MARTINI, A., and MARTINI, A.: Taxonomic revision of the yeast genus Kluyveromyces by nuclear deoxyribonucleic acid reassociation. Int. J. Syst. Bacteriol. 37, 380-385 (1987).

VAUGHAN-MARTINI, A., MARTINI, A., and CARDINALI, G.: Elec­trophoretic karyotyping as a taxonomical tool in the genus Saccharomyces. Antonie van Leeuwenhoek 63, 145-156 (1993).

VOGEL, R. E: Microbial ecology of cereal fermentations. Food Technol. Biotechnol. 35, 51-54 (1997).

WILMOTTE, A., VAN DE PEER, Y., GORIS, A., CHAPELLE, S., DE BAERE, R., NELISSEN, B., NEEFS, J.-M., HENNEBERT, G. L., and DE WACHTER, R.: Evolutionary relationships among higher fungi inferred from small ribosomal subunit RNA sequence analysis. System. Appl. Microbiol. 16,436-444 (1993).

YARROW, D.: Candida milleri sp. nov. Int. J. Syst. Bacteriol. 28, 608-610 (1978).

Corresponding author: VESA MANTYNEN, University of Helsinki, Department of Ap­plied Chemistry and Microbiology, Biocenter, P.O. Box 56, FIN-00014 University of Helsinki, Finland Tel.: 358-9-70859304; Fax: 358-9-70859322; e-mail: Vesa.Mantynen@Helsinki.Fi