ducing similar band patterns) using Hae III (Fig. 2, Table
i.e., brewing strain mixed with other closely related spe-
cies, in order to determine at what level contaminants can
be detected.
II). Similar results within Saccharomyces sensu stricto us-
3
ing Hae III were obtained by Esteve-Zarzoso et al.
It was also possible to distinguish Sacch. paradoxus
from the sensu stricto members using Msp I digestion
(
Fig. 5, Table V). This is in accordance with the rather
isolated position of Sacch. paradoxus when compared
with the other members of the “cerevisiae” cluster based
on PCR-RFLP and ITS sequence analyses as reported by
The authors wish to thank the National Research Foundation
in South Africa for funding.
8
Montrocher et al.
In addition, the Saccharomyces sensu stricto group
could easily be differentiated from the Saccharomyces
sensu lato group when comparing band patterns after Sau
1
2
. Barnett, J. A., Journal of General Microbiology, 1977, 99, 183.
. Deak, T. and Beuchat, L. R., Handbook of Food Spoilage
Yeasts, Boca Raton: CRC Press, 1996, Chapter 7.
. Esteve-Zarzoso, B., Belloch, C., Uruburu, F. and Querol, A.,
International Journal of Systematic Bacteriology, 1999, 49,
329.
3A1 digestion (Fig. 4, Table IV). It was also possible to
3
distinguish the brewing strains from all the other Sac-
charomyces species tested on the basis of the two extra
bands that differentiated the brewing yeasts from Sacch.
cerevisiae (Fig. 6). These results show promise in the con-
struction of a quality control system where it is necessary
to distinguish brewing strains from closely related yeasts.
However, more commercial brewing strains and strains
from other yeast genera known to be contaminants should
be analysed to make a thorough evaluation if the brewing
strains remain unique on the basis of this characteristic. It
is also important to apply this method to mixed cultures,
4. Guillamón, J.M., Sabaté, J., Barrio, E., Cano, J. and Querol, A.,
Archives of Microbiology, 1998, 169, 387.
5
. Innes, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J., PCR
Protocols: A Guide to Methods and Applications, New York:
Academic Press, 1990.
6. Kurtzman, C.P. and Fell, J.W., The Yeasts-A Taxonomic Study,
4th ed., Amsterdam: Elsevier Science, 1998.
7. Messner, R. and Prillinger, H., Antonie van Leeuwenhoek, 1995,
7, 363.
6
8. Montrocher, R., Verner, M.-C., Briolay, J., Gautier, C. and Mar-
meisse, R., International Journal of Systematic Bacteriology,
1998, 48, 295.
9. Nguyen, H.-V., Lepingle, A. and Gaillardin, C., Systematic and
Applied Microbiology, 2000, 231, 71.
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1
0. Phaff, H. J., Miller, M. W. and Mrak, E. M., The Life of Yeasts.
nd ed., Harvard University Press: Cambridge, 1978.
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1
1. Valente, P., Gouveia, F.C., de Lemos, G.A., Pimentel, D., van
Elsas, J.D., Mendonca-Hagler, L.C. and Hagler, A.N., FEMS
Microbiology Letters, 1996, 137, 253.
1
2. White, T. J., Bruns, T., Lee, S. and Taylor, J., Amplification and
direct sequencing of fungal ribosomal RNA genes for phylo-
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tion, M.A. Innis, D.H. Gelfand, J.J Sninksky and T.J. White,
Eds., Academic Press: New York, 1990.
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Manuscript accepted for publication March 2002)
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%44)2(-<ꢀ
Appendix. List of abbreviations and terminology used
ꢆꢃꢃ
CBS
Cfo
Centraalbureau voor Schimmelcultures
Restriction enzyme obtained from Clostridium
formicoaceticum.
ꢇꢃꢃ
CTAB
DNA
rDNA
EDTA
Hae
Hexadecyl trimethyl ammonium bromide
Deoxyribose nucleic acid
Ribosomal deoxyribose nucleic acid
2-ethylene-diamine-tetra-acetate
Restriction enzyme obtained from Haemophilus
aegyptius
ITS
Internal transcribed spacer
Msp
DNTP
PCR
Restriction enzyme obtained from Moraxella sp.
Deoxyribose nucleoside triphosphate
Polymerase chain reaction
FIG. 6. Restriction pattern of the PCR-amplified rDNA region of
Saccharomyces cerevisiae and the two brewing strains digested
with Msp I restriction enzyme. a- is the standard ladder with
bands corresponding to 200, 300, 400, 500, 1000, 1500 base
pairs. b- Sacch. cerevisiae, c- brewing strain UOFS Y-0494, d-
brewing strain UOFS Y-0532.
ppm
RFLP
Sau 3A
parts per million
Restriction fragment length polymorphisms
Restriction enzyme obtained from Staphylococcus
aureus
Tris, 2-ethylene-diamine-tetra-acetate buffer
Yeast extract 1%, Peptone 2%, Glucose 2%
T E buffer
YPD medium