Biotechnology & Biotechnological Equipment

ISSN: 1310-2818 (Print) 1314-3530 (Online) Journal homepage: http://www.tandfonline.com/loi/tbeq20

Phenotypic and Genotypic Characterization of

Probiotic Yeasts

K. Rajkowska & A. Kunicka-Styczyńska

To cite this article: K. Rajkowska & A. Kunicka-Styczyńska (2009) Phenotypic and Genotypic
Characterization of Probiotic Yeasts, Biotechnology & Biotechnological Equipment, 23:sup1,
662-665, DOI: 10.1080/13102818.2009.10818511

To link to this article: http://dx.doi.org/10.1080/13102818.2009.10818511

© 2009 Taylor and Francis Group, LLC

Published online: 15 Apr 2014.

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 PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF PROBIOTIC
YEASTS

K. Rajkowska and A. Kunicka-Styczyńska

Technical University of Lodz, Wolczanska 171/173, 90-924 Lodz, Poland
Correspondence to: K. Rajkowska

ABSTRACT
Yeast classification is traditionally based on their physiological and biochemical profiles. However, molecular methods have
been recently successfully applied to yeast strain typing and identification. The aim of this research was to characterize
probiotic yeast strains by classical (morphology, reproduction, biochemical features) and molecular (karyotype analysis)
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methods. Nine strains of Saccharomyces boulardii were examined: two strains from ATCC Collection and seven probiotic
strains isolated from medicines. Although there were some variations in biochemical utilization of carbon and nitrogen
compounds, all the strains were identified as Saccharomyces cerevisiae according to their morphological characteristics and
assimilation profiles. Chromosomal patterns of yeasts consisted of 13 – 14 bands ranging from 222 to 2251 kb, what indicated
their classification to the genus S. cerevisiae. According to current nomenclature the tested yeasts should be referred as S.
cerevisiae var. boulardii.

Keywords: biochemical profiles, karyotype, morphology, criteria are often unable to discriminate at a subspecies level
probiotics, yeast, and provide doubtful identification (4,23,26,27). However,
molecular methods have also recently become available.
Introduction Among them karyotyping, restriction analysis of mtDNA,
Probiotic yeasts are non-pathogenic strains belonging to the PCR-based techniques have been successfully applied to
genus Saccharomyces cerevisiae var. boulardii (20). yeast strain typing and identification (10,13,21,22). The aim
Probiotic cultures have been used as both a preventive and of this research was to characterize probiotic yeasts strains by
therapeutic agent for the treatment of a variety of diarrheal classical (morphology, reproduction, biochemical features)
diseases. S. boulardii is reported to be effective in the and molecular (karyotype analysis) methods.
treatment for diarrhea in adults and children infected with
Clostridium difficile, for diarrhea in human Materials and methods
immunodeficiency virus-infected patients and for acute and Yeast strains
chronic diarrhea in children and adults (16,20). Potential In this study nine strains of Saccharomyces boulardii were
mechanisms of probiotic activity are based on secretion of considered: two strains from the American Type Culture
proteases or inhibitory proteins, stimulation of Collection and seven strains isolated from medicines. All the
immunoglobulin A, acquisition and elimination of secreted strains used in this study are listed in Table 1. Isolates were
toxins (8,16). In vitro and in vivo studies have indicated that kept on yeast extract-peptone-dextrose (YPD) agar slants and
S. boulardii is able to prevent intestinal infection caused by maintained at -20°C in YPD broth with 20% (v/v) glycerol.
Escherichia coli, Salmonella Typhimurium, Staphylococcus Microscopical examination for yeast cells
aureus, Pseudomonas aeruginosa, Proteus vulgaris, Yersinia In order to determine morphology of yeasts cells and
enterocolitica and Candida albicans (5,16). Furthermore, reproduction type the cultures were examined
oral administration of S. boulardii leads to a distinct increase microscopically (2). Vegetative cells were observed after 3
in the activity of the disaccharidases lactase, sucrase and days of incubation at 25°C in YPD medium.
maltase in the brush border membrane (3). Filamentous growth was determined in slide cultures put
Yeast classification is traditionally based on into a Petri dish with potato glucose agar. Ascospores
morphological and physiological criteria. These conventional formation was examined after incubation in Gorodkowa agar,

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120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY SPECIAL EDITION/ON-LINE
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 acetate agar and malt-yeast-glucose-peptone (YM) agar every concentration of 0.8% and the electrophoresis buffer was 0.5
week up to 90 days. x TBE cooled to 10ºC. Electrophoresis was carried out at 6
V/cm for 26 h with a switching time increasing from 110 to
TABLE 1
220 s. After electrophoresis the gel was stained with ethidium
Origins of yeast strains investigated
bromide for visualization of chromosomal DNA. A standard
Yeast strains Origin Source commercial set of S. cerevisiae YNN295 chromosomes (Bio-
S1 Enterol® 250 Biocodex Rad) was used for comparison.
S2 Hamadin® N Dr. Willmar Schwabe
S3 Omniflora® Akut Novartis
Results and Discussion
S4 Perenterol® forte UCB All yeast strains were identified as Saccharomyces cerevisiae
S5 Perocur® forte Hexal according to their morphological characteristics, ability to
S6 Santax® S Asche assimilate carbon and nitrogen compounds and sugar
S7 Yomogi® Ardeypharm fermentation patterns.
S8 MYA-796 ATCC Vegetative cells of tested yeasts were cylindrical, of
dimensions 2–3μm x 5–8μm. The yeasts produced no
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S9 MYA-797 ATCC
filaments in slide cultures on potato glucose medium.
Biochemical tests However, it was shown that S. boulardii strains are able to
The strains were inoculated on YPD slants after activation. adopt a filamentous mode of growth upon exposure to a poor
The suspension of yeast cells in 0.85% NaCl with a turbidity nitrogen source (6).
equal to 2 McFarland scale was prepared. 100 μl of this All strains reproduced by budding, but they were unable
suspension was transferred into API C medium and used to to sporulate even after 90 days of incubation on sporulation
inoculate the API 20 C AUX kits (bioMerieux). The tests media. The sporulation deficiency of S. boulardii was also
were performed according to the instructions of the demonstrated in other papers (6,14,15). This finding could be
manufacturer. After incubation the reactions were read by explained by yeast chromosom IX trisomy, different copy
comparing them to growth controls. numbers for a set of genes or sequence divergence of CDC16,
The assimilation of nitrogen compounds was determined DMC1 and MND2 (6).
in liquid carbon base medium (Difco) with potassium nitrite All tested strains assimilated glucose, maltose, raffinose
or ethylamine addition at final concentration of 5 mM (2). (Table 2). However, there were differences in utilization of
The growth was assessed spectrophotometrically in relation glycerol, galactose, cellobiose and maltose. The narrowed
to negative (without inoculation) and positive (with NH4+) biochemical profiles of S. boulardii with Gal+, Mal+, Raf+
control cultures. phenotype was presented by McCullough et al. (14), which is
API-ZYM tests partly in agreement with our results. The ability of the yeasts
The strains were inoculated on YPD slopes after activation. to assimilate glucose, raffinose and maltose confirmed the
The growth was collected in distilled water and the dense observations of Hayford and Jespersen (11) for 48 isolates of
suspension formed (5 of McFarland scale) was used to S. cerevisiae. In our study only one strain isolated from
inoculate the API-ZYM kits (bioMerieux). The tests were Enterol® was able to assimilate galactose. This finding is
performed according to the instructions of the manufacturer. partially accord with states that S. boulardii does not use
Enzyme activity was graded from 0 to 5 by comparing the galactose as a carbon source (15). However, the galactose
colour developed within 5 min to the API-ZYM colour utilization has been shown to be variable among S. cerevisiae
reaction chart. strains (1) and as such, it is an unacceptable marker for
Analysis of nuclear chromosomes species identification. On the base of assimilation profiles all
Chromosomes of yeast strains were isolated and separated by the tested yeasts were classified as Saccharomyces cerevisiae
pulsed-field gel electrophoresis PFGE. The typical conditions with 96 – 99% of probability, even though some variations in
for preparation of chromosomal DNA from the biochemical patterns were obtained. The results are in
Saccharomyces yeasts were followed (24). A CHEF-DR II agreement with Querol et al. (23) who found that biochemical
apparatus (Bio-Rad) was used for separation of tests do not provide sufficient evidence to distinguish
chromosomes. The electrophoresis gel was an agarose between strains of S. cerevisiae.

BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE 663 XI ANNIVERSARY SCIENTIFIC CONFERENCE

SPECIAL EDITION/ON-LINE 120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY
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 TABLE 2
Assimilation profiles of yeasts

Strains S1 S2 S3 S4 S5 S6 S7 S8 S9
D-glucose + + + + + + + + +
Glycerol + + + + + + +
Calcium
2-keto-
gluconate
L-arabinose
D-xylose
Adonitol
Xylitol
D-galactose +
Inositol
D-sorbitol
Compounds
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Methyl-αD-
glucopyrano
side
N-acetyl-
glucosamine
D-cellobiose + +
D-lactose
D-maltose + + + + + + + + +
D- + + + + + + + + +
saccharose
D-trehalose + +
D-
melezitose
D-raffinose + + + + + + + + +
Potassium Fig. 1. Electrophoretic patterns of yeast chromosomal DNA. M – marker S.
nitrite cerevisiae YNN295 (Bio-Rad)
Ethylamine
+ assimilation, no signs − no assimilation
The karyotypes of S. boulardii are very similar to those of S.
API-ZYM tests revealed lipolitic and proteolytic activities of cerevisiae, although chromosomal length polymorphism is
yeasts. All strains exhibited alkaline phosphatase, acid observed in S. cerevisiae strains (7,19,21). MacFarland (15)
phosphatase and naphthol-AS-BI-phosphohydrolase activity. classified S. boulardii outside S. cerevisiae species on the
In addition all strains were able to hydrolyse 2-naphthyl basis of metabolic and molecular parameters. Similar results
butyrate and 2-naphthyl caprylate, indicating esterase and were obtained by Cardinali and Martini (4) using
esterase lipase activity. Strong leucine arylamidase activity comparative electrophoretic karyotyping and multivariate
was also detected in all the tested strains. Similar results were analysis of the polymorphism observed in PFGE
obtained for Saccharomyces isolates from infant faeces, Feta electrophoresis. However, Vaughan-Martini et al. (27)
cheese (22) and blue veined cheese (10). concluded that none of the species of genus Saccharomyces
The chromosomal patterns of eight strains (S2–S9) did could be distinguish by the consistent presence or absence of
not show polymorphism and consisted of 13 bands sized a unique band or cluster of bands. Some taxonomic studies
222–2251 kb (Fig. 1). (2,17,25) have indicated that S. boulardii should be rather
Only the karyotype of the strain isolated from Enterol® was considered as a strain of S. cerevisiae. DNA-DNA
distinctive with additional chromosome 1884 kb. All the reassociation studies indicated that this yeast was distinct
strains belong to the genus Saccharomyces since they show from S. cerevisiae and from other species of Saccharomyces
13-14 bands of a size varying between 200 and 2,300 kb genus (4). This conclusion was subsequently challenged
(4,19,27). based on analysis of DNA polymorphism (18) and reports of
95% DNA homology between S. cerevisiae and S. boulardii
(26). Finally, the results obtained using PCR with species-

XI ANNIVERSARY SCIENTIFIC CONFERENCE 664 BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE

120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY SPECIAL EDITION/ON-LINE
45 YEARS FACULTY OF BIOLOGY
 specific primers showed that all strains of S. boulardii could 10.
indeed be assigned to the species of S. cerevisiae (12). This 11. Hansen T.K., Jakobsen M. (2001) Int J Food Microbiol,
conclusion is reflected in current nomenclature and the name 69, 59-68.
S. boulardii is invalid for the International Code of Botanical 12. Hayford A.E., Jespersen L. (1999) J Appl Microbiol,
Nomenclature (ICBN). According to official taxonomic 86, 284-294.
definitions strains, appearing in descriptions of commercially 13. Josepa S., Guillamon J.M., Cano J. (2000) FEMS
available therapeutic products as S. boulardii, comprise the Microbiol Lett, 193, 255-259.
subtype of the Saccharomyces cerevisiae species and should 14. Malgoire JY, Bertout S, Renaud F., Bastide J.M.,
be referred as S. cerevisiae var. boulardii. Mallie M. (2005) J Clin Microbiol, 43, 1133-1137.
In conclusion, phenotypic and genotypic traits showed 15. McCullough M.J., Clemons K.V., McCusker J.H.,
diversity among tested strains and confirmed yeasts Stevens D.A. (1998) J. Clin Microbiol, 36, 2613-2617.
classification to Saccharomyces cerevisiae var. boulardii.
16. McFarland LV (1996) Clin Infect Dis, 22, 200-201.
Acknowledgments 17. McFarland L.V., Bernasconi P. (1993) Microb Ecol, 6,
157-171.
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This research was supported by the State Committee for 18. Mitterdorfer G., Mayer H.K., Kneifel W., Viernstein
Scientific Research as a grant No N312 043 32/2614. H. (2002) J Appl Microbiol, 18, 521-530.
19. Monar O, Messner R, Prillinger H, Stahl U, Slavikova
E (1995) Syst Appl Microbiol, 18, 136-145.
20. Naumov G.I., Nguyen H.V., Naumova E.S., Michel V.,
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