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Letters in Applied Microbiology 2001 Oct;33(4):251-255

Utilization of prebiotic carbohydrates by yeasts of therapeutic relevance

 G. Mitterdorfer¹, W. Kneifel¹ and H. Viernstein²

¹Department of Dairy Research and Bacteriology, University of Agricultural Sciences, Vienna, and ²1nstitute of Pharmaceutical Technology, University of Vienna, Austria 2001/80: received 12 March 2001 and accepted 16 July 2001

ABSTRACT

Aims: To investigate 17 strains of therapeutically relevant strains of Saccharomyces cerevisiae (including 10 strains of so-called S. boulardii) isolated from various pharmaceutical products, feed supplements and brewer's yeast for their capability of utilizing selected carbohydrates of prebiotic importance.

Methods and Results: Automated turbidimetric measurements and conventional test combinations were used to examine the basic sugar assimilation profiles of the test strains. It was shown that none of the so-called S. boulardii strains utilized galactose and palatinose. Among the prebiotic substrates, the yeasts indicated a pronounced preference for metabolizing the fructo-oligosaccharides.

Conclusions: Yeast strains of therapeutic relevance can be successfully combined with certain prebiotics in synbiotic formulations.

Significance and Impact of the Study: The results of this study may serve as a basis for the development of new pharmaceutical preparations for medical therapy and a better understanding of intestinal micro-ecology.
 

INTRODUCTION

Living micro-organisms are widely used for several thera­peutic purposes and their beneficial effects are rooted in history. Usually, certain strains of lactic acid bacteria and bifidobacteria (probiotics) are prevailingly applied in pharmaceutical preparations, feed additives and so-called functional foods. Besides bacteria, yeasts also possess some medicinal efficiency, and the beneficial properties of strains of Saccharomyces cerevisiae are well documented (Pothoulakis et al. 1993; Rodrigues et al. 1996). In addition to their nutritive value (e.g. provision of vitamins of the B group), yeasts offer the advantage of exerting pronounced resistance during gastrointestinal passage as well as of modulating a disturbed gastrointestinal microecology (Girola and Ventura 1995; Bleichner et al. 1997). Yeast preparations have also been successfully applied in combination with antibiotics to treat Clostridium dicile-related diarrhoea (McFarland et al. 1994) and to re-establish a normal gut function after long­term antibiotic therapy (Surawicz et al. 1989). Furthermore, they also offer some capacity in the prevention of traveller's diarrhoea (Kollaritsch et al. 1993).

Being not in accordance with the current nomenclature, strains of so-called Saccharomyces boulardii can be regarded as the most prominent representatives of `probiotic yeast' within the community of biotherapeutic S. cerevisiae strains. Today, a considerable number of pharmaceutical prepara­tions (capsules, powders, tablets, pellets) containing yeast cells are commercially available, and are marketed mainly via pharmacies and health stores. While the beneficial potential of lactic acid bacteria has recently been shown to be enhanced by the introduction of prebiotic carbohydrates (mainly of fructo- and galacto-oligosaccharidic nature) meeting the so-called synbiotic concept (Gibson 1998; Fooks et al. 1999), similar combinations with biotherapeutic yeasts have not yet been considered. Taking into account the above-mentioned beneficial effects attributed to yeasts, the question has been raised whether the synbiotic concept could also be applied for yeast-oligosaccharide combina­tions. This study was undertaken to examine the capability of yeasts used in pharmaceutical preparations of medical importance to utilize a selection of prebiotic carbohydrates, with reference to their growth behaviour in the presence of basic sugars.
 

MATERIALS AND METHODS Saccharomyces strains and culture conditions

Seventeen isolates of S. cerevisiae were harvested by means of fractionated loop streak cultures on Sabouraud 2% dextrose agar medium (Merck, Darmstadt, Germany) obtained from homogenates in sterile saline peptone solu­tions of different preparations and products (Table 1). After isolation, all strains were maintained as glycerol cultures at -85°C. Before testing, cultures were activated by means of three repetitive culture steps in Sabouraud 2% (w/v) glucose broth (Merck) under aerobic conditions at 30°C. Using the same basal medium, 24-hour-old cultures (surface plate cultures for screening test kits, liquid cultures for the growth curve monitoring) were used for screening carbo­hydrate utilization.

Utilization of carbohydrates

The API ID 32 C test kit (Biomerieux, Marcy PEtoile, France) was used as a basic screening method for carbohy­drate assimilation. Tests were performed according to the manufacturer's recommendations. Results from this screen­ing were confirmed using the YT MicroPlate^TM identifica­tion system (Biolog, Hayward, CA, USA), in connection with the Microstation equipment and also following the supplier's guidelines.

The fungal strains were examined for their capability of utilizing a selection of carbohydrate preparations with prebiotic properties (Table 2) in comparison with basic mono- and oligosaccharides. All reference sugars were of analytical grade and were purchased from Merck. For the utilization screening of prebiotic sugars, an automated turbidometer (BioScreen^R C analyser, Labsystems, Helsinki, Finland) was used, which facilitates continuous optical density measurements (600 nm) at the microtitre plate level. Fungal growth curves were monitored in Sabouraud broth containing 1% (w/v) portions of the corresponding sugars, and observed continuously during an incubation period of 48 h at 30°C under aerobic conditions. Ten replicates were performed with each strain and each carbohydrate, and mean values were calculated.
 

RESULTS

Basic carbohydrate utilization patterns

Data obtained from both the API and the YT Microplate tests were compiled to provide one combined graph visualizing the typical fermentation patterns (Fig. 1). All yeast strains utilized glucose, maltose, raffinose (two excep­tions), sucrose and trehalose. Only seven strains were able to metabolize galactose and palatinose. Arabinose, cellobiose, sorbitol and i3--xylose were utilized by two of the strains and tagatose by only one strain.

Growth in media with prebiotic carbohydrates

All yeast strains were examined for their capacities for utilizing sugars of prebiotic importance.

Fig. 2 Growth kinetics of yeast strain no. 10 (for explanation of strain code see Table 1) in Sabouraud medium containing selected carbohydrates at 1% (w/v) and monitored by the BioScreen^R C analyser

The basal growth medium was supplemented with the carbohydrates listed in Table 2, and their growth behaviour was calculated with reference to the results obtained with glucose as a carbon source. Typical growth curves were obtained, as shown for example in Fig. 2 for strain no. 10, which indicated that maximum growth was achieved after an incubation time of 48 h at 30°C. Therefore, the growth behaviour of the strains was further compared on this basis. It can also be seen from Fig. 2 that although this strain had a less pronounced slope of its exponential growth phase when Raftilose was used, higher optical density levels were finally obtained when compared with glucose. After approximately 30 h, the medium supplemented with Raftilose had a higher optical density due to higher cell counts than in the glucose­containing substrate. However, the growth kinetics of the test strains differed individually and in relation to the carbon source considered.

In Fig. 3, the growth behaviour of the individual strains is depicted in relation to the optical density values obtained in the Sabouraud glucose (1% w/v) medium at 48 h of incubation. It is evident from the distribution of the bars that both fructo-oligosaccharide preparations (Raftilose and Frutafit) yielded, in general, higher cell counts than the other sugars. With three strains (numbers 10, 15 and 16), pronouncedly higher growth rates were obtained with Raftilose than with glucose. Neither the galacto-oligosac­charide nor the guar gum product and lactulose produced fungal growth intensities comparable with those measured with glucose, and only reached the 40% level compared with glucose (=100%).

Fig. 3 Growth response patterns of the yeast isolates (for explanation of strain codes see Table 1) in Sabouraud medium containing different carbohydrates of prebiotic importance, with reference to their growth intensity in Sabouraud glucose (1% w/v) medium (growth intensity = 100%). (n), Raftilose; (q), Frutafit; (q), Elix'or; (n), Solufiber; (®), Lactulose
 

DISCUSSION

Although S. boulardii is a commonly used name for probiotic yeast, a reliable differentiation from S. cerevisiae has hitherto not been demonstrated. According to the findings of McFar­land (1996), the missing potential of utilizing galactose could be taken as a key criterion for the differentiation between conventional S. cerevisiae and S. boulardii. Our own observa­tions confirmed these findings and palatinose utilization may obviously be considered as a typical differential characteristic. However, McCullough et al. (1998) have shown that galactose can be metabolized by some of the so-called S. boulardii strains as well. Because of this uncertainty, molecular biological techniques offer a powerful tool for clarifying the genetics of discrimination among these fungi (Piarroux et al. 1999).

With the exception of the above-mentioned observations, the utilization patterns of the yeast strains regarding basic sugars as well as prebiotic substances did not exhibit any species-specific tendencies, but indicated some pronounced preferences with regard to glucose, fructose, sucrose and the fructo-oligosaccharide-based products. Distinctly weaker utilization properties were obtained with the galacto-oligo­saccharide preparation, regardless of whether or not galactose

was fermented or assimilated, and with guar gum hydrolysate and lactulose. These findings do not agree with the utilization capacities established for bacteria of therapeutic relevance, which have shown rather strain-dependent util­ization patterns (Kneifel et al. 2000). The present study supports the assumption that yeast of therapeutic relevance can be combined with fructo-oligosaccharides in so-called synbiotic formulations, since it may be expected that not only the probiotic yeast will be stimulated but also, the resident positive bacterial microflora of the colon (Gibson 1998). Positive effects of dietary fibre from Psyllium on the survival characteristics of S. boulardii during gastrointestinal passage have also been shown by Elmer et al. (1999). Based on the above findings, it is concluded that synbiotic formulations consisting of fructo-oligosaccharides and strains of S. cerevisiae (S. boulardii) could be advantageously applied in medical therapy.

REFERENCES

Bleichner, G., Blehaut, H., Mentec, H. and Moyse, D. (1997) Saccharomyces boulardii prevents diarrhoea in critically ill tube-fed patients. Intensive Care Medicine 23, 517-523.

Elmer, G.W., Martin, S.W., Horner, K.L., McFarland, L.V. and Levy, R.H. (1999) Survival of Saccharomyces boulardii in the rat gastrointestinal tract and effects of dietary fibre. Microbial Ecology in Health and Disease 11, 29-34.

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McCullough, M.J., Clemons, K.V., McCusker, J.H. and Stevens, D.A. (1998) Species identification and virulence attributes of Saccharo­myces boulardii (nom. inval.). ,journal of Clinical Microbiology 36, 2613-2617.

McFarland, L.V. (1996) Saccharomyces boulardii is not Saccharomyces cerevisiae. Clinical Infections and Diseases 22, 200-201.

McFarland, L.V., Surawicz, C.M., Greenberg, R.N. et al. (1994) A randomized Placebo-controlled trial of Saccharomyces boulardii in combination with standard antibiotics for Clostridium docile disease. ,journal of the American Medical Association 271, 1913-1918.

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Pothoulakis, C., Kelly, C.P., Joshi, M.A. et al. (1993) Saccharomyces boulardii inhibits Clostridium docile Toxin A binding and entero­toxicity in rat ileum. Gastroenterology 104, 1108-1115.

Rodrigues, A.C.P., Nardi, R.M., Bambirra, E.A., Vieira, E.C. and Nicoli, J.R. (1996) Effect of Saccharomyces boulardii against experi­mental oral infection with Salmonella typhimurium and Shigella flexneri in conventional and gnotobiotic mice. ,journal of Applied Bacteriology 81, 251-256.

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