Diversity of yeasts involved in the fermentation of tchoukoutou, an opaque sorghum beer from Benin

1 Département de Nutrition et Sciences Alimentaires, Faculté des Sciences Agronomiques, Université d’Abomey-Calavi, 01 BP 526 Cotonou, Bénin. 2 Product Design and Quality Management group, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands. 3 Department of Biology, Rutgers University, Camden, New Jersey 08102, USA. 4 Laboratory of Food Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, P. O. Box 8129, 6700 EV Wageningen, The Netherlands.


INTRODUCTION
Traditional alcoholic beverages are obtained in sub-Saharan Africa from carbohydrate-rich products (Sefa-Dedeh et al., 1999). Opaque sorghum beers are popular alcoholic beverages in Africa. The fermentation is often spontaneous and uncontrolled. The beverages often consumed in an actively fermenting state have short shelf-lives (Odunfa, 1985). In the West Africa region they are known as tchoukoutou in Benin, dolo in Burkina-Faso, pito in Ghana, and burukutu or otika in Nigeria *Corresponding author. E-mail: polykap@yahoo.fr. Tel: (+229) 97870734. (Odunfa, 1985;Glover et al., 2005;Kayodé et al., 2005). The beers have a sour taste, a relatively high dry matter content (5-13 g 100 ml -1 ) and low alcohol content (2-3 ml 100 ml -1 ), which make them suitable beverages for adults and teenagers (Agu and Palmer, 1998;Briggs et al., 2004). They are largely consumed by the poorest people and significantly contribute to the diet of millions of consumers. The beers are mostly prepared with Guinea corn (Sorghum bicolor) but other cereals such as millet or maize can be used as adjunct or as substitutes (Kayode et al., 2005). The manufacturing process consists of malting (soaking, germination, sun drying), brewing (mashing, boiling, filtration) and fermentation (Haggblade and Holzapfel, 1989). Depending on the geographic location, variations may occur in the process (Odunfa, 1985;Haggblade and Holzapfel, 1989).
Lactic acid bacteria and yeasts were identified during the fermentation of several cereal products in Africa (Jespersen et al., 1994;Muyanja et al., 2002;Mugula et al., 2003;Vieira-Dalodé et al., 2007;Muyanja et al., 2010). The yeast species most often reported in African traditional food and beverages belong to the species Saccharomyces cerevisiae. (Jespersen, 2002). Few studies investigated the micro-organisms involved in African opaque beers. The microorganisms isolated from the beer consist essentially of yeasts and lactic acid bacteria (LAB) (Van der Aa Kühle et al., 2001;Demuyakor and Ohta, 1991;Sefa-Deheh et al., 1999;Sanni and Lönner, 1993;Faparusi et al., 1973). However, the species isolated and their frequency distributions in the product vary according to the regional location. Saccharomyces cerevisiae (33%), Kluyeromyces spp, Candida spp., and Torulaspora delbrueckii are predominant yeasts in Ghanaian opaque beer (Demuyakor and Ohta, 1991;Sefa-Deheh et al., 1999). In Burukutu Nigeria, Sanni and Lönner (1993) found the predominance of Candida spp., Geotrichum candidum, S. cerevisiae, Kloeckera apiculata or Torulaspora delbrueckii. Van der Aa Kühle et al. (2001) demonstrated the predominance of Saccharomyces spp (of which 45% were identified as Saccharomyces cerevisiae) in the fermentation of sorghum beer from Ghana and Burkina-Faso. Very limited information exists on the microbiological attributes of the Beninese opaque beers. The objectives of this study are two-fold. Firstly, to characterise the Benin opaque sorghum beer in terms of its physical and microbiological attributes. Secondly, to characterise and identify the yeasts involved in the fermentation of the beer.

Sampling
Samples of actively fermenting (about 12 h of fermentation) opaque sorghum beer were collected from twelve commercial processing sites in northern Benin. The processors (one per site) were selected on the basis of their rich beer brewing tradition. The samples were collected in screw-capped bottles, packed in an insulated icebox, transported to the laboratory and analysed immediately for microbiological analysis (Hounhouigan et al., 1993).

Physico-chemical analysis
Dry matter was determined according to American Association of Cereal Chemistry (AACC) methods (AACC, 1984). Titratable acidity pH and were determined as described by Nout et al. (1989). The refractive index was measured using a refracto meter (Sopelem 9596, France).

Identification of yeast
For identification of yeasts, isolates from three representative production sites were purified by successive sub-culturing on malt extract agar (MEA, CM 59, Oxoid). Preliminary confirmation was based on microscopic observation. The isolates were tested for the fermentation of sucrose, lactose, glucose and raffinose, as well as the assimilation of selected nitrogen sources i.e. nitrate, ethylamine, L-lysine, cadaverine, and creatine. The assimilation of carbon sources was performed using API 20 C AUXstrips (BioMérieux, Lyon, France) according to the manufacturer's instructions. The Diazonium Blue B reaction, a test to differentiate between ascomycetous and basidiomycetous yeasts, was performed as described by Kurtzman et al. (2003).

Data analysis
Mean values and standard deviation are reported. The data were analysed using the statistical program SPSS 11.0. The on-line available software (http:www.cbs.knaw.nl) of Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands was used for identification of yeasts.

Physico-chemical characteristics of the Benin opaque beer
The mean value of the pH was 3.67 and the titratable acidity of the beer averaged to 0.7 (% as lactic acid). The dry mater content of the beer is high but variable (15-20%) and averaged to 18.1%. The refractive index averaged to 7.0 (Table 1). It has been demonstrated that at pH < 4.0 in food products, the growth of diarrhea causing pathogens is inhibited (Motarjemi and Nout, 1996). Thus it can be expected that the tchoukoutou beer is inherently safe from a microbiological point of view. The Benin opaque beer resembles pito, the Ghanaian opaque beer, in terms of acidity content (Sefa-Dedeh et al., 1999) but its refractive index is higher.

Microbial content
The mean counts of lactic acid bacteria and yeasts were respectively 9.1 and 9.1 log cfu/ml. The Enterobacteriaceae were not detectable in the beer (log cfu/ml< 1) ( Table 2). The yeast concentration in tchoukoutou is relatively higher than reported in pito beer (Glover et al., 2005) but close to values reported for burukutu, another opaque sorghum beer from Nigeria

Phenotypic characters of yeast isolates
After preliminary microscopic confirmation all 40 yeasts were subjected to morphology, fermentation and assimilation tests. None of the isolates could ferment lactose whereas the majority fermented glucose (100%), sucrose (95%) and raffinose (90%). Only 5% of the isolates assimilated nitrate, ethylamine or creatine, whereas 15% assimilated L-lysine. All isolates were ascomycetous yeasts as revealed by Diazonium Blue B test (Table 3).
On the basis of their fermentation profile and their nitrogen assimilation pattern the 40 yeasts could be grouped into 5 distinct clusters with the majority (63%) present in one group which showed a metabolism profile typical of Saccharomyces spp.

Assimilation profile and identification of yeasts isolates
Based on their assimilation of carbon compounds, sixteen assimilation profiles were distinguished. All yeasts assimilated glucose and maltose (100%), 42.5% assimilated sucrose, 23% assimilated raffinose and only 2.5% could assimilate trehalose. None of them assimilated arabinose, sorbitol and methyl-αDglucopyranoside (Table 4). On the basis of their phenotypic characteristics, the 40 yeasts were found to belong to four genera and seven species of yeast ( Figure  1). Clearly, Saccharomyces cerevisiae (68%) predominates in the Benin opaque sorghum beer. Our result ressembles findings by Konlani et al. (1996) who reported a prevalence of 55-90% for S. cerevisiae in sorghum beer from Togo and Burkina-Faso, two regions close to our study area. Van der Aa Kühle et al., 2001 also identified a large number (45%) of the yeasts involved in the fermentation of opaque beers from Burkina-Faso and Ghana as S. cerevisiae. From a study carried out on pito in Northern Ghana, Glover et al. (2005) identified 72% of 247 isolates as S. cerevisiae based on their assimilation profiles. Twenty seven percent of isolates had narrow assimilation profiles atypical of the specie and could not be clearly identified.

Conclusion
Based on the phenotypic characterisation, S. cerevisiae was found to be the predominant yeast species in the fermentation of Benin opaque sorghum beer. There is a need of genotyping the isolates for a best characterisation of the yeasts involved. In view of controlling the quality of tchoukoutou, the predominant species identified can be selected and used as single or mixed starter cultures for a more predictable fermentation outcome.