Phenotypic characters of yeasts isolated from kpete-kpete , a traditional starter of a Benin opaque sorghum beer

1 Laboratoire de Valorisation et de Gestion de la Qualité de Bio ingrédients Alimentaires, Université d‟Abomey-Calavi, 01 BP 526 Cotonou, Bénin. 2 Department of Biology and Center for Computational and Integrative Biology, Rutgers University, 315 Penn St., Camden, NJ 08102, USA. 3 Laboratoire de Biochimie Microbienne et de Biotechnologie Alimentaires (LMBA), Faculté des Sciences Agronomiques, Université d‟Abomey-Calavi; 01 BP 526; Cotonou, Benin.


INTRODUCTION
Fermented beverages play a major role in the diet of African people.The most studied African alcoholic beverages are opaque beers often produced from sorghum (Chamunorwa et al., 2002;Jespersen, 2003;Maoura et al., 2005;Lyumugabe et al., 2010;Lyumugabe et al., 2012).Opaque sorghum beers are consumed at various festivals and African ceremonies (for example, marriage, birth, the handing over of a dowry, etc.) and constitute a source of economic return for the women beer producers.They are known as tchoukoutou in Benin, dolo in Burkina-Faso, pito in Ghana and burukutu, otika or sekete in Nigeria, Impeke in Burundi (Odunfa, 1985;Sanni and Lönner, 1993;Kayode et al., 2005).These beers are very rich in calories, B-group vitamins and essential amino acids such as lysine (Lyumugabe et al., 2012) and inexpensive.Therefore, they are largely consumed by the poorest people and contribute to their dietary needs (Kayodé et al., 2012).Due to their low alcohol content (2-3% v/v) and the large quantity of suspended solids (5-7%), many consumers consider fermented sorghum beers to be more a food than a beverage (Pattison et al., 1998).The beers are mostly produced at household level or at small industrial scale with varying quality and stability (Sanni and Lönner, 1993;Zulu et al., 1997).Basically, the processing of African opaque sorghum beers involves malting, souring, boiling, mashing, straining and alcoholic fermentation (Kayode et al. 2005;Odunfa, 1985;Haggblade and Holzapfel, 1989).Depending on country and region, variations occur in the beer process (Jespersen, 2003).The fermentation remains a critical step in the process (Kayode et al., 2012).Beneficial effects of fermentation include improvement of flavor and texture, reduced loss of raw materials, reduced cooking time, improved bioavailability of micronutrients and elimination of toxic and anti-nutritional factors (Sanni and Lönner, 1993;Iwuoha and Eke, 1996;Padmaja, 1995;Sindhu and Khetarpaul, 2001).
In Benin, Lactic acid bacteria and yeasts have been reported (Kayodé et al., 2007) to be the major microorganisms involved in the fermentation of tchoukoutou.Kpete-kpete is the traditional starter used for the fermentation of tchoukoutou.Based on its fermenting properties, producers use it to ferment the sorghum wort during the manufacturing process.It is generally harvested from the bottom of a previous fermenting beer resulting from 13 to 14 h overnight fermentation.However, the microorganisms contained in kpete-kpete used for the fermentation of tchoukoutou have not yet been investigated.Especially, works reporting on the species of yeasts contained in such starter are hard to come by.The increasing interest for moving from uncontrolled conditions towards regulated processing conditions, and thus ensuring quality safety and product stability, makes the application of starter cultures and thereby identification and classification of the strains involved necessary (Van der Aa Kühle et al., 2001).
The present study was conducted to determine the physicochemical and microbiological characteristics of the traditional starter kpete-kpete, and to identify the different species of yeasts involved using phenotypic analysis tools.

Sampling
Twenty four (24) samples (500 mL) of kpete-kpete, the traditional starter of tchoukoutou, were collected from eight of the most important production sites of tchoukoutou in northern Benin.The processors (one per site) were selected on the basis of their rich beer brewing tradition.The samples were collected in screwcapped bottles, packed in an insulated icebox, transported to the laboratory and analyzed immediately for microbiological analysis (Hounhouigan et al., 1993).

Physico-chemical analysis
Dry matter was determined according to the AACC method (AACC, 1984).The pH was determined using a digital pH meter (HI 8418; Hanna instruments, Limena, Italy) calibrated with buffers at pH 4.0 and 7.0 (WTW, Weilheim, Germany).The titratable acidity, expressed as lactic acid, was performed by using the method described by Nout et al. (1989).The refractive index was measured using a refracto meter (Sopelem 9596, France).

Identification of yeast
The identification of yeast strains was performed according to the method described by Yarrow, (1998) and Kurtzman et al. (2011).The isolates from eight representative sites were purified by successive sub-culturing on oxytetracycline glucose yeast agar (OGYA, CM0545, Basingstoke Hampshire, England) made selective by addition of oxytetracycline.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 that is, 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
For the analytical data, mean values as well as 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 Centraal bureauvoor Schimmel cultures (Central Bureau of Fungal Cultures), Utrecht, the Netherlands was used for identification of yeasts.

Physico-chemical characteristics and yeast content of kpete-kpete
The mean value of yeast counts was 9.24 log cfu/mL  (2006).That difference could be due to the fact that there is a significant difference between the dry matter of both products: 16.6% for the kpete-kpete and 10.0% for tchoukoutou (Kayodé, 2006).Data from Table 1 show there is a significant difference (p < 0.05) between the counts of yeasts from the eight communities.Previous studies performed on African opaque sorghum beers established that the frequencies of microbial species vary according to the region and the ingredients used for the brewing (Demuyakor and Ohta, 1991;Ekundayo, 1969;Faparusi et al., 1973;Nout, 1980;Odunfa, 1985;Sanni and Lönner, 1993;Sefa-Dedeh et al., 1999).Mean values of pH was 3.58.Data analysis showed there is a significant (p < 0.05) difference between samples from the various production sites.However, there is no significant difference (p<0.05) for the titratable acidity, dry matter and refractive index between the samples from the eight areas.On the basis of the titratable acidity, the dry matter and the refractive index, the starters collected from various sites appear to be similar.However, the different starters seem different on the basis of their yeasts content and their pH values.

Phenotypic characteristics of yeasts isolates
Results (Table 2) show that a minority of the strain could ferment raffinose (24.5%), whereas the great majority fermented sucrose (85.7%) and glucose (100%); but none of the isolates could ferment lactose.These results are in close agreement with data reported by Kayode et al. (2011) for yeast strains isolated from tchoukoutou.The nitrogen assimilation test revealed that a minority of the isolates assimilated ethylamine (16.3%),L-lysine (32.7%) and cadaverine (42.9%) whereas the majority assimilated sulfate of ammonium (67.3 %) and nitrate (55.1%).The diazonium blue B test (Table 2) revealed that 22.4% of the isolates were basidiomycetous whereas 77.6% were ascomycetous.On the basis of their fermentation profile and the nitrogen assimilation pattern, the 49 yeasts could be grouped into 18 distinct clusters.14.3% were in the first cluster, 8.2% were in the second cluster, 8.2% in the fourth cluster and the rest are distributed in the 15 other clusters.
Researches on improvement of traditional sorghum beers revealed that S. cerevisiae is of a vital importance for making an effective starter culture.Sefa-Dedeh et al. (1999) used a pure culture of S. cerevisiae and a mixture culture consisting of S. cerevisiae and other strains such as Kloeckera apiculata and Candida tropicalis, to produce a pito beer containing high ethanol content.Also, Orji et al. (2003) found that S. cerevisiae in combination with Lactobacillus plantarum, as a starter culture, also led to the satisfactory production of a pito beer.N'Guessan et al. ( 2010) successfully used S. cerevisiae in combination with C. tropicalis as starter cultures for the alcoholic fermentation of the tchapalo beer.

Conclusion
S. cerevisiae was identified as the predominant specie of yeast in the traditional starter kpete-kpete on the basis of the phenotypic characterization.In order to refine the identification process, the molecular characterization is found to be necessary.This would be an important step towards the elaboration of a starter culture for the fermentation of African opaque sorghum beers.Such approach would lead to an improvement of the fermentation process and the quality of local African beers.

Table 1 .
Physicochemical and microbiological characteristics of the traditional starter kpete-kpete.Coefficient of variation, *Values with the same letter in the same column are not significantly different ( P< 0.05) (Table1).The yeast concentration in kpete-kpete is higher than the counts of yeast (7.8-8.5 log ufc/g) in the sorghum beer tchoukoutou as reported by Kayodé et al. a