Isolation and molecular identification of yeast strains from “Rabilé” a starter of local fermented drink

1 Centre de Recherche en Sciences Biologiques, Alimentaires et Nutritionnelles (CRSBAN). Département de Biochimie – Microbiologie (DBM), Unité de Formation et de Recherche en Sciences de la Vie et de la Terre (UFR/SVT), Université Ouaga I Pr Joseph KI-ZERBO, 03 BP 7131, Ouagadougou 03, Burkina Faso. 2 Laboratoire de biologie moléculaire appliquée (LBMA), Faculté des Sciences et Techniques, Université des Sciences, Des Techniques Et Des Technologies De Bamako, BP : E 3206, Colline de Badalabougou, Bamako, Mali.

Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License of population are consumers (Bationo et al., 2015).Local fermented drink as "dolo" results from the fermentation of Sorghum bicolor.It is mainly produced by women (Maoura et al., 2005) using various processes depending on the geographic location.The manufacturing process consists of three phases: Malting, mashing and fermentation (Kayodé et al., 2012).The fermentation step is the most important step of the process (Djegui et al., 2014).However, this fermentation is uncontrolled and takes place in poor hygienic conditions (Benjamin et al., 2015) and its success depends on the accurate knowledge of the processor in terms of the starter handling (Kayodé et al., 2012).In Burkina Faso, "Rabilé" is used as traditional starter culture for the production of sorghum beer ("dolo").This starter is obtained by drying the pellet from the fermentation of "dolo" at room temperature.It was reported that "Rabilé" is also largely used as ingredient in sauce and food cooking (Konlani et al., 1996b)."Rabilé" brings to those people a wide range of nutritional benefits and contributes to their dietary need, as it is mainly constituted of yeast, lactic acid bacteria and various metabolites resulting from fermentation process.Indeed, brewer's yeast is an important source of group B vitamins and minerals such as Ca, P, K, Mg, Cu, Fe, Zn, Mn and Cr, in addition to its profile balanced in amino acids (Bekatorou et al., 2006;Feldmann, 2012).Despite its common use in diet, very limited information exists on microbiological and nutritional characteristics of "Rabilé" in particular its yeast diversity.The present study was focused on isolation and identification of yeast strains from "Rabilé" using conventional and molecular methods.

Sampling
Samples of dried yeast harvested "Rabilé" from sorghum beer were collected from commercial sites of four localities of Burkina Faso (Banfora, Fada N'Gourma, Ouagadougou and Tenkodogo) as indicate in Figure 1.In each locality, 25 g of ferment were purchased from five local beer producers.Once at laboratory, the 20 samples collected were stored at 4°C for yeasts isolation.

Yeast strains enumeration and isolation
An amount of 10 g from each sample was crushed in blender suspended and mixed in 90 ml of sterile diluents (physiological water).Serial 10-fold dilution was carried out and yeast was isolated on Sabouraud Agar (Biomerieux) with addition of chloramphenicol at 30°C.Chloramphenicol is an antibiotic used to inhibit growth of other microorganisms and allow only the growth of yeast.Colony counts were carried out using a colony counter (IUL Instruments).Total counts of yeasts population were expressed as log colony forming units per gram (log cfu/g) of "Rabilé".The representative colony forming units were recorded and purified twice on MYGP Agar (Malt extract, yeast extract, glucose and peptone).A total of 20 selected yeasts was grown on the YEPD liquid media and stored 4°C.

Morphological, physiological and biochemical identification
Morphological and physiological characteristics determined, were color of colonies on solid media.Cell shape and mode of vegetative growth were determined by microscopic observation (Bonciu et al., 2010;Stoicescu et al., 2011).Ascospores were highlighted on the media of sporulation Mac Clary (Nishida et al., 2004).Fermentation and assimilation tests of carbon compounds, nitrate, and sodium acetate were carried out according to Guiraud et al. (1984).

Microorganism preparation and genomic DNA extraction
The 20 representative isolated yeast strains were grown on MYPD agar at 30°C for 72 h.For each strain culture a loop full was collected for DNA extraction.Genomic DNA was extracted and purified according to CTAB extraction method used by Kumar et al. (2014).

Specific-PCR
DNA amplification was performed in a reaction volume of 25 µl, containing 0.4 µM of each primer (MWG Operon Eurofins, USA), 2 ng/µl of genomic DNA, 0.8 mM deoxynucleotides (dATP, dCTP, dGTP, dTTP), 4 mM of MgCl2, 0.04 U/µl Taq polymerase and buffer 1 X (Invitrogen, China).Sequences of the primers pairs used separately are shown in Table 1.The amplification was performed with an automatic thermocycler (MJ Research PTC-200) using program with an initial denaturation step at 94°C for 5 min, amplification reaction was performed in 35 cycles of denaturing at 95°C for 1 min, annealing at 60°C for 1 min, elongation at 72°C for 2 min and a final extension step at 72°C for 10 min.Amplified DNA fragments were separated on 3% agarose gel.After electrophoresis, gels were scanned under ultraviolet ray.All fragments were analyzed with Kodak 1D 3.5.software to determine the size of the bands obtained.

RFLP analysis
An aliquot (10 µl) of PCR product was digested separately with four restriction endonucleases EcoR I, Hind III, Apa I and BamH I (Invitrogen, China) to generate restriction fragments.Reaction mixture consisted of 1 µl enzyme, 2 µl buffer, 10 µl amplicon and 7 µl pure water, to a total volume of 20 µl.Digestion was carried out at 37°C for 1 h according to the manufacturer's instructions (Invitrogen, China).Restriction fragments were visualized by ethidium bromide staining and UV transillumination.Identification was carried out using specific standards.CECT database (http://cectvirt11.uv.es/searchdb/) was used to verify suitable restriction enzymes as Hind III for selection and for identification of S. cerevisiae.

Yeast amount and distribution of isolated yeast
Table 2 shows the yeast counts obtained for "Rabilé" samples from four localities of Burkina Faso.Yeast counts ranged from 9.49 to 10.35 log cfu/g respectively for samples from Tenkodogo and Fada N'Gourma.A total of 20 strains were isolated from collected samples.The origin of these strains is given in Table 2.

Morphological, physiological and biochemical identification
Strains in solid and liquid allowed to notice morphological, physiological and biochemical characteristics as described in the Table 3.The phenotypical yeast identification was based on criteria of Guiraud et al. (1984).In solid media, all strains appear white colonies except strains CRSBANYF1, CRSBANYF 4 , CRSBANYO 3 , CRSBANYT 3 and CRSBANYT 4 which presented red coloring.Under microscope, vegetative cells had an oval form and divided by budding.An asexual reproduction leading to the formation of asque (4 ascospores) was observed in strains CRSBANYB 3 , CRSBANYF 3 , CRSBANYB 1 , CRSBANYB 4 , CRSBANYF 2 , CRSBANYT 2 and CRSBANYB 5 .

Yeast PCR-RFLP characterization
In order to verify profile difference between isolated yeast strains, PCR-RFLP was used.ITS-5.8S-rDNAregion was amplified with primers ITS1 and ITS4 then digested with restriction enzymes (EcoR I, Hind III, Apa I and BamH I).
The amplicons obtained (Figure 3) and the restriction fragments (Table 5) indicated three profiles relating three specific species of strains.PCR-RFLP indicated existence of a large polymorphism between the three groups of yeasts isolated as well by the size of amplicons and then their restriction profile.The first group of species, initially identified was S. cerevisiae yielded amplicons of 880 bp and presented on their ITS-5.8S-DNArregion one restriction site for EcoR I and Apa I enzymes.The second group of species identified was R. mucilaginosa with amplicons of 630 bp and had two and one restriction sites respectively for EcoR I and Hind III enzymes.The third group of species gave 500 bp amplicons and had only one restriction site for the enzyme EcoR I.The use of CECT database (http://cectvirt11.uv.es/searchdb/) has shown the amplicons of 850 bp only for S. cerevisiae with restriction enzyme as Hind III.

DISCUSSION
The yeast concentration of "Rabilé" obtained (Table 2),   was higher than those reported by Kayodé et al. (2012) on dried traditional starter of African opaque sorghum beers but closed to values reported in "Kpètè kpètè" by Djêgui et al. (2015) and "Otchè" by Djegui et al. (2014) which are liquid traditional starter of African sorghum beers.The number of viable yeasts obtained could justify the wild use of "Rabilé" as a traditional starter of sorghum beer in Burkina Faso.However, further investigation would be necessary to determine the long shelf life of this starter.
Phenotypical and molecular characterization revealed respectively three genera and two different species among isolated strains from "Rabilé".As indicated in Table 5, 35% of isolates belong to S. cerevisiae and 25% to R. mucilaginosa.The high prevalence of S. cerevisiae in "Rabilé" from Banfora, Candida from Ouagadougou and R. mucilaginosa from Fada N'Gourma and Tenkodogo demonstrates a biodiversity of yeast in the local starter "Rabilé" responsible to traditional fermentation.This is in agreement with the idea according to which the composition of the yeast population responsible for the spontaneous fermentation of sorghum beer could be related to the regional location (van der Aa Kühle et al., 2001).Due to the number of yeast selected strains their geographic repartition need to be complete by other investigation.In many studies, S. cerevisiae has been reported as responsible for the spontaneous fermentation of sorghum beer (Konlani et al., 1996a;Naumova et al., 2003;Lyumugabe et al., 2014).S. cerevisiae is fully accepted for human consumption and is the most common food grade yeast (Bekatorou et al., 2006).Contrary the presence of R. mucilaginosa in sorghum beer could be dangerous because it has been reported to cause Onychomycosis (Da Cunha et al., 2009;Jimoh et al., 2011).It was reported that certain yeasts involved in sorghum beer production were phenotypically different from reference strains (van der Aa Kühle et al., 2001).PCR-RFLP using EcoR I, Hind III, Apa I and BamH I, did not allow to prove this difference at the molecular level and to discriminate at subspecies level.Nevertheless, it permitted to confirm the results of specific-PCR.The results obtained are in accordance with the studies of several authors in which RFLP method was found useful for differentiation of yeast at species level (Esteve-Zarzoso et al., 1999;Granchi et al., 1999).The high prevalence of S. cerevisiae would indicate a predictive characteristic of good brewer's starter.

Table 1 .
Primers used for specific-PCR.

Table 2 .
Yeasts content of "Rabilé" and distribution of isolated yeast strains.

Table 3 .
Morphological, physiological and biochemical identification of isolated yeast strains.

Table 4 .
Molecular identification of strains.

Table 5 .
Size of ITS-5.8S-DNArregion and RFLP restriction pattern with enzymes EcoR I, BamH I, Hind III and Apa I.