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Genetic Analysis of Saccharomyces Cerevisiae Strains Isolated from Palm Wine in Eastern Nigeria

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Genetic Analysis of Saccharomyces Cerevisiae Strains Isolated from Palm Wine in Eastern Nigeria Genetic analysis of Saccharomyces cerevisiae strains isolated from Palm Wine in Eastern Nigeria. Comparison with other African strains. O.U. Ezeronye, Jean Luc Legras To cite this version: O.U. Ezeronye, Jean Luc Legras. Genetic analysis of Saccharomyces cerevisiae strains isolated from Palm Wine in Eastern Nigeria. Comparison with other African strains.. Journal of Applied Microbi- ology, Wiley, 2009, 106 (5), pp.1569-1578. 10.1111/j.1365-2672.2008.04118.x. hal-02668435 HAL Id: hal-02668435 https://hal.inrae.fr/hal-02668435 Submitted on 31 May 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Journal of Applied Microbiology ISSN 1364-5072 ORIGINAL ARTICLE Genetic analysis of Saccharomyces cerevisiae strains isolated from palm wine in eastern Nigeria. Comparison with other African strains O.U. Ezeronye1 and J.-L. Legras2 1 Michael Okpara University of Agriculture, Umudike, Nigeria 2 INRA ⁄ ULP, UMR Sante´ de la Vigne et Qualite´ du Vin, Colmar, France Keywords Abstract microsatellite, molecular polymorphism, palm wine, yeast. Aims: To study the yeast diversity of Nigerian palm wines by comparison with other African strains. Correspondence Methods and Results: Twenty-three Saccharomyces cerevisiae strains were Jean-Luc Legras, INRA ⁄ ULP, UMR Sante´ de la obtained from palm wine samples collected at four locations in eastern Nigeria, Vigne et Qualite´ du Vin, 28 rue de and characterized using different molecular techniques: internal transcribed Herrlisheim, BP 20507, Colmar Cedex 68021, spacer restriction fragment length polymorphism and sequence analysis, pulsed France. E-mail: [email protected] field gel electrophoresis, inter delta typing and microsatellite multilocus analy- This paper is dedicated to the memory of sis. These techniques revealed that palm wine yeasts represent a group of Prof. O.U. Ezeronye (1954–2007). closely related strains that includes other West African isolates (CBS400, NCYC110, DVPG6044). Population analysis revealed an excess of homozygote 2008 ⁄ 0975: received 9 June 2008, revised 11 strains and an allelic richness similar to wine suggestive of local domestication. October 2008 and accepted 17 October 2008 Several other African yeast strains were not connected to this group. Ghana doi:10.1111/j.1365-2672.2008.04118.x sorghum beer strains and other African strains (DBVPG1853 and MUCL28071) displayed strikingly high relatedness with European bread, beer or wine strains, and the genome of strain MUCL30909 contained African and wine-type alleles, indicating its hybrid origin. Conclusions: Nigerian palm wine yeast represents a local specific yeast flora, whereas a European origin or hybrid was suspected for several other Africa isolates. Significance and Impact of the Study: This study presents the first genetic characterization of an autochthonous African palm wine yeast population and confirms the idea that human intervention has favoured yeast migration. incision into the phloem vessel of the tree. The palm sap Introduction is transparent and contains fermentable sugars at levels Palm wine is an alcoholic drink obtained by fermenting up to 90 g ⁄ l (Faparusi 1981; Eze and Ogan 1988; the sugary sap of the oil palm tree Elaeis guianeensis or of Obahiagbon and Osagie 2007), whereas palm wine is raffia palms (Raphia hookeri and Raphia vinifera). This milky-white and effervescent because of the presence of alcoholic beverage is extremely popular in Nigeria and live bacteria and yeasts (Okafor 1975) resulting from West and Central Africa, where it plays an important role natural spontaneous fermentation. Palm wine contains up in popular culture. to 3–4% alcohol, depending on the stage of fermentation Raffia palm sap is obtained by tapping the palm, which at which it is consumed. leads to the death of the tree. When the Raffia palm Numerous studies have been carried out on different begins to blossom, the palm wine tapper climbs up the aspects of palm wine production, microbiological quality tree and excises the new inflorescence, making a deep and preservation (Okafor 1978; Ojimelukwe 2001; Morah ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 1 Genetic analysis of palm wine Saccharomyces cerevisiae O.U. Ezeronye and J.-L. Legras 1995; Agu et al. 1999; Uzochukwu et al. 1999; Amoa- the genetic diversity of palm wine yeasts that were briefly Awua et al. 2007; Ogbulie et al. 2007). These studies described by Legras et al. (2007). Twenty-three isolates revealed considerable diversity amongst yeast species, were obtained from 13 palm wine fermentations and resulting from the natural flora of the palms, visiting identified using internal transcribed spacer PCR-restric- insects such as Drosophilia melanogaster and the tapping tion fragment length polymorphism (ITS PCR-RFLP) and equipment employed (Faparusi 1973). Faparusi (1973) sequencing. Electrophoretic karyotyping, interdelta typing showed that yeasts are present in the stalk, spathe and and microsatellite (SSRs) typing (Blondin and Vezinhet floret of palm trees. Some of the yeasts and fungi identi- 1988; Yamamoto et al. 1991; Ness et al. 1993 modified by fied in palm wine include Schizosaccharomyces pombe, Legras and Karst 2003; Legras et al. 2005) were used to Pichia pastoris, Candida species (Faparusi 1974) and assess the diversity of yeast isolates. These analyses members of the Saccharomyces sensu stricto group. How- allowed us to draw some conclusions concerning yeast ever, by comparison with other yeasts, Saccharomyces populations in palm wine from various parts of eastern cerevisiae strains were rarely found on the floret (Faparusi Nigeria, compared with some other African strains. 1973). As well as the fungal microflora, bacteria also contribute significantly to the final quality of palm wine. Material and methods Lactic acid bacteria are responsible for the lowering the pH from about 7–4 (thus gradually souring the taste) and Yeast strains also for synthesis of the gums that give palm wine its spe- cific texture. Twenty-three Nigerian palm wine yeast isolates were A similar, marked diversity of yeast genera has been obtained from 13 fresh palm wine samples collected ran- reported in the context of traditional wine making and domly from different locations in the eastern states of cider fermentation in Europe. Furthermore, the large Nigeria during the years 2002 and 2004, on the first day number of studies carried out during the past 20 years of alcoholic fermentation. Conventional mycological have also revealed the broad diversity of S. cerevisiae techniques were used to isolate the yeasts (Beech and strains isolated during fermentation (Versavaud et al. Davenport 1971; Barnett et al. 1983). Table 1 shows the 1995; Esteve-Zarzoso et al. 2000; Sabate et al. 2002; different yeast strains used during the study and their Morrissey et al. 2004) that may explain their variable origins. The other strains analysed (wine, laboratory or physiological properties. However, only a few genetic beer strains) were obtained from our own collection, studies have so far focused on yeast strains isolated from institutional collections (MUCL, CBS) or from Prof. from palm wine, revealing a degree of diversity with Ed J. Louis’ laboratory, Nottingham University (strains respect to genetic and physiological properties (Owu- from the Saccharomyces Genome Resequencing Project ama and Saunders 1990; Ezeronye and Okerentugba (SGRP), http://www.sanger.ac.uk/Teams/Team71/durbin/ 2000, 2002). sgrp/index.shtml). We performed microsatellite multilo- Understanding the biological process of palm wine cus typing on four clones of MUCL30909 and obtained fermentation is essential if improvements are to be the same profile, thus confirming the purity of the strain. made to traditional technologies. As a consequence, assessing the diversity of the local yeasts responsible for DNA extraction alcoholic fermentation is a prerequisite. All taxonomic studies to date on palm wine yeast microflora have Yeast cultures were grown in 10 ml YPD medium for been performed using conventional yeast identification 24 h in a shake flask incubator at 28°C, and DNA methods based on the morphology, sporulation and extraction was performed as described by Legras and physiological activity of yeasts. The recent development Karst 2003. of PCR-based techniques has provided opportunities for yeast strain identification and enables a substantial Species identification reduction in the time required when compared to other molecular techniques (Legras and Karst 2003; Lopez Species were identified by amplification and RFLP anal- et al. 2003). And despite the global trends and develop- ysis of the 5Æ8S-ITS region (Esteve-Zarzoso et al. 1999). ments in yeast taxonomy, little molecular work has PCR products were digested without further purification been done on palm wine yeast (Fay and Benavides with three restriction enzymes: HaeIII, Hinf1 and Dra1 2005). (MBI Fermentas, Lithuania). Amplified PCR products In a recent study (Legras et al. 2007), we described the and their restriction
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