- Email: [email protected]
Diversity of Saccharomyces cerevisiae strains isolated from Borassus akeassii palm wines from Burkina Faso in comparison to other African beverages
Diversity of Saccharomyces cerevisiae strains isolated from Borassus akeassii palm wines from Burkina Faso in comparison to other African beverages Franc¸ois Tapsoba, Jean-Luc Legras, Aly Savadogo, Sylvie Dequin, Alfred Sababenedyo Traore PII: DOI: Reference:
S0168-1605(15)30065-9 doi: 10.1016/j.ijfoodmicro.2015.07.010 FOOD 6981
To appear in:
International Journal of Food Microbiology
Received date: Revised date: Accepted date:
21 May 2015 26 June 2015 5 July 2015
Please cite this article as: Tapsoba, Fran¸cois, Legras, Jean-Luc, Savadogo, Aly, Dequin, Sylvie, Traore, Alfred Sababenedyo, Diversity of Saccharomyces cerevisiae strains isolated from Borassus akeassii palm wines from Burkina Faso in comparison to other African beverages, International Journal of Food Microbiology (2015), doi: 10.1016/j.ijfoodmicro.2015.07.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Diversity of Saccharomyces cerevisiae strains isolated from Borassus akeassii palm wines from Burkina Faso in comparison to other African beverages
Laboratory of Microbiology and Biotechnology, Research Center in Biological, Food and
SC R
1
IP
and Alfred Sababenedyo TRAORE1, 2
T
François TAPSOBA1, 3*, Jean-Luc LEGRAS3,4,5, Aly SAVADOGO2 , Sylvie DEQUIN3,4,5
Nutrition Sciences (CRSBAN), Department of Biochemistry and Microbiology, University of Ouagadougou, Burkina Faso
Laboratory of Food Technology, Department of Biochemistry and Microbiology, University
NU
2
3
MA
of Ouagadougou, Burkina Faso INRA, UMR1083 Sciences pour l’Œnologie, 2 Place Viala, F-34060 Montpellier, France Montpellier SupAgro, UMR1083 Sciences pour l’Œnologie, 2 Place Viala, F-34060
D
4
Montpellier University, UMR1083 Sciences pour l’Œnologie, 2 Place Viala, F-34060
CE P
5
TE
Montpellier, France
Montpellier, France.
AC
*Corresponding author: Email: [email protected]; [email protected] ABSTRACT
In South-West of Burkina Faso, palm wine is produced by spontaneous fermentation of the sap from a specific palm tree Borassus akeassii and plays an important role in people's lives. Saccharomyces cerevisiae is the main agent of this alcoholic fermentation but little is known about the diversity of the isolates from palm. In this work, 39 Saccharomyces cerevisiae strains were isolated from palm wines samples collected from 14 sites in Burkina Faso, as well as 7 isolates obtained from sorghum beer (Dolo) from 3 distant sites. Their diversity was analyzed at 12 microsatellite loci, and compared to the genotypes obtained for other African 1
ACCEPTED MANUSCRIPT yeast populations isolated from Cocoa hulks from Ghana, sorghum beer from Ivory Coast, palm wine from Djibouti Republic, and to our database of strains from miscellaneous origins
T
(bread, beer, wine, sake, oaks…). The ploidy of these strains has been assessed as well by
IP
flow cytometry. Our results show that B. akeassii palm wine contains a specific yeast
SC R
population of diploid strains, different from Dolo produced in the same area and from other palm wine strains from Ivory Coast, Nigeria, or Djibouti Republic. In contrast, Dolo strains appeared as a group of related and mainly tetraploid strains despite being isolated from
MA
NU
different countries.
Key words: Palm wine, Borassus akeassii, Diversity, Saccharomyces cerevisiae,
CE P
INTRODUCTION
TE
D
Microsatellite typing, Flow cytometry
Palm wine is an alcoholic beverage produced in different regions, especially in the tropical regions of America, Asia and Africa (Miège, 1985). In West Africa, it is consumed by more
AC
than 10 million people (Ukhum et al., 2005). This wine represents an indispensable beverage for most ceremonies in traditional African societies, and also a source of income for many people. According to the country or region, this beverage is called by different names (Santiago-Urbina and Ruíz-Terán, 2014). It is produced by natural and uncontrolled fermentation of the sap of local palm trees such as Elaeis guineensis, Raphia hookeri, Raphia vinifera in Nigeria, Borassus akeassii Bayton in Burkina Faso, and Hyphaene thebaica in Djibouti Republic (Ezeronye and Legras, 2009; Bayton and Ouédraogo, 2009; Bayton et al., 2006, Yaméogo et al., 2008). The sweet sap of these palm trees is fermented spontaneously and transformed into a milky-white, flaky and effervescent beverage because of the presence 2
ACCEPTED MANUSCRIPT of live bacteria and yeasts (Okafor, 1975, Ogbulie et al., 2007). Most of the authors have reported that Saccharomyces cerevisiae is the predominant yeast of palm wines fermentations
T
(Aidoo et al., 2006; Amoa-Awua et al., 2006; Ouoba et al., 2012), but the global
IP
characterization of species present in palm wine microflora revealed little information on the
SC R
diversity of the S. cerevisiae strains isolated in Africa. In a former study, the diversity of palm wine S. cerevisiae strains isolated in Nigeria was evaluated and revealed a specific population (Ezeronye and Legras, 2009) with a high level of genetic diversity (Ezeronye and Legras,
NU
2009) which was also observed for S. cerevisiae strains isolated from Cameroun palm wines
MA
(Stringini et al. 2009). S. cerevisiae is also one of the main species isolated from other African beverages such as Dolo, a sorghum beer widely prepared in Ghana, Togo, Burkina Faso or Ivory Coast (Konlani et al. 1996, Van der AA Kühle et al., 2001, Maoura et al. 2005, Kayodé
TE
D
et al., 2011; Al Safadi et al., 2010). Interestingly, the yeast population isolated from Dolo in Ivory Coast was shown polyploid and found to follow a specific sexual life cycle (Al Safadi et
CE P
al., 2010). Lastly, the recent genome sequencing of 38 S. cerevisiae strains (Liti et al., 2009) or Rad tag sequencing (Cromie et al., 2013) revealed specific groups of strains associated to 2
AC
west African strains (NCYC119 and DBVPG6044 isolated from Ginger beer, and Bili wine respectively) and to Ghana cacao strains, but little is known about the relatedness of many of these African S. cerevisiae strains. In this study, 39 S. cerevisiae strains were isolated from Borassus akeassii palm wine as well as 7 isolates from Dolo in Burkina Faso. The diversity of these isolates was explored with microsatellite typing and compared with strains from different other African beverages, such as sorghum beer isolates from Ghana, Ivory Coast or palm wine from Djibouti Republic or Nigeria. The measure of the cell DNA content of these isolates confirms also the differences between palm wine and Dolo isolates.
3
ACCEPTED MANUSCRIPT MATERIAL AND METHODS 1. Palm wine and Dolo sample collection,
T
Samples of palm wine of B. akeassii were collected in 14 different sites in Burkina Faso, from
IP
November 2013 to February 2014, at Banfora (Western Burkina, 400 km from Ouagadougou)
SC R
where palm wine is largely produced and consumed. In each site, 3 samples of 250 ml of fresh palm wine were transferred in sterile plastic containers which were immediately immersed in
NU
an isothermal box, and brought to the laboratory and maintained at 4° C before the analysis to reduce fermentation rate (Bassir, 1962). Once in the laboratory, 15 ml each of samples were
MA
centrifuged at 3000 rpm and the supernatants were stored at - 20 ° C until HPLC analysis. In addition to these palm wine isolates, 15 isolates from sorghum beer (Dolo) from
D
Ouagadougou and rural area in Banfora, and Koudougou (100 km west from Ouagadougou)
CE P
2. Biochemical analysis
TE
were obtained.
Total titrable acidity (TTA) was determined as described by Amoa-Awua et al. (2006) and
AC
expressed as lactic acid percentage. The pH of each sample was measured directly with 10 ml of each sample using a pH-meter (WTW 82362) calibrated with buffer solutions of pH4 and pH7 at 25° C. Different organic acids, sugars, glycerol and ethanol contents were determined by High-Performance Liquid Chromatography (Camarasa et al., 2011)
3. Microbial population analysis of palm fermented sap and yeast isolation On receipt at the laboratory, samples were left at room temperature (30°C) for 4 to 6 hours, and 10 ml of each sample were mixed with 90 ml of sterile peptone solution. Serial dilution was performed with the same solution and 100 µl of the appropriate decimal dilutions 4
ACCEPTED MANUSCRIPT were plated in duplicate on Plate count agar (PCA), Sabouraud agar supplemented with chloramphenicol (0.5 g/l) (Biokar, France) for estimation of total viable counts (TVC), total
T
yeasts (TY) and a lysine medium adapted from Heard and Fleet (1986): Yeast Nitrogen Base
IP
(YNB, Difco) without amino acid without ammonium: 1.7 g/l, Lysine 1g/l) for the estimation
SC R
of non-Saccharomyces yeasts (NSY). All plates were incubated aerobically at 30º C ± 2 for 24 h-72 h. Colony forming units per ml (CFU/ml) were estimated.
For total yeasts, 44 distinct colonies were randomly picked from these 42 palm wine samples
NU
and purified twice by streaking on the same culture medium. Pure cultures were grown on
MA
agar medium at 30°C for 24 h and kept at 4°C for further use. For Dolo, 15 strains were isolated using the same procedure as described above, and 7 were identified as S. cerevisiae.
TE
D
All the strains isolated in here are listed in Table 1.
4. Microbial characterization of yeast isolates
CE P
Yeasts species were differentiated by amplification and RFLP analysis of the 5-8S-ITS region according to (Esteve-Zarzoso et al., 1999). Amplification of the rRNA region spanning the 5.8
AC
rRNA gene and the ITS1 and ITS2 was performed from a cell suspension prepared from a loopful of a fresh yeast colony suspended in 100 µl of water milliQ and heated at 80 °C for 20 min. The restriction analysis of the PCR products was performed with the restriction endonucleases Hinf I and Hae III MBI (Fermentas, Lithuania). PCR products and their restriction fragments were respectively separated on 2 % agarose gel containing 2 µl/ml ethidium bromide, with 0.5 X Tris-Borate-EDTA buffer (TBE: 90 mmol/l Tris-Borate, 1 mmol/l EDTA). After electrophoresis at 100 Volts, gels were visualized and scanned with a Gel 100 Kodak camera. Sizes were estimated by comparison with 100 bp ladder (biolabs Gibco-BRL).
5
ACCEPTED MANUSCRIPT This restriction analysis enabled us to assemble groups of strains with identical patterns. For 11 strains with identical pattern, this ITS1-ITS2 region was amplified and the corresponding
T
PCR products were purified with QIAquick® PCR Purification kit (Qiagen) according to the
IP
manufacturer’s instructions and submitted to sequencing (Eurofin). The sequences were
SC R
compared to those deposited in GenBank, using the BLAST algorithm at NCBI
4.1.S. cerevisiae microsatellite analysis
NU
(https://blast.ncbi.nlm.nih.gov/Blast.cgi).
MA
Strains of S. cerevisiae were grown in Yeast Extract Peptone Dextrose Agar (YEPD-Agar) medium (10 g/l yeast extract, 20 g/l peptone, 20 g/l dextrose, 20 g/l agar) for DNA extraction.
D
DNA was extracted using the rapid method described by Lõoke et al. (2011) and
TE
microsatellite loci analysis was performed according the method used by Legras et al. (2007). Amplification was performed at 12 loci combined in two multiplex of 6 loci, and the size of
CE P
fluorescent amplicons was measured on a ABI3100 (Applied Biosystems) capillary electrophoresis device, using Gene Scan HD400Rox size standard (Applied Biosystems). The
AC
size of each amplicons was then further converted into allelic bins. These genotypes were compared to data obtained in previous work on strains from other origins. The detailed origins of these strains are given in supplementary table S1. Genetic analysis was performed using R software version 3.1.2. The Bruvo distance among strains was calculated using the package Polysat 1.1.2 (Clark et al., 2011). Population structure was assessed using Discriminant Analysis of Principal Components (DAPC) of the package adegenet (Jombart et al., 2008). For the few aneuploidy or tetraploid stains (i.e. bread isolates), two of the scored alleles were chosen randomly per locus.
6
ACCEPTED MANUSCRIPT 5. Ploidy of S. cerevisiae DNA cell content was measured using a flow cytometer (C6) according to Haase et al. (2002)
T
modified by Delobel and Tesnière (2014). Approximately 106 cells were stained with Sytox
IP
green (1.25 μM). Acquisition was performed on 20,000 events observed with a gating on
SC R
forward scatter/side scatter signal. The flow rate was set to approximately 2,000 events per second (medium flow, 35µl/min; core, 16µm). Doublet cells were eliminated by gating for fluorescence area versus height on a linear scale. Finally, the median fluorescence of the
NU
G0/G1 peak was recorded, and ploidy estimated by comparison with fluorescence measure for
MA
control strains By4741 and By4742. 6. Other Data Analysis
CE P
RESULTS
TE
D
Other statistical analyses were performed using R software version 3.1.2.
1. Isolation of S. cerevisiae Palm wine isolates and Dolo isolates
AC
The mean biochemical composition of the different palm wines used for yeast sampling in this study is presented in table 2 after 72 hours of fermentation. These results show that Borassus akeassii palm wines have a lower ethanol content and a higher pH than those of Elais guineensis palm wines, but they present lactic acid contents and acetic acid contents similar to Elais guineensis palm wines, (Amoa-Awua et al., 2006). It must be reminded that palm wine is sold in an active state of fermentation and all these parameters are changing which explain the high variations of the values. Forty-four (44) yeasts were isolated from these different palm wines of B. akeassii. Restriction analysis of the 5.8S rRNA and ITS1 and ITS2 region using two restriction enzymes (Hae III, Hinf I) (Esteve-Zarzoso et al., 1999) 7
ACCEPTED MANUSCRIPT revealed that 39 isolates presented the typical RFLP profile of Saccharomyces cerevisiae, that was confirmed by sequencing for 2 strains. In addition, 15 isolates were obtained from 15
T
sorghum beer samples and 7 isolates were characterized as S. cerevisiae using the same
SC R
IP
approach.
2. Diversity of S. cerevisiae from different Palm wines
NU
In order to evaluate the diversity of these 39 S. cerevisiae isolates of palm wine and 7 from sorghum beer of Burkina Faso, their genotype was obtained at 12 microsatellite loci. These
MA
genotypes were compared to those from other African isolates: 6 sorghum strains from Ivory Coast and 4 from Ghana (Al Safadi et al., 2010, Van der AA Kuhle et al., 2001, Legras et al.,
D
2007), 12 cocoa hulk isolates of Ghana (Jespersen et al., 2005), and other isolates from other
TE
palm wines: 5 Hyphaene thebaica palm wine isolates of Djibouti Republic, 19 Elaeis
CE P
guianeensis palm wine isolates from Nigeria and one from Ivory coast (already genotyped, Ezeronye et Legras, 2009). These genotypes were also compared simultaneously a set of 172 genotypes from our database representing miscellaneous origins (bread, beer, wine, sake,
AC
cheese oaks,…) (Legras et al., 2007). The genotyping enabled the differentiation of 238 S. cerevisiae strains, among which, 38 of 39 from Burkina palm wines and 7 sorghum beer isolates of Burkina Faso. A dendrogramm obtained from a pairwise individual distance matrix calculated with Bruvo’s distance and Neighbor-Joining clustering is given in figure 1. This figure presents different clusters clearly associated to the source from which these strains have been isolated: one cluster of sorghum beer isolates (in pink) containing all strains from Ghana, Burkina Faso, and Ivory Coast, whereas palm wine isolates from different countries were divided into 4 clusters. Interestingly the two identical palm wine isolates were not obtained
8
ACCEPTED MANUSCRIPT from the same palm wine sample, and more generally we could not detect any correlation between clustering and the palm wine source.
T
In order to confirm the detection of population structure a discriminant principal component
IP
analysis has been performed on a sub set of 252 genotypes (out of 264) representatives of the
SC R
different main groups (Jombart et al., 2010). The differentiation of the different groups is represented in figure 2 based on 80 % of the global variation obtained from the 50 first PCA axis. The two first axis enable the differentiation of sorghum beer isolates (different shades of
NU
pink in the lower part of figure 2a) from palm wine: Nigeria palm wine strains and a cluster of
MA
miscellaneous African strains (brown and red-brown) in the upper part of figure 2a whereas Djibouti palm wine (in light brown) and Burkina Faso palm wine isolates (dark blue) are in the center of the right part of figure 2a. Last Ghana (bright green) and Burkina Faso palm
TE
D
wine (dark blue) isolates were better separated from other groups according to axis 3 as they
CE P
are found in the right and in the lower left part of figure 2b respectively. 3. Ploidy of S. cerevisiae strains The measure of the DNA cell content revealed different ploidies for these African isolates
AC
(Table 3). Ivory Coast Dolo isolates (and the two Ghana isolates presented a DNA content close to 4 indicating that this population contains mainly tetraploid strains, which explains the 4 alleles observed at several microsatellite loci. Burkina Faso Dolo isolates were more heterogeneous, with 3 strains to 3.5 n and 4 diploid (4 strains). In contrast, palm wine isolates of Burkina Faso presented ploidies close to 2 for the majority of strains (30 strains), but 5 were detected as triploid, and 2 tetraploid. A Kruskal-Wallis rank sum test indicated that these populations do not have similar ploidies (p-value < 0.0002).
9
ACCEPTED MANUSCRIPT DISCUSSION Despite the wide expansion of the production of palm wine in Africa, little is known about the
T
diversity of S. cerevisiae isolated from these beverages. A former study performed on Elaeis
IP
guianeensis palm wine isolates from Nigeria (Ezeronye and Legras, 2009) indicated a specific
SC R
population, and more recently the population genomic survey of S. cerevisiae diversity (Liti et al., 2009), revealed the existence of an African lineage for S. cerevisiae strains. The genotyping of these African yeast populations revealed that these are well differentiated from
NU
European (bread, wine, cheese) or Asian populations. Two main factors have been shown to
MA
explain yeast genetic diversity: isolation source and geography (Cromie et al., 2013; Legras et al., 2007). Despite being isolated in Burkina Faso, palm wine and Dolo isolates were clearly
D
differentiated. They were also different from those isolated on other palm wines, sorghum
TE
beers or Cocoa hulks isolates. However, the different strains isolated from sorghum beer in Burkina Faso, Ghana, and Ivory Coast, were clustered very closely in Discriminant Analysis
CE P
of Principal Components (DAPC) and in our tree, which suggests that these strains are related. This implies that isolation source is the main factor explaining population structure
AC
for Dolo. The inoculation of Dolo wort is achieved from a previous brew through different manners: directly by a fraction or the foam of a previous brew, or by a woven belt in which is trapped dried yeast cells from a former fermentation (Sefa-Dedeh et al., 1999). Human population migration and this specific inoculation may explain this yeast population structure. On the contrary to Dolo, isolates obtained from the different palm wines from Borassus akeassii Bayton in Burkina Faso, Elaeis guineensis in Nigeria and Ivory Coast, and Hyphaene thebaica in Djibouti republic, belong to different populations, as can be attested from the different clusters observed in DAPC analysis, and from the individuals tree. In addition, two other isolates from West Africa (NCYC110, and DBVPG6044), isolated from beverages prepared from other plants (Zingiber officinale and Osbeckia grandiflora), or strains isolated 10
ACCEPTED MANUSCRIPT from cocoa hulks in Ghana are not related to any of the former populations, suggesting a wide diversity of S. cerevisiae in Africa. The differences between palm wine S. cerevisiae
T
populations might result from geography but also from the palm tree these beverages are
IP
produced from. CBS400, an Elaeis guineensis palm wine isolate from Ivory coast is not
SC R
related to those isolated from those from Nigeria which suggests that geography may explain also part of the diversity of these populations, but should be confirm through a much wider sampling.
NU
Ploidy is another trait differentiating sorghum beer and palm wine isolates. A ploidy of 2n
MA
was observed for most palm wine isolates with some triploid or tetraploid strains, whereas sorghum beer isolates where mainly tetraploid. As bread or beer strains, which are not related to these sorghum beer strains are also polyploid, we can hypothesized that polyploidy may
TE
D
result from a selective pressure imposed by this starch based media, or by the continuous culture imposed by man caused by back sloping process (Al Safadi et al., 2010). All these
CONCLUSION
CE P
features outline the specific properties of these original African yeast populations.
AC
The present study provides first information on the diversity of Saccharomyces cerevisiae isolated from palm wine obtained from the fermented sap of B. akeassii in Burkina Faso. The characterization of the yeast population showed that B. akeassii palm wine contains a specific and diverse population of yeast, different from Dolo. But our work highlights as well the diversity of yeast populations used for African beverages, and our first results indicated that strains of these populations have different physiological properties. More work has to be done in order to understand how geographical, technological and historical factors have structured this diversity. The industrial potential value of these genetic resources has to be further evaluated for biotechnological applications. 11
ACCEPTED MANUSCRIPT ACKNOWLEDGEMENT The authors are grateful to Prof L. Jespersen, University of Copenhagen, for providing cacao
T
strains, and to Dr. S Casaregola, Cirm levures, Thiverval-Grignon, and Prof. M. Aigle
IP
(Université de Lyon 1) for providing Djibouti Republic palm wine and Dolo strains.
SC R
The corresponding author is grateful to International Foundation for Science (IFS), West African Economic and Monetary Union (UEMOA) for supporting.
NU
REFERENCES
Aidoo, K. E., Nout, M. J. R., Sarkar, P. K., 2006. Occurrence and function of yeasts in Asian
MA
indigenous fermented foods. FEMS Yeasts Res. 6:30–39. Al Safadi, R.,
eiss- ayet, M., Briolay, ., Aigle, M., 2010. A polyploid population of
TE
D
Saccharomyces cerevisiae with separate sexes (dioecy). FEMS Yeast Res. 10: 757–76 Amoa-Awua, W. K., Sampson, E., Tano-Debrah, K., 2006. Growth of yeasts, lactic and acetic
CE P
acid bacteria in palm wine during tapping and fermentation from felled oil palm (Elaeis guineensis) in Ghana. J. of Appl. Microbiol. 102 (2): 599-606.
AC
Bassir, O., 1962. Observations on the fermentation of palm-wine. West Afric.J. Biol. Appl. Chem. 6: 20 – 25.
Bayton, R. P., Ouédraogo, A., Guinko, S., 2006. The genus Borassus (Arecacea) in West Africa with a description of a new species from Burkina Faso. J. Linn Soc. 150: 419–427. Bayton, R. P., Ouédraogo, A., 2009. Discovering Africa’s newest palm. Palms. 53: 37–45. Camarasa, C., Sanchez I., Brial P., Bigey, F., Dequin, S., 2011. Phenotypic Landscape of Saccharomyces cerevisiae during Wine Fermentation: Evidence for Origin-Dependent Metabolic Traits. PLoS One. 6: e25147. 12
ACCEPTED MANUSCRIPT Clark, L. V., Jasieniuk, M., 2011. POLYSAT: A R package for polyploid microsatellite analysis. Mol. Ecol. Res. 11 (3): 562–66.
T
Cromie, G. A., Hyma, K. E., Ludlow, C. L., Garmendia-Torres, C., Gilbert, T. L., May, P.,
IP
Huang A. A., Dudley A. M., Fay, J. C., 2013. Genomic Sequence Diversity and Population
SC R
Structure of Saccharomyces cerevisiae Assessed by RAD-seq. Genes, Genom, Gen. 3: 21632171.
NU
Delobel, P., Tesnière C., 2014. A Simple FCM Method to Avoid Misinterpretation in Saccharomyces cerevisiae Cell Cycle Assessment between G0 and Sub-G1. PLoSONE. 9(1):
MA
e84645.
D
Esteve-Zarzoso, B., Belloch C., Uruburu, F., Querol A., 1999. Identification of yeasts by
TE
RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal
CE P
internal transcribed spacers. Int. J. of Systematic Bacteriol. 49: 329–337. Ezeronye, O. U., Legras, J. L., 2009. Genetic analysis of Saccharomyces cerevisiae strains
AC
isolated from palm wine in eastern Nigeria. Comparison with other African strains. J. of Appl. Microbiol.106: 1569–78. Haase, S. B., Reed, S. I., 2002. Improved flow cytometric analysis of the budding yeast cell cycle. Cell Cycle.1: 132–136. Heard, G. M., Fleet, G. H., 1986. Evaluation of selective media for enumeration of yeasts during wine fermentation. J. Appl. Microbiol. 60: 477–481. Jespersen, L., Dennis, S. N., Susanne H., Mogens, J., 2005. Occurrence and diversity of yeasts involved in fermentation of West African cocoa beans. FEMS Yeast Research. 5: 441–53. 13
ACCEPTED MANUSCRIPT Jombart, T., Devillard, S., Balloux, F., 2010. Discriminant analysis of principal components: a new method for the analysis of genetically structured populations. BMC Gen. 11:94.
T
Jombart, T., 2008. Adegenet: a R package for the multivariate analysis of genetic markers.
SC R
IP
Bioinfo. 24: 1403-1405.
Kayodé, A. P. P., Vieira-Dalodé, G., Linnemann, A. R., Kotchoni, S. O., Hounhouigan, A. J. D., Van Boekel, M. A. J. S., Nout, M. J. R. 2011. Diversity of yeasts involved in the
NU
fermentation of tchoukoutou, an opaque sorghum beer from Benin. Afr J. of Microbiol. Res.
MA
5: 2737-2742.
Konlani, S., Delgenes, J. P., Moletta, R., Traoré A., Doh, A., 1996. Isolation and
D
physiological characteristics of yeasts involved in sorghum beer production. Food Biotech.
TE
10: 29-40.
CE P
Legras, J. L., Merdinoglu, D., Cornuet, J. M., Karst, F., 2007. Bread, Beer and Wine, Saccharomyces cerevisiae diversity reflects human history. Mol. Ecol. 16: 2091–2102. Liti, G., Carter. D. M., Moses, A. M., Warringer, J., Parts, L., James, S. A., Davey, R. P.,
AC
Roberts, I. N., Burt, A., Koufopanou,V., Tsai, I. J., Bergman, C. M., Bensasson, D., O'Kelly, M. J., Van Oudenaarden, A., Barton, D. B., Bailes, E., Nguyen, A. N., Jones, M., Quail, M. A., Goodhead, I., Sims, S., Smith, F., Blomberg, A., Durbin, R., Louis, E. J., 2009. Population genomics of domestic and wild yeasts. Nat. 458: 337–341. Lõoke, M., Kristjuhan, K., Kristjuhan, A., 2011. Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques. 50: 325–8.
14
ACCEPTED MANUSCRIPT Lyumugabe, F., Gros, J., Nzungize, J., Bajyana, E., Thonart, P., 2012. Characteristics of African traditional beers brewed with sorghum malt: a review. Biotechnol. Agron. Soc.
T
Environ. 16(4): 509-530.
IP
Maoura, N., Mbaiguinam, M., Nguyen, H. V. Gaillardin, C., Pourquie, J., 2005. Identification
SC R
and typing of the yeast strains isolated from Bili Bili, a Traditional Sorghum Beer of Chad. Afr. J. of Biotech. 4 (7): 646–56.
NU
Miège, J., 1985. Palmales. In J. Bersani, J.Gall, H. Schweizer, and M. Lardy (ed.),
MA
Encyclopaedia universalis, 13:764-767. Encyclopaedia universalis publisher, Paris. Ogbulie, T. E., Ogbulie J. N., Njoku, H. O., 2007. Comparative study on the microbiology
D
and shelf life stability of palm wine from Elaeis guineensis and Raphia hookeri obtained from
TE
Okigwe, Nigeria. Afr. J. of Biotech. 6: 914-922.
CE P
Okafor, N., 1975. Microbiology of Nigeria palm wine with particular reference to bacteria. J. Appl. Bacteriol. 38: 81-88.
Ouoba, L. I. I., Kando C., Parkouda, C., Sawadogo-Lingani, H., Diawara, B., Sutherland, J.P.,
AC
2012. The microbiology of bandji, palm wine of Borassus akeassii from Burkina Faso: identification and genotypic diversity of yeasts, lactic acid and acetic acid bacteria. J. of Appl. Microbiol.113: 1364-5072. R Core Team, 2014. R: A language and environment for statistical computing, Version 3.1.2. R Foundation for Statistical Computing 2014. http://www.R-project.org. Santiago-Urbina, J. A., Ruíz-Terán, F., 2014. Microbiology and biochemistry of traditional palm wine produced around the world. Int. Food Res. J. 21: 1261-1269.
15
ACCEPTED MANUSCRIPT Sefa-Dedeh, S., Sanni, I., Tetteh, G., Sakyi-Dawson, E., 1999. Yeasts in the traditional brewing of pito in Ghana. World J Microbiol Biotechnol. 15: 593–597.
T
Stringini, M., Comitini, F., Taccari, M., Ciani, M., 2009. Yeast diversity during tapping and
SC R
IP
fermentation of palm wine from Cameroon. Food Microbiol. 26: 415–20.
Ukhum, M. E., Okolie, N. P., Oyerinde, A. O., 2005. Some mineral profiles of fresh and bottled palm wine – a comparative study. Afr. J. Biotech. 4: 829-32.
NU
Van der Aa Kühle, A., Jespersen, L., Glover, R. L., Diawara, B., Jakobsen, M., 2001.
MA
Identification and Characterization of Saccharomyces Cerevisiae strains isolated from West African Sorghum Beer. Yeast. 18: 1069–79.
D
Yaméogo, J., Bélem-Ouédraogo, M., Bayala, J., Ouédraogo, M. B., Guinko, S., 2008. Uses
TE
and commercialization of Borassus akeassii Bayton, Ouédraogo, Guinko non-wood timber
AC
Env. 12: 47-55.
CE P
products in South-Western Burkina Faso, West Africa. Biotechnologie, Agronomie, Société et
16
ACCEPTED MANUSCRIPT Figure legend : Figure 1: Genetic diversity of Burkina Faso palm wine and dolo S. cerevisiae isolates revealed
T
by microsatellite typing: neighbour joining dendrogramm obtained from the pairwise
AC
CE P
TE
D
MA
NU
SC R
IP
individual Bruvo’s distance matrix.
17
ACCEPTED MANUSCRIPT
Figure 2: Genetic diversity of different African S. cerevisiae strains from different origins
T
revealed by Discriminant Analysis of Principal Component (DAPC). a- representation of the
IP
different clusters with regard to the axis 1 and 2; b- representation of the different clusters
AC
CE P
TE
D
MA
NU
SC R
with regard to the axis 3 and 4.
18
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
19
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
20
ACCEPTED MANUSCRIPT Table 1: Description of the Burkina Faso palm wine and dolo isolates, and of the African S. cerevisiae isolates genotyped in this study Origin
Isolates codes
Reference
PWBA2
PW7 ; PW18 ; PW34
PWBA3
PW14 ; PW21 ; PW29
PWBA4
PW2 ; PW19
PWBA 5
PW6 ; PW31
PWBA 6
PW28
PW1 ; PW8
PWBA 9
TE
PWBA 10
PW5 ; PW32 ; PW36 ; PW17
CE P
PWBA 11
AC
PW4 ; PW13 ; PW15 ; PW22 ; PW44
D
PWBA 8
Faso,
IP
PW27 ; PW26
NU
PWBA1
PWBA 7
Burkina Dolo
Isolates
SC R
Sampling site
MA
Burkina Faso, palm wine (Banfora)
T
Strains isolated in this study
PW35 PW33 ;
PW37 ;
PW38 ;
PW40 ; PW41 ; PW42 ; PW43
PWBA 12
PW3 ; PW16 ; PW23 ; PW30
PWBA 13
PW10 ; PW25
PWBA 14
PW20 ; PW24
Banfora
B1, B3, B4, B5
Ouagadougou Koudougou
PW39 ;
F2, F3 T2
African strains genotyped in this study Ivory Coast, Millet beer Ghana, cocoa isolates Djibouti Republic, wine
palm
CH02 (CliB1162) , CH06 (CliB1163), CH10 (CliB1164), CH11 (CliB1165), CH13 (CliB1167), CH14 (CliB1166) MTF2546, MTF2547, MTF2548, MTF2549, MTF2550, MTF2552, MTF2553, MTF2554, MTF2555, MTF2556, MTF2557, MTF2620 DJ65 (CliB1169), DJ66 (CliB1169), DJ71, DJ73, DJ74
Al Safadi et al., 2010 Jespersen et al., 2005 M. Aigle (un published data)
21
ACCEPTED MANUSCRIPT CBS: Centraalbureau voor Schimmelcultures (CBS) - Netherlands; MUCL: Louvain,
AC
CE P
TE
D
MA
NU
SC R
IP
T
Belgium; PW: yeasts isolated from different palm wines of B. akeassii.
22
ACCEPTED MANUSCRIPT Table 2: Biochemical and microbial characteristics of the different palm wines (after 72 hours of fermentation) used for Saccharomyces cerevisiae isolation
Mean ± SD
T
Biochemical characteristics
4.79 ± 0.12
Total titrable acidity (%)
0.66 ± 0.25
Ethanol (%)
3.69 ± 0.69
SC R
Lactic Acid (g/l)
3.40 ±1.30
Malate (g/l)
Acetate (g/l)
MA
Succinate (g/l)
0.60 ± 0.30
NU
Glucose (g/l) Fructose (g/l)
Glycerol (g/l)
D
Microbial characteristics TVC (*108 UFC/ml)
TE
IP
pH
2.60 ± 4.90 0.80 ± 0.50 2.70±1.80 2.40 ± 0.40 1.30 ±0.50 Mean±SD 1.89 ± 0.70 1.71 ± 1.40
NSY (*104 UFC/ml)
2.76 ± 1.80
CE P
TY (*106 UFC/ml)
TVC: Total viable counts; TY: Total yeast cells; NSY: Non-Saccharomyces yeasts; SD:
AC
Standard deviation
23
ACCEPTED MANUSCRIPT
Mean*
Palm Wine, Burkina Faso
35
2.2
Dolo, Burkina Faso
7
Dolo (Chapallo), Ivory Coast
6
Palm wine, Djibouti Republic
7
Dolo, Ghana
2
SC R NU
MA
Isolates origin
Palm Wine, Nigeria
1
IP
Number of isolates analysed
T
Table 3: DNA content of the different Saccharomyces cerevisiae isolates measured by flow cytometry
SD
0.5
2.6
0.9
4.1
0.3
2.7
0.5
3.5 2.8
AC
CE P
TE
D
*Significance of the differences between populations tested by a Kruskal-Wallis rank sum test (pvalue <0.0002) for the first four populations. SD: Standard deviation.
24
ACCEPTED MANUSCRIPT HIGHLIGTS
Borassus akeassii palm wines in Burkina Faso present a specific S. cerevisiae population as well as palm wines produced from other palm trees.
T
Saccharomyces cerevisiae isolated from Burkina, Ghana and Ivory Coast Dolo belong to a
CE P
TE
D
MA
NU
SC R
IP
single cluster of tetraploid strains.
AC
25
S0168-1605(15)30065-9 doi: 10.1016/j.ijfoodmicro.2015.07.010 FOOD 6981
To appear in:
International Journal of Food Microbiology
Received date: Revised date: Accepted date:
21 May 2015 26 June 2015 5 July 2015
Please cite this article as: Tapsoba, Fran¸cois, Legras, Jean-Luc, Savadogo, Aly, Dequin, Sylvie, Traore, Alfred Sababenedyo, Diversity of Saccharomyces cerevisiae strains isolated from Borassus akeassii palm wines from Burkina Faso in comparison to other African beverages, International Journal of Food Microbiology (2015), doi: 10.1016/j.ijfoodmicro.2015.07.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Diversity of Saccharomyces cerevisiae strains isolated from Borassus akeassii palm wines from Burkina Faso in comparison to other African beverages
Laboratory of Microbiology and Biotechnology, Research Center in Biological, Food and
SC R
1
IP
and Alfred Sababenedyo TRAORE1, 2
T
François TAPSOBA1, 3*, Jean-Luc LEGRAS3,4,5, Aly SAVADOGO2 , Sylvie DEQUIN3,4,5
Nutrition Sciences (CRSBAN), Department of Biochemistry and Microbiology, University of Ouagadougou, Burkina Faso
Laboratory of Food Technology, Department of Biochemistry and Microbiology, University
NU
2
3
MA
of Ouagadougou, Burkina Faso INRA, UMR1083 Sciences pour l’Œnologie, 2 Place Viala, F-34060 Montpellier, France Montpellier SupAgro, UMR1083 Sciences pour l’Œnologie, 2 Place Viala, F-34060
D
4
Montpellier University, UMR1083 Sciences pour l’Œnologie, 2 Place Viala, F-34060
CE P
5
TE
Montpellier, France
Montpellier, France.
AC
*Corresponding author: Email: [email protected]; [email protected] ABSTRACT
In South-West of Burkina Faso, palm wine is produced by spontaneous fermentation of the sap from a specific palm tree Borassus akeassii and plays an important role in people's lives. Saccharomyces cerevisiae is the main agent of this alcoholic fermentation but little is known about the diversity of the isolates from palm. In this work, 39 Saccharomyces cerevisiae strains were isolated from palm wines samples collected from 14 sites in Burkina Faso, as well as 7 isolates obtained from sorghum beer (Dolo) from 3 distant sites. Their diversity was analyzed at 12 microsatellite loci, and compared to the genotypes obtained for other African 1
ACCEPTED MANUSCRIPT yeast populations isolated from Cocoa hulks from Ghana, sorghum beer from Ivory Coast, palm wine from Djibouti Republic, and to our database of strains from miscellaneous origins
T
(bread, beer, wine, sake, oaks…). The ploidy of these strains has been assessed as well by
IP
flow cytometry. Our results show that B. akeassii palm wine contains a specific yeast
SC R
population of diploid strains, different from Dolo produced in the same area and from other palm wine strains from Ivory Coast, Nigeria, or Djibouti Republic. In contrast, Dolo strains appeared as a group of related and mainly tetraploid strains despite being isolated from
MA
NU
different countries.
Key words: Palm wine, Borassus akeassii, Diversity, Saccharomyces cerevisiae,
CE P
INTRODUCTION
TE
D
Microsatellite typing, Flow cytometry
Palm wine is an alcoholic beverage produced in different regions, especially in the tropical regions of America, Asia and Africa (Miège, 1985). In West Africa, it is consumed by more
AC
than 10 million people (Ukhum et al., 2005). This wine represents an indispensable beverage for most ceremonies in traditional African societies, and also a source of income for many people. According to the country or region, this beverage is called by different names (Santiago-Urbina and Ruíz-Terán, 2014). It is produced by natural and uncontrolled fermentation of the sap of local palm trees such as Elaeis guineensis, Raphia hookeri, Raphia vinifera in Nigeria, Borassus akeassii Bayton in Burkina Faso, and Hyphaene thebaica in Djibouti Republic (Ezeronye and Legras, 2009; Bayton and Ouédraogo, 2009; Bayton et al., 2006, Yaméogo et al., 2008). The sweet sap of these palm trees is fermented spontaneously and transformed into a milky-white, flaky and effervescent beverage because of the presence 2
ACCEPTED MANUSCRIPT of live bacteria and yeasts (Okafor, 1975, Ogbulie et al., 2007). Most of the authors have reported that Saccharomyces cerevisiae is the predominant yeast of palm wines fermentations
T
(Aidoo et al., 2006; Amoa-Awua et al., 2006; Ouoba et al., 2012), but the global
IP
characterization of species present in palm wine microflora revealed little information on the
SC R
diversity of the S. cerevisiae strains isolated in Africa. In a former study, the diversity of palm wine S. cerevisiae strains isolated in Nigeria was evaluated and revealed a specific population (Ezeronye and Legras, 2009) with a high level of genetic diversity (Ezeronye and Legras,
NU
2009) which was also observed for S. cerevisiae strains isolated from Cameroun palm wines
MA
(Stringini et al. 2009). S. cerevisiae is also one of the main species isolated from other African beverages such as Dolo, a sorghum beer widely prepared in Ghana, Togo, Burkina Faso or Ivory Coast (Konlani et al. 1996, Van der AA Kühle et al., 2001, Maoura et al. 2005, Kayodé
TE
D
et al., 2011; Al Safadi et al., 2010). Interestingly, the yeast population isolated from Dolo in Ivory Coast was shown polyploid and found to follow a specific sexual life cycle (Al Safadi et
CE P
al., 2010). Lastly, the recent genome sequencing of 38 S. cerevisiae strains (Liti et al., 2009) or Rad tag sequencing (Cromie et al., 2013) revealed specific groups of strains associated to 2
AC
west African strains (NCYC119 and DBVPG6044 isolated from Ginger beer, and Bili wine respectively) and to Ghana cacao strains, but little is known about the relatedness of many of these African S. cerevisiae strains. In this study, 39 S. cerevisiae strains were isolated from Borassus akeassii palm wine as well as 7 isolates from Dolo in Burkina Faso. The diversity of these isolates was explored with microsatellite typing and compared with strains from different other African beverages, such as sorghum beer isolates from Ghana, Ivory Coast or palm wine from Djibouti Republic or Nigeria. The measure of the cell DNA content of these isolates confirms also the differences between palm wine and Dolo isolates.
3
ACCEPTED MANUSCRIPT MATERIAL AND METHODS 1. Palm wine and Dolo sample collection,
T
Samples of palm wine of B. akeassii were collected in 14 different sites in Burkina Faso, from
IP
November 2013 to February 2014, at Banfora (Western Burkina, 400 km from Ouagadougou)
SC R
where palm wine is largely produced and consumed. In each site, 3 samples of 250 ml of fresh palm wine were transferred in sterile plastic containers which were immediately immersed in
NU
an isothermal box, and brought to the laboratory and maintained at 4° C before the analysis to reduce fermentation rate (Bassir, 1962). Once in the laboratory, 15 ml each of samples were
MA
centrifuged at 3000 rpm and the supernatants were stored at - 20 ° C until HPLC analysis. In addition to these palm wine isolates, 15 isolates from sorghum beer (Dolo) from
D
Ouagadougou and rural area in Banfora, and Koudougou (100 km west from Ouagadougou)
CE P
2. Biochemical analysis
TE
were obtained.
Total titrable acidity (TTA) was determined as described by Amoa-Awua et al. (2006) and
AC
expressed as lactic acid percentage. The pH of each sample was measured directly with 10 ml of each sample using a pH-meter (WTW 82362) calibrated with buffer solutions of pH4 and pH7 at 25° C. Different organic acids, sugars, glycerol and ethanol contents were determined by High-Performance Liquid Chromatography (Camarasa et al., 2011)
3. Microbial population analysis of palm fermented sap and yeast isolation On receipt at the laboratory, samples were left at room temperature (30°C) for 4 to 6 hours, and 10 ml of each sample were mixed with 90 ml of sterile peptone solution. Serial dilution was performed with the same solution and 100 µl of the appropriate decimal dilutions 4
ACCEPTED MANUSCRIPT were plated in duplicate on Plate count agar (PCA), Sabouraud agar supplemented with chloramphenicol (0.5 g/l) (Biokar, France) for estimation of total viable counts (TVC), total
T
yeasts (TY) and a lysine medium adapted from Heard and Fleet (1986): Yeast Nitrogen Base
IP
(YNB, Difco) without amino acid without ammonium: 1.7 g/l, Lysine 1g/l) for the estimation
SC R
of non-Saccharomyces yeasts (NSY). All plates were incubated aerobically at 30º C ± 2 for 24 h-72 h. Colony forming units per ml (CFU/ml) were estimated.
For total yeasts, 44 distinct colonies were randomly picked from these 42 palm wine samples
NU
and purified twice by streaking on the same culture medium. Pure cultures were grown on
MA
agar medium at 30°C for 24 h and kept at 4°C for further use. For Dolo, 15 strains were isolated using the same procedure as described above, and 7 were identified as S. cerevisiae.
TE
D
All the strains isolated in here are listed in Table 1.
4. Microbial characterization of yeast isolates
CE P
Yeasts species were differentiated by amplification and RFLP analysis of the 5-8S-ITS region according to (Esteve-Zarzoso et al., 1999). Amplification of the rRNA region spanning the 5.8
AC
rRNA gene and the ITS1 and ITS2 was performed from a cell suspension prepared from a loopful of a fresh yeast colony suspended in 100 µl of water milliQ and heated at 80 °C for 20 min. The restriction analysis of the PCR products was performed with the restriction endonucleases Hinf I and Hae III MBI (Fermentas, Lithuania). PCR products and their restriction fragments were respectively separated on 2 % agarose gel containing 2 µl/ml ethidium bromide, with 0.5 X Tris-Borate-EDTA buffer (TBE: 90 mmol/l Tris-Borate, 1 mmol/l EDTA). After electrophoresis at 100 Volts, gels were visualized and scanned with a Gel 100 Kodak camera. Sizes were estimated by comparison with 100 bp ladder (biolabs Gibco-BRL).
5
ACCEPTED MANUSCRIPT This restriction analysis enabled us to assemble groups of strains with identical patterns. For 11 strains with identical pattern, this ITS1-ITS2 region was amplified and the corresponding
T
PCR products were purified with QIAquick® PCR Purification kit (Qiagen) according to the
IP
manufacturer’s instructions and submitted to sequencing (Eurofin). The sequences were
SC R
compared to those deposited in GenBank, using the BLAST algorithm at NCBI
4.1.S. cerevisiae microsatellite analysis
NU
(https://blast.ncbi.nlm.nih.gov/Blast.cgi).
MA
Strains of S. cerevisiae were grown in Yeast Extract Peptone Dextrose Agar (YEPD-Agar) medium (10 g/l yeast extract, 20 g/l peptone, 20 g/l dextrose, 20 g/l agar) for DNA extraction.
D
DNA was extracted using the rapid method described by Lõoke et al. (2011) and
TE
microsatellite loci analysis was performed according the method used by Legras et al. (2007). Amplification was performed at 12 loci combined in two multiplex of 6 loci, and the size of
CE P
fluorescent amplicons was measured on a ABI3100 (Applied Biosystems) capillary electrophoresis device, using Gene Scan HD400Rox size standard (Applied Biosystems). The
AC
size of each amplicons was then further converted into allelic bins. These genotypes were compared to data obtained in previous work on strains from other origins. The detailed origins of these strains are given in supplementary table S1. Genetic analysis was performed using R software version 3.1.2. The Bruvo distance among strains was calculated using the package Polysat 1.1.2 (Clark et al., 2011). Population structure was assessed using Discriminant Analysis of Principal Components (DAPC) of the package adegenet (Jombart et al., 2008). For the few aneuploidy or tetraploid stains (i.e. bread isolates), two of the scored alleles were chosen randomly per locus.
6
ACCEPTED MANUSCRIPT 5. Ploidy of S. cerevisiae DNA cell content was measured using a flow cytometer (C6) according to Haase et al. (2002)
T
modified by Delobel and Tesnière (2014). Approximately 106 cells were stained with Sytox
IP
green (1.25 μM). Acquisition was performed on 20,000 events observed with a gating on
SC R
forward scatter/side scatter signal. The flow rate was set to approximately 2,000 events per second (medium flow, 35µl/min; core, 16µm). Doublet cells were eliminated by gating for fluorescence area versus height on a linear scale. Finally, the median fluorescence of the
NU
G0/G1 peak was recorded, and ploidy estimated by comparison with fluorescence measure for
MA
control strains By4741 and By4742. 6. Other Data Analysis
CE P
RESULTS
TE
D
Other statistical analyses were performed using R software version 3.1.2.
1. Isolation of S. cerevisiae Palm wine isolates and Dolo isolates
AC
The mean biochemical composition of the different palm wines used for yeast sampling in this study is presented in table 2 after 72 hours of fermentation. These results show that Borassus akeassii palm wines have a lower ethanol content and a higher pH than those of Elais guineensis palm wines, but they present lactic acid contents and acetic acid contents similar to Elais guineensis palm wines, (Amoa-Awua et al., 2006). It must be reminded that palm wine is sold in an active state of fermentation and all these parameters are changing which explain the high variations of the values. Forty-four (44) yeasts were isolated from these different palm wines of B. akeassii. Restriction analysis of the 5.8S rRNA and ITS1 and ITS2 region using two restriction enzymes (Hae III, Hinf I) (Esteve-Zarzoso et al., 1999) 7
ACCEPTED MANUSCRIPT revealed that 39 isolates presented the typical RFLP profile of Saccharomyces cerevisiae, that was confirmed by sequencing for 2 strains. In addition, 15 isolates were obtained from 15
T
sorghum beer samples and 7 isolates were characterized as S. cerevisiae using the same
SC R
IP
approach.
2. Diversity of S. cerevisiae from different Palm wines
NU
In order to evaluate the diversity of these 39 S. cerevisiae isolates of palm wine and 7 from sorghum beer of Burkina Faso, their genotype was obtained at 12 microsatellite loci. These
MA
genotypes were compared to those from other African isolates: 6 sorghum strains from Ivory Coast and 4 from Ghana (Al Safadi et al., 2010, Van der AA Kuhle et al., 2001, Legras et al.,
D
2007), 12 cocoa hulk isolates of Ghana (Jespersen et al., 2005), and other isolates from other
TE
palm wines: 5 Hyphaene thebaica palm wine isolates of Djibouti Republic, 19 Elaeis
CE P
guianeensis palm wine isolates from Nigeria and one from Ivory coast (already genotyped, Ezeronye et Legras, 2009). These genotypes were also compared simultaneously a set of 172 genotypes from our database representing miscellaneous origins (bread, beer, wine, sake,
AC
cheese oaks,…) (Legras et al., 2007). The genotyping enabled the differentiation of 238 S. cerevisiae strains, among which, 38 of 39 from Burkina palm wines and 7 sorghum beer isolates of Burkina Faso. A dendrogramm obtained from a pairwise individual distance matrix calculated with Bruvo’s distance and Neighbor-Joining clustering is given in figure 1. This figure presents different clusters clearly associated to the source from which these strains have been isolated: one cluster of sorghum beer isolates (in pink) containing all strains from Ghana, Burkina Faso, and Ivory Coast, whereas palm wine isolates from different countries were divided into 4 clusters. Interestingly the two identical palm wine isolates were not obtained
8
ACCEPTED MANUSCRIPT from the same palm wine sample, and more generally we could not detect any correlation between clustering and the palm wine source.
T
In order to confirm the detection of population structure a discriminant principal component
IP
analysis has been performed on a sub set of 252 genotypes (out of 264) representatives of the
SC R
different main groups (Jombart et al., 2010). The differentiation of the different groups is represented in figure 2 based on 80 % of the global variation obtained from the 50 first PCA axis. The two first axis enable the differentiation of sorghum beer isolates (different shades of
NU
pink in the lower part of figure 2a) from palm wine: Nigeria palm wine strains and a cluster of
MA
miscellaneous African strains (brown and red-brown) in the upper part of figure 2a whereas Djibouti palm wine (in light brown) and Burkina Faso palm wine isolates (dark blue) are in the center of the right part of figure 2a. Last Ghana (bright green) and Burkina Faso palm
TE
D
wine (dark blue) isolates were better separated from other groups according to axis 3 as they
CE P
are found in the right and in the lower left part of figure 2b respectively. 3. Ploidy of S. cerevisiae strains The measure of the DNA cell content revealed different ploidies for these African isolates
AC
(Table 3). Ivory Coast Dolo isolates (and the two Ghana isolates presented a DNA content close to 4 indicating that this population contains mainly tetraploid strains, which explains the 4 alleles observed at several microsatellite loci. Burkina Faso Dolo isolates were more heterogeneous, with 3 strains to 3.5 n and 4 diploid (4 strains). In contrast, palm wine isolates of Burkina Faso presented ploidies close to 2 for the majority of strains (30 strains), but 5 were detected as triploid, and 2 tetraploid. A Kruskal-Wallis rank sum test indicated that these populations do not have similar ploidies (p-value < 0.0002).
9
ACCEPTED MANUSCRIPT DISCUSSION Despite the wide expansion of the production of palm wine in Africa, little is known about the
T
diversity of S. cerevisiae isolated from these beverages. A former study performed on Elaeis
IP
guianeensis palm wine isolates from Nigeria (Ezeronye and Legras, 2009) indicated a specific
SC R
population, and more recently the population genomic survey of S. cerevisiae diversity (Liti et al., 2009), revealed the existence of an African lineage for S. cerevisiae strains. The genotyping of these African yeast populations revealed that these are well differentiated from
NU
European (bread, wine, cheese) or Asian populations. Two main factors have been shown to
MA
explain yeast genetic diversity: isolation source and geography (Cromie et al., 2013; Legras et al., 2007). Despite being isolated in Burkina Faso, palm wine and Dolo isolates were clearly
D
differentiated. They were also different from those isolated on other palm wines, sorghum
TE
beers or Cocoa hulks isolates. However, the different strains isolated from sorghum beer in Burkina Faso, Ghana, and Ivory Coast, were clustered very closely in Discriminant Analysis
CE P
of Principal Components (DAPC) and in our tree, which suggests that these strains are related. This implies that isolation source is the main factor explaining population structure
AC
for Dolo. The inoculation of Dolo wort is achieved from a previous brew through different manners: directly by a fraction or the foam of a previous brew, or by a woven belt in which is trapped dried yeast cells from a former fermentation (Sefa-Dedeh et al., 1999). Human population migration and this specific inoculation may explain this yeast population structure. On the contrary to Dolo, isolates obtained from the different palm wines from Borassus akeassii Bayton in Burkina Faso, Elaeis guineensis in Nigeria and Ivory Coast, and Hyphaene thebaica in Djibouti republic, belong to different populations, as can be attested from the different clusters observed in DAPC analysis, and from the individuals tree. In addition, two other isolates from West Africa (NCYC110, and DBVPG6044), isolated from beverages prepared from other plants (Zingiber officinale and Osbeckia grandiflora), or strains isolated 10
ACCEPTED MANUSCRIPT from cocoa hulks in Ghana are not related to any of the former populations, suggesting a wide diversity of S. cerevisiae in Africa. The differences between palm wine S. cerevisiae
T
populations might result from geography but also from the palm tree these beverages are
IP
produced from. CBS400, an Elaeis guineensis palm wine isolate from Ivory coast is not
SC R
related to those isolated from those from Nigeria which suggests that geography may explain also part of the diversity of these populations, but should be confirm through a much wider sampling.
NU
Ploidy is another trait differentiating sorghum beer and palm wine isolates. A ploidy of 2n
MA
was observed for most palm wine isolates with some triploid or tetraploid strains, whereas sorghum beer isolates where mainly tetraploid. As bread or beer strains, which are not related to these sorghum beer strains are also polyploid, we can hypothesized that polyploidy may
TE
D
result from a selective pressure imposed by this starch based media, or by the continuous culture imposed by man caused by back sloping process (Al Safadi et al., 2010). All these
CONCLUSION
CE P
features outline the specific properties of these original African yeast populations.
AC
The present study provides first information on the diversity of Saccharomyces cerevisiae isolated from palm wine obtained from the fermented sap of B. akeassii in Burkina Faso. The characterization of the yeast population showed that B. akeassii palm wine contains a specific and diverse population of yeast, different from Dolo. But our work highlights as well the diversity of yeast populations used for African beverages, and our first results indicated that strains of these populations have different physiological properties. More work has to be done in order to understand how geographical, technological and historical factors have structured this diversity. The industrial potential value of these genetic resources has to be further evaluated for biotechnological applications. 11
ACCEPTED MANUSCRIPT ACKNOWLEDGEMENT The authors are grateful to Prof L. Jespersen, University of Copenhagen, for providing cacao
T
strains, and to Dr. S Casaregola, Cirm levures, Thiverval-Grignon, and Prof. M. Aigle
IP
(Université de Lyon 1) for providing Djibouti Republic palm wine and Dolo strains.
SC R
The corresponding author is grateful to International Foundation for Science (IFS), West African Economic and Monetary Union (UEMOA) for supporting.
NU
REFERENCES
Aidoo, K. E., Nout, M. J. R., Sarkar, P. K., 2006. Occurrence and function of yeasts in Asian
MA
indigenous fermented foods. FEMS Yeasts Res. 6:30–39. Al Safadi, R.,
eiss- ayet, M., Briolay, ., Aigle, M., 2010. A polyploid population of
TE
D
Saccharomyces cerevisiae with separate sexes (dioecy). FEMS Yeast Res. 10: 757–76 Amoa-Awua, W. K., Sampson, E., Tano-Debrah, K., 2006. Growth of yeasts, lactic and acetic
CE P
acid bacteria in palm wine during tapping and fermentation from felled oil palm (Elaeis guineensis) in Ghana. J. of Appl. Microbiol. 102 (2): 599-606.
AC
Bassir, O., 1962. Observations on the fermentation of palm-wine. West Afric.J. Biol. Appl. Chem. 6: 20 – 25.
Bayton, R. P., Ouédraogo, A., Guinko, S., 2006. The genus Borassus (Arecacea) in West Africa with a description of a new species from Burkina Faso. J. Linn Soc. 150: 419–427. Bayton, R. P., Ouédraogo, A., 2009. Discovering Africa’s newest palm. Palms. 53: 37–45. Camarasa, C., Sanchez I., Brial P., Bigey, F., Dequin, S., 2011. Phenotypic Landscape of Saccharomyces cerevisiae during Wine Fermentation: Evidence for Origin-Dependent Metabolic Traits. PLoS One. 6: e25147. 12
ACCEPTED MANUSCRIPT Clark, L. V., Jasieniuk, M., 2011. POLYSAT: A R package for polyploid microsatellite analysis. Mol. Ecol. Res. 11 (3): 562–66.
T
Cromie, G. A., Hyma, K. E., Ludlow, C. L., Garmendia-Torres, C., Gilbert, T. L., May, P.,
IP
Huang A. A., Dudley A. M., Fay, J. C., 2013. Genomic Sequence Diversity and Population
SC R
Structure of Saccharomyces cerevisiae Assessed by RAD-seq. Genes, Genom, Gen. 3: 21632171.
NU
Delobel, P., Tesnière C., 2014. A Simple FCM Method to Avoid Misinterpretation in Saccharomyces cerevisiae Cell Cycle Assessment between G0 and Sub-G1. PLoSONE. 9(1):
MA
e84645.
D
Esteve-Zarzoso, B., Belloch C., Uruburu, F., Querol A., 1999. Identification of yeasts by
TE
RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal
CE P
internal transcribed spacers. Int. J. of Systematic Bacteriol. 49: 329–337. Ezeronye, O. U., Legras, J. L., 2009. Genetic analysis of Saccharomyces cerevisiae strains
AC
isolated from palm wine in eastern Nigeria. Comparison with other African strains. J. of Appl. Microbiol.106: 1569–78. Haase, S. B., Reed, S. I., 2002. Improved flow cytometric analysis of the budding yeast cell cycle. Cell Cycle.1: 132–136. Heard, G. M., Fleet, G. H., 1986. Evaluation of selective media for enumeration of yeasts during wine fermentation. J. Appl. Microbiol. 60: 477–481. Jespersen, L., Dennis, S. N., Susanne H., Mogens, J., 2005. Occurrence and diversity of yeasts involved in fermentation of West African cocoa beans. FEMS Yeast Research. 5: 441–53. 13
ACCEPTED MANUSCRIPT Jombart, T., Devillard, S., Balloux, F., 2010. Discriminant analysis of principal components: a new method for the analysis of genetically structured populations. BMC Gen. 11:94.
T
Jombart, T., 2008. Adegenet: a R package for the multivariate analysis of genetic markers.
SC R
IP
Bioinfo. 24: 1403-1405.
Kayodé, A. P. P., Vieira-Dalodé, G., Linnemann, A. R., Kotchoni, S. O., Hounhouigan, A. J. D., Van Boekel, M. A. J. S., Nout, M. J. R. 2011. Diversity of yeasts involved in the
NU
fermentation of tchoukoutou, an opaque sorghum beer from Benin. Afr J. of Microbiol. Res.
MA
5: 2737-2742.
Konlani, S., Delgenes, J. P., Moletta, R., Traoré A., Doh, A., 1996. Isolation and
D
physiological characteristics of yeasts involved in sorghum beer production. Food Biotech.
TE
10: 29-40.
CE P
Legras, J. L., Merdinoglu, D., Cornuet, J. M., Karst, F., 2007. Bread, Beer and Wine, Saccharomyces cerevisiae diversity reflects human history. Mol. Ecol. 16: 2091–2102. Liti, G., Carter. D. M., Moses, A. M., Warringer, J., Parts, L., James, S. A., Davey, R. P.,
AC
Roberts, I. N., Burt, A., Koufopanou,V., Tsai, I. J., Bergman, C. M., Bensasson, D., O'Kelly, M. J., Van Oudenaarden, A., Barton, D. B., Bailes, E., Nguyen, A. N., Jones, M., Quail, M. A., Goodhead, I., Sims, S., Smith, F., Blomberg, A., Durbin, R., Louis, E. J., 2009. Population genomics of domestic and wild yeasts. Nat. 458: 337–341. Lõoke, M., Kristjuhan, K., Kristjuhan, A., 2011. Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques. 50: 325–8.
14
ACCEPTED MANUSCRIPT Lyumugabe, F., Gros, J., Nzungize, J., Bajyana, E., Thonart, P., 2012. Characteristics of African traditional beers brewed with sorghum malt: a review. Biotechnol. Agron. Soc.
T
Environ. 16(4): 509-530.
IP
Maoura, N., Mbaiguinam, M., Nguyen, H. V. Gaillardin, C., Pourquie, J., 2005. Identification
SC R
and typing of the yeast strains isolated from Bili Bili, a Traditional Sorghum Beer of Chad. Afr. J. of Biotech. 4 (7): 646–56.
NU
Miège, J., 1985. Palmales. In J. Bersani, J.Gall, H. Schweizer, and M. Lardy (ed.),
MA
Encyclopaedia universalis, 13:764-767. Encyclopaedia universalis publisher, Paris. Ogbulie, T. E., Ogbulie J. N., Njoku, H. O., 2007. Comparative study on the microbiology
D
and shelf life stability of palm wine from Elaeis guineensis and Raphia hookeri obtained from
TE
Okigwe, Nigeria. Afr. J. of Biotech. 6: 914-922.
CE P
Okafor, N., 1975. Microbiology of Nigeria palm wine with particular reference to bacteria. J. Appl. Bacteriol. 38: 81-88.
Ouoba, L. I. I., Kando C., Parkouda, C., Sawadogo-Lingani, H., Diawara, B., Sutherland, J.P.,
AC
2012. The microbiology of bandji, palm wine of Borassus akeassii from Burkina Faso: identification and genotypic diversity of yeasts, lactic acid and acetic acid bacteria. J. of Appl. Microbiol.113: 1364-5072. R Core Team, 2014. R: A language and environment for statistical computing, Version 3.1.2. R Foundation for Statistical Computing 2014. http://www.R-project.org. Santiago-Urbina, J. A., Ruíz-Terán, F., 2014. Microbiology and biochemistry of traditional palm wine produced around the world. Int. Food Res. J. 21: 1261-1269.
15
ACCEPTED MANUSCRIPT Sefa-Dedeh, S., Sanni, I., Tetteh, G., Sakyi-Dawson, E., 1999. Yeasts in the traditional brewing of pito in Ghana. World J Microbiol Biotechnol. 15: 593–597.
T
Stringini, M., Comitini, F., Taccari, M., Ciani, M., 2009. Yeast diversity during tapping and
SC R
IP
fermentation of palm wine from Cameroon. Food Microbiol. 26: 415–20.
Ukhum, M. E., Okolie, N. P., Oyerinde, A. O., 2005. Some mineral profiles of fresh and bottled palm wine – a comparative study. Afr. J. Biotech. 4: 829-32.
NU
Van der Aa Kühle, A., Jespersen, L., Glover, R. L., Diawara, B., Jakobsen, M., 2001.
MA
Identification and Characterization of Saccharomyces Cerevisiae strains isolated from West African Sorghum Beer. Yeast. 18: 1069–79.
D
Yaméogo, J., Bélem-Ouédraogo, M., Bayala, J., Ouédraogo, M. B., Guinko, S., 2008. Uses
TE
and commercialization of Borassus akeassii Bayton, Ouédraogo, Guinko non-wood timber
AC
Env. 12: 47-55.
CE P
products in South-Western Burkina Faso, West Africa. Biotechnologie, Agronomie, Société et
16
ACCEPTED MANUSCRIPT Figure legend : Figure 1: Genetic diversity of Burkina Faso palm wine and dolo S. cerevisiae isolates revealed
T
by microsatellite typing: neighbour joining dendrogramm obtained from the pairwise
AC
CE P
TE
D
MA
NU
SC R
IP
individual Bruvo’s distance matrix.
17
ACCEPTED MANUSCRIPT
Figure 2: Genetic diversity of different African S. cerevisiae strains from different origins
T
revealed by Discriminant Analysis of Principal Component (DAPC). a- representation of the
IP
different clusters with regard to the axis 1 and 2; b- representation of the different clusters
AC
CE P
TE
D
MA
NU
SC R
with regard to the axis 3 and 4.
18
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
19
AC
CE P
TE
D
MA
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
20
ACCEPTED MANUSCRIPT Table 1: Description of the Burkina Faso palm wine and dolo isolates, and of the African S. cerevisiae isolates genotyped in this study Origin
Isolates codes
Reference
PWBA2
PW7 ; PW18 ; PW34
PWBA3
PW14 ; PW21 ; PW29
PWBA4
PW2 ; PW19
PWBA 5
PW6 ; PW31
PWBA 6
PW28
PW1 ; PW8
PWBA 9
TE
PWBA 10
PW5 ; PW32 ; PW36 ; PW17
CE P
PWBA 11
AC
PW4 ; PW13 ; PW15 ; PW22 ; PW44
D
PWBA 8
Faso,
IP
PW27 ; PW26
NU
PWBA1
PWBA 7
Burkina Dolo
Isolates
SC R
Sampling site
MA
Burkina Faso, palm wine (Banfora)
T
Strains isolated in this study
PW35 PW33 ;
PW37 ;
PW38 ;
PW40 ; PW41 ; PW42 ; PW43
PWBA 12
PW3 ; PW16 ; PW23 ; PW30
PWBA 13
PW10 ; PW25
PWBA 14
PW20 ; PW24
Banfora
B1, B3, B4, B5
Ouagadougou Koudougou
PW39 ;
F2, F3 T2
African strains genotyped in this study Ivory Coast, Millet beer Ghana, cocoa isolates Djibouti Republic, wine
palm
CH02 (CliB1162) , CH06 (CliB1163), CH10 (CliB1164), CH11 (CliB1165), CH13 (CliB1167), CH14 (CliB1166) MTF2546, MTF2547, MTF2548, MTF2549, MTF2550, MTF2552, MTF2553, MTF2554, MTF2555, MTF2556, MTF2557, MTF2620 DJ65 (CliB1169), DJ66 (CliB1169), DJ71, DJ73, DJ74
Al Safadi et al., 2010 Jespersen et al., 2005 M. Aigle (un published data)
21
ACCEPTED MANUSCRIPT CBS: Centraalbureau voor Schimmelcultures (CBS) - Netherlands; MUCL: Louvain,
AC
CE P
TE
D
MA
NU
SC R
IP
T
Belgium; PW: yeasts isolated from different palm wines of B. akeassii.
22
ACCEPTED MANUSCRIPT Table 2: Biochemical and microbial characteristics of the different palm wines (after 72 hours of fermentation) used for Saccharomyces cerevisiae isolation
Mean ± SD
T
Biochemical characteristics
4.79 ± 0.12
Total titrable acidity (%)
0.66 ± 0.25
Ethanol (%)
3.69 ± 0.69
SC R
Lactic Acid (g/l)
3.40 ±1.30
Malate (g/l)
Acetate (g/l)
MA
Succinate (g/l)
0.60 ± 0.30
NU
Glucose (g/l) Fructose (g/l)
Glycerol (g/l)
D
Microbial characteristics TVC (*108 UFC/ml)
TE
IP
pH
2.60 ± 4.90 0.80 ± 0.50 2.70±1.80 2.40 ± 0.40 1.30 ±0.50 Mean±SD 1.89 ± 0.70 1.71 ± 1.40
NSY (*104 UFC/ml)
2.76 ± 1.80
CE P
TY (*106 UFC/ml)
TVC: Total viable counts; TY: Total yeast cells; NSY: Non-Saccharomyces yeasts; SD:
AC
Standard deviation
23
ACCEPTED MANUSCRIPT
Mean*
Palm Wine, Burkina Faso
35
2.2
Dolo, Burkina Faso
7
Dolo (Chapallo), Ivory Coast
6
Palm wine, Djibouti Republic
7
Dolo, Ghana
2
SC R NU
MA
Isolates origin
Palm Wine, Nigeria
1
IP
Number of isolates analysed
T
Table 3: DNA content of the different Saccharomyces cerevisiae isolates measured by flow cytometry
SD
0.5
2.6
0.9
4.1
0.3
2.7
0.5
3.5 2.8
AC
CE P
TE
D
*Significance of the differences between populations tested by a Kruskal-Wallis rank sum test (pvalue <0.0002) for the first four populations. SD: Standard deviation.
24
ACCEPTED MANUSCRIPT HIGHLIGTS
Borassus akeassii palm wines in Burkina Faso present a specific S. cerevisiae population as well as palm wines produced from other palm trees.
T
Saccharomyces cerevisiae isolated from Burkina, Ghana and Ivory Coast Dolo belong to a
CE P
TE
D
MA
NU
SC R
IP
single cluster of tetraploid strains.
AC
25