Abstract
Study on the biodiversity and characterization of yeasts isolated
from natural environments of Cameroon and Namibia
Stringini Marzia
Corso di Dottorato di Ricerca in
Scienze Biomolecolari Applicate, VII ciclo
Universita Politecnica delle Marche
Keys words: yeast - biodiversity- ecology - palm wine - tropical environmental enrichment cultures - PCR-DGGE - molecular methods - molecular typing extracellular enzymatic activity - killer yeasts - proteolytic -β-glucosidase - amylase chitinase activity – yeasts fermentation
In the present study, we have investigated the occurrence of yeast flora on
several agricultural products coming from crop-growing environments in Cameroon,
to provide better knowledge of the biodiversity of yeast flora, and to thus define the
impact of this biodiversity on food products. The yeast biodiversity was investigated
using traditional culture-dependent methods, along with culture-independent
methods. The culture-dependent approach was carried out using both direct and
enrichment procedures, to detect the broadest possible presence of yeast species.
A total of 151 strains belonging to 26 different yeast species were isolated and
identified using restriction pattern analysis of the internal transcribed spacer region
5.8S-ITS and sequence analysis of D1/D2 domain of 26S rRNA gene. The
enrichment isolation procedures carried out in high-sugar media allowed the
recognition of fermentative species such as Saccharomyces cerevisiae and
Torulaspora delbrueckii, which have previously not been detected using direct
isolation methodology.
The results of culture-independent method using DGGE patterns and sequencing of
the DNA bands revealed a lower number of yeast species when compared with the
culture-dependent methodology even if allowed the identification of several yeast
species not detected by traditional microbiological procedures such as Candida
tropicalis and Hanseniaspora uvarum. Thus, these multiphasic approaches to study
yeast biodiversity (culture-dependent and -independent methods) have allowed us to
get a more complete picture of the microbial diversity in these natural environments.
Subsequently, we have investigated the occurrence of yeast flora during
tapping and fermentation of palm wine from Cameroon. The yeast diversity was
investigated using both traditional culture-dependent and culture-independent
methods. Moreover, to characterize the isolates of the predominant yeast species
(Saccharomyces cerevisiae) at the strain level, primers specific for δ sequences and
minisatellites of genes encoding the cell wall were used. The results confirm the
broad quantitative presence of yeast, lactic acid bacteria and acetic acid bacteria
during the palm wine tapping process, and highlight a reduced diversity of yeast
species using both dependent and independent methods. Together with the
predominant species S. cerevisiae, during the tapping of the palm wine the other
species found were Saccharomycodes ludwigii and Zygosaccharomyces bailii. In
addition, denaturing gradient gel electrophoresis (DGGE) analysis detected
Hanseniaspora uvarum, Candida parapsilopsis, Candida fermentati and Pichia
fermentans. In contrast to the progressive simplification of yeast diversity at the
species level, the molecular characterization of the S. cerevisiae isolates at the strain
level showed a wide intraspecies biodiversity during the different steps of the tapping
process. Indeed, 15 different biotypes were detected using a combination of three
primer pairs, which were well distributed in all of the samples collected during the
tapping process, indicating that a multistarter fermentation takes place in this
particular natural, semi-continuous fermentation process.
The final objective of this research has been to investigate the extracellular
enzymatic activity profile of yeasts isolated from tropical environments of Cameroon
and Namibia. This screening survey could constitute the first approach in selecting
yeast strains of environmental origin potentially exploitable as enzyme producers. In
this study, 181 yeasts (151 isolated from crop-growing environments in Cameroon
representing 26 different yeast species and, 30 isolated from Namibia, representing 7
yeast species) were screened for amylases, proteases, β-glucosidase, chitinases and
killer activity.
The results demonstrated that, a higher number of yeasts exhibited proteolytic
activity and, a lower number of yeast species showed amylases (C. solani, Kodamaea
laetipori, L. elongisporus, P.guilliermondii, T. delbrueckii e S. cerevisiae), βglucosidase (Aureobasidium pullulans, S. cerevisiae, Kodamaea laetipori, L.
elongisporus, P.guilliermondii e S. cerevisiae), killer and chitinases (only in one
yeast strain) activity. The extracellular protease production was observed in P.
anomala, D. anomala, P. kluyeri, T. delbrueckii, R. mucilaginosa, Y. lipolytica, C.
montana, C. apicola, P. fermentans, P. guilliermondii, S. cerevisiae, Kodamaea
laetipori, L. elongisporus, Cryptococcus albidus.