identification and Population Dynamics of Yeasts in Sourdough Fermentation Processes by PCR-Denaturing Gradient Gel Electrophoresis
Christiane B. Meroth, Walter P. Hammes, and Christian Hertel*
Institute of Food Technology, University of Hohenheim, D-70599 Stuttgart, Germany
*Corresponding author. Mailing address: Institute of Food Technology, University of Hohenheim, Garbenstr. 28, D-70599 Stuttgart, Germany. Phone: 49 711 459 4255. Fax: 49 711 459 4199. E-mail:
hertel@uni-hohenheim.de.
Received May 30, 2003; Accepted September 15, 2003.
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Abstract
Four sourdoughs (A to D) were produced under practical conditions, using a starter obtained from a mixture of three commercially available sourdough starters and baker's yeast. The doughs were continuously propagated until the composition of the microbiota remained stable. A fungi-specific PCR-denaturing gradient gel electrophoresis (DGGE) system was established to monitor the development of the yeast biota.
The analysis of the starter mixture revealed the presence of Candida humilis, Debaryomyces hansenii, Saccharomyces cerevisiae, and Saccharomyces uvarum. In sourdough A (traditional process with rye flour), C. humilis dominated under the prevailing fermentation conditions. In rye flour sourdoughs B and C, fermented at 30 and 40°C, respectively, S. cerevisiae became predominant in sourdough B, whereas in sourdough C the yeast counts decreased within a few propagation steps below the detection limit. In sourdough D, which corresponded to sourdough C in temperature but was produced with rye bran, Candida krusei became dominant. Isolates identified as C. humilis and S. cerevisiae were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. The yeast species isolated from the sourdoughs were also detected by PCR-DGGE. However, in the gel, additional bands were visible. Because sequencing of these PCR fragments from the gel failed, cloning experiments with 28S rRNA amplicons obtained from rye flour were performed, which revealed Cladosporium sp., Saccharomyces servazii, S. uvarum, an unculturable ascomycete, Dekkera bruxellensis, Epicoccum nigrum, and S. cerevisiae. The last four species were also detected in sourdoughs A, B, and C.
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Characterization of complex microbiota as they occur in food fermentation processes is facilitated by the development and application of sensitive and powerful molecular methods but is still a challenge. The microorganisms contributing to the characteristic properties of the food during the course of the fermentation process should be known in order to allow control of the process by selection of the appropriate technological condition and by using defined cultures. In a previous study, we reported the monitoring of lactic acid bacterium (LAB) population dynamics during the fermentation process in four continuously propagated sourdoughs by a LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) system (25). PCR-DGGE detects the 90 to 99% most numerous species of a community without discriminating living from dead cells or cells in a noncultivable state. The study revealed fluctuations within the LAB population, and under different ecological conditions, characteristic species prevailed.
Because yeasts fulfill several important *func tion s in bread making, the knowledge of their composition is also essential (15). They contribute to leavening (38) and produce metabolites such as alcohols, esters, and carbonyl compounds which contribute to the development of the characteristic bread flavor (7, 9, 16, 20, 21). Furthermore, the enzymatic activities of yeasts by enzymes such as proteases, lecithinase, lipases, α-glucosidase, β-fructosidase, and invertase have an influence on the dough stickiness and rheology as well as on the flavor, crust tonalidad, crumb texture, and firmness of the bread (2, 6, 24). As these activities are species or even strain specific, a special interest arose to control the yeast biota by adjusting the fermentation conditions to the ecological requirements of the desired microorganisms.
In studies of the sourdough yeast microbiota, traditional cultivation methods in combination with phenotypic (physiological and biochemical) and/or genotypic (randomly amplified polymorphic DNA [RAPD]-PCR and restriction fragment length polymorphism [RFLP] analysis) identification methods have commonly been used (8, 10, 19, 28, 31). These studies focused on the characterization of ripe doughs and revealed the presence of 23 yeast species belonging especially to the genera Saccharomyces and Candida (5, 27, 32). No data are available on the competitiveness of yeasts; thus, the effects of ecological factors and process conditions on the development of yeast biota during sourdough fermentation processes are virtually unknown.
To gain insight into the role of yeasts, we monitored changes of yeast population dynamics during sourdough fermentation processes by investigating samples of four previously described sourdoughs (25). For this purpose, a fungi-specific PCR-DGGE system based on the 28S rRNA gene was established. In addition, strains of the various yeast species were isolated by culturing and were identified phenotypically and by partial 28S rRNA sequencing. Their origins were traced back to the s