Biomedical Engineering Reference
In-Depth Information
5.3
Isolation of Sourdough Yeasts and Lactic Acid Bacteria
5.3.1
Isolation of Sourdough Yeasts
Yeast isolation from mature sourdoughs is relatively uncomplicated as a stable
sourdough usually harbors a homogeneous yeast population as part of the resident
microbiota. However, the follow-up of different developmental stages of a sour-
dough or the search for minor components requires a strategy that is optimized
towards the detection of subdominant components. Detailed information on food
yeast isolation was provided by Deak [
197
]. Here, only a brief discussion of the
currently practiced yeast isolation methods from sourdough is given, including
some references to more complete methodological resources. After sample homog-
enization and dilution, yeast growth is suitably effected on solid media. This
sequence of manipulations should be performed with minimal delay to avoid the
settling of yeast cells and cell death. Diluents, usually distilled water, peptone water,
saline, or Ringer solution, may influence the resulting cell counts, with peptone
water having resulted in the highest cell recovery [
198
] . Overviews of classical
growth media and isolation techniques for yeasts and foodborne yeasts are given by
Yarrow [
199
] and Beuchat [
200
], respectively. The cultivation media used in sour-
dough analyses are rich media containing complex compounds such as peptone
(e.g., Sabouraud agar), tryptone (e.g., Wallerstein Laboratory nutrient agar), yeast
extract (e.g., yeast extract peptone dextrose agar), malt (e.g., yeast and malt extract
agar, wort agar), and potato infusion (e.g., potato dextrose agar), together with an
additional component to inhibit bacterial growth. Most often chloramphenicol is
used as an antibiotic that can be added to the medium before sterilization without
losing its activity. Acidification of the medium is sometimes used to restrict bacte-
rial growth, while this is known to affect growth of some yeasts (namely of the
genus
Schizosaccharomyces
). However, the acidity of the sourdough lets it appear
unlikely for acid-sensitive strains to be present in a ripe sourdough. In general, most
yeasts show good vegetative growth at room temperature, although some may grow
at subzero temperatures and others up to 45 °C [
201
] . Cardinal growth temperatures
of yeasts are species- and strain-specific. The most suitable incubation temperature
for sourdough yeasts would be the temperature at which the sourdough is in its most
active state. The most frequently applied temperatures are 25-30 °C. First yeast
growth can under such conditions usually be observed after 2-3 days, while daily
inspection of the plates for 5 up to 10 days is recommended to allow full differentia-
tion of colony morphology and detection of more slowly growing components.
Other than the consideration of growth conditions, the selection of yeast colonies
for further characterization and identification is the most important factor influencing
the completeness of a diversity survey. Even though sourdough samples often pres-
ent a homogeneous yeast population, one needs to bear in mind that the colony
morphology of different yeast species is often very similar. Each observed morpho-
type should therefore be sampled more than once. A logical strategy would be to
recover a number of colonies of each type that represents a reasonable percentage
