Biomedical Engineering Reference
In-Depth Information
4.6.1
Determination of the Number of Lactic Acid Bacteria
and Yeasts
The standard plate count is used to estimate the cell density of both lactic acid bacteria
and yeasts. Suitable culture media to assess the number of sourdough lactic acid bacteria
include SDB (Sour Dough Bacteria) medium, as originally described by Kline and
Sugihara (
25
); or MRS (de Man, Rogosa, Sharpe) medium (
51
) , with modi fi cations
concerning the pH and the nutrient composition, particularly to include maltose and
fructose as carbon sources and electron acceptors, respectively, and cysteine as reducing
agent (
26-
28
); Homohiochii medium, especially recommended to enumerate obligately
heterofermentative lactic acid bacteria (
29
); SFM (San Francisco medium), containing
wheat or rye bran especially useful for lactic acid bacteria as well as lactobacilli (
30
) .
To all the above media cycloheximide (100 ppm) can be added to inhibit the
growth of yeast when requested.
Yeast are generally enumerated on WL medium (
31
) ; YPDA medium (
32,
33
) ;
Sabouraud dextrose agar (
34,
35
) ; YGC agar (
14
); and Malt extract agar (
36
) .
To inhibit bacterial growth, all the above media generally contain 50-100 ppm of
chloramphenicol.
4.6.2
Phenotypic and Genetic Analyses to Identify and Type
Lactic Acid Bacteria and Yeasts
Sourdough lactic acid bacteria and yeasts are generally identified using a polyphasic
approach, combining both phenotypic and genotypic assays.
Besides morphological and physiological tests, specific biochemical assays are
available to identify at species level
Weissella
spp
.
,
Pediococcus
spp
.
and
Enterococcus
spp. as well as the predominant sourdough lactic acid bacteria,
which are often members of the genus
Lactobacillus
(
19
). In that case, the minia-
turized commercial test (e.g. API 50 CHL, Biomerieux, France) or automated
assay (e.g. Biolog system) are frequently applied after a preliminary evaluation
based on cell morphology, Gram stain, catalase test, growth at 15°C and 45°C,
CO
2
production from glucose, and NH
3
release from arginine (
37
) . Nevertheless,
the high biochemical diversity in the above-mentioned genus, means that the
identification scheme must include some chemotaxonomic analysis (e.g. SDS-
PAGE,
Sodium Dodecyl Sulphate-PolyAcrylamide Gel Electrophoresis
of total or
cell-wall associated proteins) (
38,
39
) or, mainly, genotypic tests such as 16S
rRNA gene sequencing (
30
). Moreover, on the basis of specifically designed prim-
ers targeting species-specific sequences, many rapid identification systems rely-
ing on PCR (e.g. multiplex-PCR, Intergenic Spacer Region-based PCR) have
been recently applied to sourdough lactic acid bacteria identification (
27,
40,
41
) .
Among genotypic systems, PCR-RAPD
(Random Ampli fi ed Polymorphic DNA)
as well as RFLP
(Restriction Fragment Length Polymorphism)
or PFGE (
Pulsed
Field Gel Electrophoresis
) have been successfully applied for the evaluation of
intra-specific differences (e.g. strain typing) (
38,
42,
43
) .
