Handbook of Meat Processing

186 Chapter 9

is not taken into consideration. This weak-

ness can be addressed by the application of

techniques such as PCR - DGGE, which

instead of relying on culturing of the bacteria,

incorporates the direct extraction of the DNA

from the food sample. Furthermore, another

technique that has not yet been applied in

fermented sausages is fl uorescence in situ

hybridization (FISH). In this technique, the

direct detection of a microorganism in a food

sample is achieved by using specifi c probes

that allow spatial distribution studies to take

place.

The microorganisms that are most fre-

quently encountered are Lactobacillus curva-

tus , Lb. plantarum , Lb. sakei , Staphylococcus

carnosus , St. saprophyticus , and St. xylosus

(Table 9.1 ). These microorganisms seem to

be autochthonous in this ecosystem and have

the capacity to prevail during fermentation.

The competitiveness of Lb. sakei has been

new, powerful, and reliable techniques has
enabled the detailed study of this complex
ecosystem. Table 9.1 lists techniques used
for the identifi cation of technological micro-
biota in fermented sausages throughout the
world. The phenotypic approach (i.e., the
identifi cation based on assimilation – fermen-
tation – growth challenges) has been the fi rst
to be applied and is still in use, despite its
drawbacks regarding reliability and accu-
racy, even at species level. The most fre-
quently applied and most reliable technique
is sequencing of the 16S - rRNA gene. This
technique has been applied either in combi-
nation with other techniques, such as SDS -
PAGE of whole cell proteins, RAPD - PCR,
or PFGE, or directly to the isolated microor-
ganism. Although this approach provides
accurate identifi cation at strain level, an
equally important part of the microbiota,
namely the viable but not culturable fraction,

Table 9.1. Microbial diversity in spontaneously fermented sausages throughout the world

Species Origin of spontaneously

fermented sausages

Identifi cation approach

Lb. alimentarius Greece

Hungary

Sequencing of 16S - rRNA gene 5

PCR - DGGE 7

Phenotypic 10

Lb. bavaricus Hungary Phenotypic 10

Lb. brevis Greece

Croatia

Italy

Spain

Species specifi c PCR 1

Phenotypic 10

PCR - DGGE 1, 16

SDS - PAGE - sequencing of 16S - rRNA gene 20

Lb. casei Italy Species specifi c PCR 1

PCR - DGGE 1

RAPD - PCR — sequencing of 16S - rRNA gene 21

Lb. casei/paracasei Greece Sequencing of 16S - rRNA gene 5

PCR - DGGE 7

Lb. cellobiosus Serbia Phenotypic 10

Lb. collinoides Serbia Phenotypic^10

Lb. curvatus Italy

Greece

Hungary

Croatia

Bosnia and Herzegovina

Spain

Argentina

Species specifi c PCR 1, 12

PCR - DGGE 1, 3, 7, 14, 15, 16, 22, 23

Sequencing of 16S - rRNA gene 5

Phenotypic 10

RAPD - PCR - Species specifi c PCR 13

RAPD - PCR - sequencing of 16S - rRNA gene 14, 21

SDS - PAGE - sequencing of 16S - rRNA gene 20

Lb. delbrueckii spp.

bulgaricus

Serbia Phenotypic 10