Download the contribution of molecular methods for a better comprehension of

Biodiversity and dynamics of meat
fermentations: the contribution of
molecular methods for a better
comprehension of a complex microbial
ecosystem
Luca Cocolin
Di.Va.P.R.A., Università di Torino, via Leonardo
da Vinci 44, 10095 Grugliasco, Torino
[email protected]
Main microorganisms involved in the fermentation of
the sausages and responsible for the sensory
characteristics of the final product
Yeasts
Lactobacillus spp.
Pediococcus spp.
Candida spp.
Debaryomyces spp.
Bacteria
Staphylococcus spp.
Kocuria spp.
Penicillium spp.
Moulds
Experimental approaches to study the ecology of
fermented sausages
Pyrosequencing
Significance of molecular targets in the study of
fermented sausages microbiota
RNA
DNA
DIVERSITY
ACTIVITY
Viable not culturable
state?
In situ molecular methods in the study of fermented
sausages microbiota
Methods
Denaturing Gradient Gel
Elecrophoresis (DGGE)
Single Strand
Conformation
Polymorphism (SSCP)
Fluorescence in Situ
Hybridization (FISH)
Quantitative Polymerase
Chain Reaction (qPCR)
Microarrays
Aims
Diversity and
activity
Diversity and
activity
Activity and
localization
In situ gene
expression
DGGE separation
PCR products with different
nucleotide sequence
B
C
D
E
+
Denaturants
Electric field
A
B
C
D
E
A
LAB
CNC
Klijn et al. 1991, AEM, 57, 3390-3393
Direct profiling of microbial populations during the
natural fermentation of sausages
Use of molecular methods for strain
characterization
Differentiation of strains belonging to
the same species
Definition of subclones or
subpopulation
Important in strain selection for the
production of commercial starters
RFLP, RAPD,
ERIC, REP,
Sau-PCR
C
L
U
47%
45%
36%
39%
Lb. plantarum
S. xylosus
Exploitation of molecular methods to assess
the capability of CNC to dominate sausage
fermentations
Alternative starter cultures with respect to S.
xylosus and S. carnosum
Staphylococcus vitulinus
Macrococcus caseolyticus
Alternative starter cultures with respect to S.
xylosus and S. carnosum
M. caseolyticus
S. vitulinus
Sv
Mc
Mc
Commercial starter
Cs
Conclusions
• The PCR-DGGE protocol allows differentiation of members of
Lactobacillus, Staphylococcus and Kocuria spp.
• A direct monitoring of the fermentation is possible
• The protocol can be applied to identify strains that have been
isolated by culture dependent methods or can be directly
performed in food
• The method can be performed in 8 hours and it represents a
reliable tool to follow “on line” the transformation process
• By applying these methods new information became available
on the ecology and behavior of micro-organisms in food
• They offer essential tools to carry out multiphasic approaches
to study micro-organisms in food
• They cannot substitute traditional methods but only improve the
results obtained !!!
Acknowledgements
Grants:
- PRIN (ex 40%) - Ministry of University, Rome, Italy
- EU Project (V FP) - Safety on traditional fermented
sausages: development of protective cultures and
bacteriocins
- EU Project (Leonardo da Vinci) - MeatTips
Thanks to:
Production plans:
- Kalliopi Rantsiou
- Paola Dolci
- Rosalinda Urso
- Giuseppe Comi
- Salumificio Luvison Snc
- Salumificio Uanetto Snc
- Raspini Spa
www.etat.gr/meat-tips/