A molecular approach to monitor the dynamic changes in the main populations involved in fermentation of Italian sausages was used. This approach exploited the potential of PCR to amplify, with suitable primers, regions conserved within the domain Eubacteria,
as well as the discriminatory power of DGGE to differentiate DNA molecules on the basis of differences in their sequences (20
). Fermentation of sausages is a well-known microbial process, and ecological studies during ripening date back to the 1970s (22
). These studies, based on traditional methods, described the changes in populations during ripening. LAB are the main population involved in the decrease in pH. Also involved are representatives of the Micrococcaceae
, which neutralize the organic acids from LAB activity, produce peptides and amino acids due to their proteolytic activity, and induce the release of various aromatic substances related to their ability to produce lipases (10
In the last few years the possibility of using molecular approaches and direct sampling of the DNA and/or RNA in complex microbial systems has opened up areas of research that were already being studied but were not completely understood because of the biases related to the traditional methods. With traditional techniques only easily culturable organisms are counted, and often microorganisms for which selective enrichment and subculturing is problematic or impossible cannot be characterized.
In this paper we describe a PCR-DGGE protocol for detecting the microbial changes during natural fermentation of sausages. The first step was optimization of the method by using standard cultures obtained from international collections to determine the experimental conditions for amplification by PCR and differentiation by DGGE. Different sets of primers were selected from those available and used for the PCR-DGGE analysis. Only primers P1 and P2 (19
) were considered suitable for obtaining good differentiation among Lactobacillus
, and Kocuria
spp. without band comigration for different species. In mixed populations, individual members were identified by PCR-DGGE when the concentrations were more than 104
CFU/g, which allowed detection of species at a threshold level during fermentation (data not shown). Results were obtained with two different denaturant gradients in the DGGE gels. A 30 to 50% denaturant gradient was optimal for differentiation of Lactobacillus
spp., whereas gels with a 40 to 60% denaturant gradient could be used to distinguish the Staphylococcus
The method was used to monitor the population dynamics during natural fermentation of sausages. Both DNA and RNA were sampled directly in order to determine the levels of expression of the 16S rDNA of the most prominent bacteria, which may reflect their contributions to the fermentation process. Gels were visually inspected to identify the bands representing the populations involved in the fermentation. To circumvent the biases inherent in subjective interpretation, the presence of the bands was confirmed by direct sequencing. When the results obtained from both traditional plating and DGGE were analyzed, it became evident that the fermentation was characterized by strong LAB activity. In DNA and RNA DGGE gels, multiple bands were visible for the first 3 days of fermentation, when different species, most of which were related to Staphylococcus spp., were identified. From the 10th day of maturation only the LAB bands were present. The main difference detected by sampling RNA rather than DNA was the presence of natural meat contaminants, such as Brochothrix thermosphacta, Enterococcus sp., Leuconostoc mesenteroides, and Brevibacillus sp., which were not present after the third day. Staphylococcus species, recognized as proteolytic agents due to their ability to produce proteases, were found only in the meat mixture before sausages were filled and after 3 days. The only Staphylococcus species represented in the DGGE gel after 3 days was S. xylosus, which produced a specific band in the gel until the end of fermentation. It is important to emphasize that a corresponding S. xylosus band was not found when the RNA amplicons were analyzed, since band 12 was present only at zero time and 3 days and then disappeared. This could be explained by the large quantity of LAB RNA, which restricted amplification of RNA from different species present.
The presence of multiple copies of the rRNA operon, as described previously for other microorganisms (8
), made evaluation of the profiles obtained from single cultures difficult, but it did not affect interpretation of the fingerprints obtained from the total DNA and RNA extracted directly from sausages during fermentation.
The profiles obtained by DGGE agreed with the results obtained by traditional methods. The LAB population was the largest population during fermentation. The LAB strains isolated at the different steps of fermentation were all identified by PCR-DGGE as L. sake
and L. curvatus
. These results were in complete agreement with the profiles obtained when both DNA and RNA amplicons were analyzed, where the bands identified as Lactobacillus
belonged to the two species mentioned above. When the DNA was sampled, a single band referred to L. plantarum
was found only at zero time. The PCR product was probably generated from dead cells, since no L. plantarum
cells were isolated at zero time and no specific signal was detected in the RNA amplicons. Moreover, the specific band obtained from DNA disappeared after the first sampling. S. xylosus
might have been the only Staphylococcus
species present, as previously described by other authors (5
), justifying the band present in the gels in which DNA was analyzed.
The characteristic increase in pH that follows the initial decrease due to acid production by LAB is usually caused by proteolytic activity attributed mainly to endogenous muscle cathepsins in the initial phase (29
) and to the ability of staphylococci to produce proteases in the second stage (6
). In our opinion, in this study the increase in pH after 10 days of ripening could not be explained by the Micrococcaceae
activity because of the low number of cells present but could be explained by attributing an extracellular proteinase activity to LAB. As previously described (12
), L. sake
and L. curvatus
are able to use muscle sarcoplasmatic proteins as substrates, which results in peptide production that could play a role in the increase in the pH. L. sake
and L. curvatus
are the only two species isolated from sausages that remained stable throughout the fermentation, and they were responsible for the proteolytic activity that resulted in a final pH of 5.56.
The DGGE approach was first used in environmental ecology studies, such as sea sediments (23
), hot springs (13
), or wastewater treatment plants (14
), and in studies of the populations present in the rumen (21
) or gastrointestinal contents (30
). Only in the last year was the same approach used to study microbial systems such as food fermentation, in which many microorganisms are difficult to cultivate or are thought to be nonculturable. DGGE has been used to monitor the microbial dynamics during production of the Mexican fermented maize dough pozol (2
) and to monitor the dynamic changes during wine fermentation (4
). By applying the method to the natural fermentation of sausages, we were able to determine that LAB, represented by L. sake
and L. curvatus
, were the main organisms responsible for the physical and organoleptic changes that occurred during fermentation of the sausages tested. Micrococcaceae
strains had restricted importance during production compared to LAB. Their high levels and acid production made the LAB the only active population, as determined by both DNA and RNA DGGE analyses, in transformation of fermented sausages from Friuli Venezia Giulia, making them potential starter cultures for this kind of production. Moreover, the ability to monitor the population by PCR-DGGE could provide real time information concerning the state of fermentation. Since the results are available 8 h after sampling, immediate technological adjustments can be made when they are required.