Cultureindependent Approaches

Identification at genus/species level can be achieved by using culture-independent techniques such as PCR-DGGE/TGGE/SSCP. These methods have the advantage of providing identification and monitoring of a microbiota at species level without isolating the microorganisms on culture media. Instead of isolating bacteria of each sample from milk, natural starter, intermediate of production or the final cheese, direct DNA extraction can be achieved to provide a mixture of nucleic acids from most of the microorganisms present in the original dairy matrix. PCR amplification is subsequently required, and the most commonly employed target for identification at species level is the DNA encoding for ribosomal RNA. In most cases, for the identification of Bacteria, portions of the 16S rRNA gene are used. The 16S rRNA gene is conserved and allows the development of PCR primers that can be used for all Bacteria. However, it also contains variable regions, whose variability of sequence is species-specific in most cases. Therefore, the result of PCR amplification will be a portion of the 16S rRNA gene from all the microbial species whose DNA was extracted in situ, and the sequence of the amplicon is likely to vary from species to species. This sequence variation will allow separation of the fragments according to formation of discrete regions of thermal (TGGE) or chemical (DGGE) denaturation or the formation of different single strand conformations (SCCP), but the final output of the analysis will always be a fingerprint. The fingerprint will be made of a number of bands corresponding (in most but not all cases) to as many microbial species and will represent the microbiological identity of the milk, starter, intermediate of production or cheese analyzed. The final identification of each species can then be obtained by the purification and sequencing of each band and by comparison with the available data bases (Gene Bank, www.ncbi.nlm.nih. gov/blast/).

2.1.1 Diversity and Dynamics of Natural and Selected Starter Cultures

The microbes occurring in the cheese may arise from the raw milk, from the environment and tools of production, or can be added as selected starter cultures under controlled conditions. Another interesting possible source of bacteria exists in natural starter cultures, currently employed in much traditional cheese making where fermentation is assured by the back-slopping of milk or whey cultures from previous preparations.

The studies of selected and natural starter cultures share common interests such as the knowledge of the fate of microorganisms present in the culture at the beginning of fermentation, and their interaction with background microbiota. These interactions are recognized to be fundamental in selecting the microorganisms actually dominating the process and contributing to the principal rheological and sensorial attributes of the cheese. In addition, the study of the accessory microbiota is also important since the bacteria occurring at lower loads, along with the dominant bacteria, can potentially contribute to the development of product flavor and taste, thanks to specific metabolic pathways (Beresford, et al. 2001).

Of the above-cited fingerprinting techniques the most commonly employed in dairy microbiology is PCR-DGGE. We will be giving some examples of how this has been used to study the diversity and dynamics of microbial communities in cheese production.

The PCR-DGGE approach has been exploited to directly identify microbial species occurring in natural whey cultures (NWCs) used as starter for water buffalo Mozzarella cheese manufacture (Ercolini, et al. 2001). Both thermophilic and mesophilic LAB were identified by sequencing of the V3 region of the 16S rRNA

gene from DGGE fragments of NWCs profiles, namely Lb. delbrueckii, Lb. crispatus, Lactococcus (L.) lactis and Streptococcus (St.) hemophilus. Moreover, the occurrence of contaminants such as Alishewanella fetalis could also be highlighted. In the same study, a novel PCR-DGGE approach was developed to rapidly check the diversity of the bacterial community after cultivation on specific or non-specific culture media. Briefly, after colony counting has been performed, the colonies from the plates can be collected in "bulks" and subjected to DNA extraction and PCR-DGGE analysis (Ercolini, et al. 2001). Consequently, a DGGE fingerprint can be obtained for each plate, dilution, and culture medium. This method to investigate the cultivable microbial community has been shown to have good potential in food microbiology (Ercolini, et al. 2001; Ercolini, et al. 2003a; Ercolini, et al. 2004; Ercolini 2004). Firstly, it provides an alternative to traditional tools for identification. Qualification of the dominant species could be achieved by sequencing of the DGGE bands arising from the patterns corresponding to the highest dilutions, in spite of the isolation of single colonies followed by purification and biochemical identification (Ercolini 2004). Analysis of DGGE profiles obtained from bulk cells provides an image of the cultivable community, while simultaneously allowing ecological information to be obtained. Ercolini, et al. (2001) counted a population of mesophilic streptococci of 108 cfu/ml in NWCs for Mozzarella cheese production, but realized, after bulk PCR-DGGE analysis of all the dilutions, that the only species reaching the value of 108 was the thermophilic St. thermophilus and that mesophilic cocci were only present at levels of 104 cfu ml-1.

PCR-DGGE fingerprinting can also be useful to trace process dynamics during cheese making. The approach can be used to track the starter during production by examining the fingerprints of samples from raw material, through intermediate of production until the final product. This is important in both traditional and industrial dairy production. In the latter case the use of selected starter cultures ensures controlled fermentation and a standard quality of the final product. Analysis of DGGE fingerprints of the samples during manufacture can be important to ascertain that the starter culture is actually dominating the fermentation and can be of help in highlighting the occurrence of contaminating bacteria. On the other hand, in the case of traditional cheese production, one can trace the evolution of the contributing microbiota during the whole production and assess whether the raw milk or the natural whey/milk culture microflora actually contributes to cheese production. It can also show which microbial species of the natural starter survives fermentation, processing and the possible stresses imposed by the technology of production (pH, thermal stress, etc.). In a recent study the fate of the natural whey culture for the manufacture of traditional water buffalo Mozzarella cheese was investigated by PCR-DGGE (Ercolini, et al. 2004). The analysis of DGGE fingerprints from the intermediate samples during cheese production was shown to be useful to check the natural starter effectiveness and to determine the contribution of different groups of LAB during fermentation leading to the final Mozzarella cheese. All the DGGE profiles of dairy samples taken during manufacture were analyzed: raw milk, NWC, raw milk after NWC addition, curd before and after ripening, drained whey, stretched curd and final product (Ercolini, et al. 2004). A single glance at the succession of the fingerprints (Fig. 2.1) explains all that occurred in the process: the raw milk had a complex profile, but none of the species occurring in the milk were present in the profiles of the other samples. As soon as the NWC was added to the milk, the profile changed into the NWC fingerprint, which was displayed by all further samples until the final water buffalo Mozzarella cheese (Fig. 2.1). In other words, in this specific manufacture, the NWC was the main performer in the fermentation, giving high loads of bacteria to the raw milk, concealing the raw milk microbiota in the fingerprints, but probably giving strength to the fermentation and allowing the process to be properly carried out in respect of tradition. In this case, the microbial succession could be registered as "pictures" of microbial groups involved in premium quality production. This procedure may find useful applications for the monitoring of non-premium quality products where poor quality arises from the lack of development of the NWC. This procedure can be easily applied to dairy plants, allowing process development and starter effectiveness to be checked by analyzing dairy samples by PCR-DGGE. In comparison with traditional culture-dependent microbiological analyses, molecular approaches can be considered a step forward for the innovation of tracing systems in food technology, and may play an important role in the quality control of traditional

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