244
Microbial Population Dynamics during aging of Cheddar cheese
The dynamics of bacterial microflora changes in Cheddar cheese during ageing are largely unknown. Normally, the unwanted bacteria may die out and not survive through 60 days of storage of hard and semi-hard cheeses. However, the beneficial flavor producers such as starter and adjunct LAB may survive longer, as may some unwanted bacteria that have a competitive advantage due to shorter growth times and fewer nutritional requirements. Traditional estimates of bacterial populations depended on the ability to grow bacteria from cheese on specific media. While this is possible for the dominant bacterial species, it still does not define the breadth of the bacterial diversity adequately. A slightly broader picture of bacterial classes can be obtained by culture-independent techniques such as quantitative PCR analysis (qPCR) or phylogenetic microarrays, which are based on the levels of a gene of a particular organism estimated from DNA extracted from cheese. Challenges for DNA extraction such as interference from dairy components such as milk fat and protein in not allowing DNA separation and inability to lyse bacteria inside solid matrices have been tackled. We recently demonstrated that probiotic bacteria survive up to 6 mo of aging without population reduction by measuring their levels using qPCR. However, the high number of simultaneous assays needed to define the diversity of bacteria and the necessity for prior knowledge of bacteria that may exist in cheese are both limitations for qPCR’s or microarrays’ applicability to cheese. Currently, the bacterial ecology research arena largely depends on sequencing of amplified 16s ribosomal gene segments at a very high throughput to characterize the populations in diverse extreme environments. This approach surmounts some of the drawbacks of array-based technologies where we only target short (11 probes that are each 20-25 bases long), discontinuous portions of the gene sequences, whereas with sequencing, we seek to identify bacteria based on 200 base-long continuous DNA sequences. Our objective was to study the changes in bacterial populations in cheese during manufacture and aging using the 16s pyrosequencing approach. While lactococci and lactobacilli were the dominant microbial species (P<0.05) as expected, the presence of hitherto unknown species was also identified. This demonstrates that 16s DNA sequencing is a nonrestrictive approach towards surveying both known bacterial types and relatively unknown or previously unexpected cheese microflora members.
Keywords: Cheddar cheese, mciroflora, lactococcus, lactobacillus