Abstract
Microbial communities play an important role in cheese ripening and determine the flavor and taste of different cheese types to a large extent. However, under adverse conditions human pathogens may colonize cheese samples during ripening and may thus cause severe outbreaks of diarrhoea and other diseases. Therefore in the present study we investigated the bacterial community structure of three raw ewe's milk cheese types, which are produced without the application of starter cultures during ripening from two production sites based on fingerprinting in combination with next generation sequencing of 16S rRNA gene amplicons. Overall a surprisingly high diversity was found in the analyzed samples and overall up to 213 OTU97 could be assigned. 20 of the major OTUs were present in all samples and include mostly lactic acid bacteria (LAB), mainly Lactococcus, and Enterococcus species. Abundance and diversity of these genera differed to a large extent between the 3 investigated cheese types and in response to the ripening process. Also a large number of non LAB genera could be identified based on phylogenetic alignments including mainly Enterobacteriaceae and Staphylococcacae. Some species belonging to these two families could be clearly assigned to species which are known as potential human pathogens like Staphylococcus saprophyticus or Salmonella spp. However, during cheese ripening their abundance was reduced. The bacterial genera, namely Lactobacillus, Streptococcus, Leuconostoc, Bifidobacterium, Brevibacterium, Corynebacterium, Clostridium, Staphylococcus, Thermoanerobacterium, E. coli, Hafnia, Pseudomonas, Janthinobacterium, Petrotoga, Kosmotoga, Megasphaera, Macrococcus, Mannheimia, Aerococcus, Vagococcus, Weissella and Pediococcus were identified at a relative low level and only in selected samples. Overall the microbial composition of the used milk and the management of the production units determined the bacterial community composition for all cheese types to a large extend, also at the late time points of cheese ripening.
Highlights
Cheeses of raw ewe’s milk are generally characterized by a stronger taste and a richer flavor compared to cheeses made from pasteurized milk [1,2]
Croatian raw ewe’s milk cheeses are hard cheeses that are produced by traditional techniques without pasteurization and application of starter cultures, and they are characterized by an aging process of 90–120 days
T-RFLP analysis of cheese bacterial communities based on 16S rRNA gene amplicons
Summary
Cheeses of raw ewe’s milk are generally characterized by a stronger taste and a richer flavor compared to cheeses made from pasteurized milk [1,2]. Croatian raw ewe’s milk cheeses are hard cheeses that are produced by traditional techniques without pasteurization and application of starter cultures, and they are characterized by an aging process of 90–120 days This aging time is rather long and the low pH and water activity (aw) of ripened ewe’s milk cheeses usually does not support the growth of pathogens if the cheeses are completely mature [4]. In comparison to culture dependent methods, culture independent methods based on DNA/RNA analysis are less time consuming, more sensitive, more specific and more accurate They have not often been used to study microbial diversity in food matrices, except for fingerprinting approaches based on 16S rRNA gene amplification flowed by denaturing gradient gel electrophoresis (DGGE) or clone library construction [6,9,14,15,16]
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