Yunita, Dewi
(2016)
The role of non-starter bacterial consortia in mould-ripened cheese.
PhD thesis, University of Nottingham.
Abstract
Stichelton is a blue-veined raw milk cheese which is made following the Stilton cheese making process. In a previous study, a preliminary examination of its microflora during production was examined by traditional culture methods and initial PCR DGGE profiling. The aim of this study was to complete the profiling of Stichelton cheese and examine the contribution of its microbiota components to product characteristics.
Stored samples of cheese production isolates were sequenced and in addition whole population DNA was extracted directly from a fully ripened Stichelton cheese (12 weeks) and from bulk cell suspensions collected on various media. The V3, V4V5 and V6V8 regions of 16S rDNA were amplified by PCR and separated by DGGE using 20 – 80 % urea formamide denaturing gradient. While Lactobacillus casei/paracasei, Staphylococcus equorum, Bacillus sp., Brevibacterium sp., Halomonas sp., Acinetobacter sp., Alkalibacterium sp. and Corynebacterium casei were only found by the molecular method, traditional culture detected a large number of potentially raw milk microbiota.
Lactococcus lactis was detected in the raw milk sample and along the process by both methods. The L. lactis subsp. lactis which was detected in the core of matured Stichelton was shown by PFGE to be from the raw milk and not the starter culture used in Stichelton production.
S. equorum was found in the crust of cheese pre-piercing and in all parts of the cheese post-piercing by the molecular approach only. This suggested this organism was introduced originally via handling. Five S. equorum isolated from Stilton, Danish Blue and Reblochon could grow up to 10% salt but did not tolerate low pH levels suggesting S. equorum in Stichelton must have been introduced by handling before or during ripening as if it was present in the early fermentation then it would die as fermentation progressed due to pH sensitivity.
A model cheese system made with commercial UHT milk was used to examine the interaction between mixed Lc. lactis, P. roqueforti and S. equorum B2 isolated from the Stilton crust. S. equorum was either added 1.5 h after the addition of L. lactis or it was smeared on the surface of the cheese immediately after un-moulding. The viable counts and pH were analysed throughout the process, while texture, water activity and flavour volatiles using GCMS SPME were determined for one month ripened cheeses only. The results showed S. equorum survived in the cheese following either method of introduction and that in cheeses without P. roqueforti addition, the presence of incorporated and surface-spread S. equorum could inhibit the surface growth of a contaminant Penicillium. It also slowed the growth of starter P. roqueforti in cheeses made with this mould. A paler coloured crust, firmer textured cheese and a low amount of alcohols were shown in the model cheeses made with surface-smear S. equorum. Conversely, addition of S. equorum in the initial process made the cheese core softer and produced low amounts of acids. Ethanol, 3-methyl-1-butanol and 2-pentanone were the main flavour compounds in the model cheeses examined.
The antifungal activity of the isolate was confirmed in laboratory media. Its ability to prevent Penicillium surface growth could be beneficial for white cheeses where this is an undesirable flaw. The results showed that the sporulation inhibitory effect on P. roqueforti was because of an antifungal agent produced by S. equorum, but it was not acid, bacteriocin or H2O2. Further study is needed to detect the antifungal agent. Overall, the study has expanded the understanding of the role non-starter bacteria may have in contributing to cheese ripening.
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