Monitoring lactococcal phages during milk fermentation

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Slavin, Rebecca
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University College Cork
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Complex undefined starter cultures are widely used for the production of Dutch- style cheese such as Gouda and Edam. They exhibit more resistance to phage infection in comparison to defined starter cultures. Thus, of recent interest in the dairy research area is the search for potential phage-resistant strains from complex blends, which are more compatible to the dairy industry expectations. This includes isolating strains with inherent phage resistant properties. A commercially available, LD, mesophilic, complex blend, referred to as Blend X in this thesis, was deconstructed and characterised by two approaches. A culture independent approach was employed to isolate 43 strains from the blend, followed by an extensive genotypic characterisation to classify the strains at subspecies level, but also to genetically distinguish closely related strains. Routine methods such as 16S rRNA sequencing confirmed the Lactococcus lactis species. This was followed by use of the histidine operon PCR to confirm subspecies lactis and cremoris. Further genotypic differentiation was supported through sequencing of the purR gene, a core gene encoding a purine biosynthesis repressor. Interestingly, this approach resulted not only in differentiation of the subspecies but also in differentiation of the biovariant diacetylactis. Subsequent sequencing of the soft-core epsD gene of these isolates allowed differentiation at strain level. A combination of sequence data from the purR and epsD analysis allowed the identification of 12 genetic lineages in Blend X. The presence of lactococcal phages in the fermentation industry can have detrimental effect on both the acidification capacity and the quality of the final product. Blend X is employed in many production plants around Europe; therefore, it is not unusual for the blend to encounter problems regarding phage contamination. Ten whey samples from problematic production vats were collected, from which 40 phages were isolated and purified. Restriction analysis revealed a high degree of genetic diversity amongst the 40 phages, all of which were of the 936 phage species. By determining the host range relationship, two L. lactis subsp. lactis bv. diacetylactis phage-resistant strains were recovered from Blend X Finally, the population dynamics of Blend X over fermentation and in the presence of phage during fermentation was monitored using a targeted amplicon sequencing approach. For this experiment, two narrow host range phages derived from the ten whey samples were selected to target specific strains within Blend X, and a reconstructed Blend X (RBX), created from a strains isolated from the complex blend, where a strain from each genetic lineage was selected. The phages were spiked into a Blend X and RBX fermentation and the population dynamics of both of the blends was monitored by a culture- independent approach, by targeting three genes; the 16S rRNA gene, purR and epsD. Targeted sequencing of the 16S rRNA gene allowed discrimination of microbial composition to genus level only. This revealed that in Blend X, Leuconostoc species are present at 17% at the beginning of fermentation and present at 25% at the end of fermentation. In comparison, targeting the purR gene enabled us to distinguish the lactoccocal strains at a subspecies level, determining that the L. lactis subsp. cremoris is the dominant strain throughout the fermentation. The presence of phages appeared to make no impact on the population dynamics at a genus or subspecies level but a shift in population dynamics was observed at strain level. Targeted sequencing of the soft core epsD gene identified 60 OTUs in the blend and revealed shifts in microbial composition at OTU level in the presence of narrow host-range phages. Substantial differences where observed from the start to the end of fermentations, where phages were infected into the blends to target specific lineages. Thus, targeting the epsD gene resulted in an effective method to monitor the impact of lactoccocal phage during milk fermentation.
Bacteriophage , Starter cultures
Slavin, R. 2019. Monitoring lactococcal phages during milk fermentation. MRes Thesis, University College Cork.
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