Full-Length SSU rRNA Gene Sequencing Allows Species-Level Detection of Bacteria, Archaea, and Yeasts Present in Milk.

Full-length SSU rRNA gene sequencing allows species-level identification of the microorganisms present in milk samples. Here, we used bulk-tank raw milk samples of two German dairies and detected, using this method, a great diversity of bacteria, archaea, and yeasts within the samples. Moreover, the species-level classification was improved in comparison to short amplicon sequencing. Therefore, we anticipate that this approach might be useful for the detection of possible mastitis-causing species, as well as for the control of spoilage-associated microorganisms. In a proof of concept, we showed that we were able to identify several putative mastitis-causing or mastitis-associated species such as Streptococcusuberis, Streptococcusagalactiae, Streptococcusdysgalactiae, Escherichiacoli and Staphylococcusaureus, as well as several Candida species. Overall, the presented full-length approach for the sequencing of SSU rRNA is easy to conduct, able to be standardized, and allows the screening of microorganisms in labs with Illumina sequencing machines.

Amplicon-sequencing of raw milk microbiota: impact of DNA extraction and library-PCR.

The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy® PowerFood® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers. KEY POINTS: • Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota. • Reduction of eukaryotic DNA by enzymatic digestion. • Shift of detected microbiome caused by high cycle numbers in library-PCR.

Fundicoccus ignavus gen. nov., sp. nov., a novel genus of the family Aerococcaceae isolated from bulk tank milk.

Three strains of a Gram-stain-positive, catalase-negative, facultative anaerobic, and coccoid species were isolated from German bulk tank milk. Phylogenetic analyses based on the 16S rRNA gene sequences indicated that the three strains (WS4937T, WS4759 and WS5303) constitute an independent phylogenetic lineage within the family Aerococcaceae with Facklamia hominis CCUG 36813T (93.7-94.1 %) and Eremococcus coleocola M1831/95/2T (93.5 %) as most closely related type species. The unclassified strains demonstrated variable growth with 6.5 % (w/v) NaCl and tolerated pH 6.5-9.5. Growth was observed from 12 to 39 °C. Their cell-wall peptidoglycan belongs to the A1α type (l-Lys-direct) consisting of alanine, glutamic acid and lysine. The predominant fatty acids were C16 : 1 ω9c, C16 : 0 and C18 : 1 ω9c and in the polar lipids profile three glycolipids, a phospholipid, phosphatidylglycerol, phosphoglycolipid and diphosphatidylglycerol were found. The G+C content of strain WS4937T was 37.4 mol% with a genome size of ~3.0 Mb. Based on phylogenetic, phylogenomic and biochemical characterizations, the isolates can be demarcated from all other genera of the family Aerococcaceae and, therefore, the novel genus Fundicoccus gen. nov. is proposed. The type species of the novel genus is Fundicoccus ignavus gen. nov., sp. nov. WS4937T (=DSM 109652T=LMG 31441T).

Foam-stabilizing properties of the yeast protein PAU5 and evaluation of factors that can influence its concentration in must and wine.

The absence of the yeast protein seripauperin 5 (PAU5) from Saccharomyces cerevisiae has been suggested as a biomarker for the occurrence of gushing in sparkling wine as samples lacking PAU5 were found to be more susceptible to gushing. In this study, further characterization of PAU5 regarding its foam-stabilizing properties was performed to elucidate whether PAU5 has foam-stabilizing properties and therefore, to elucidate a direct influence on the gushing potential of sparkling wines. PAU5 was successfully purified from non-gushing sparkling wine using reversed-phase high-performance liquid chromatography (RP-HPLC). Pure protein was added to grape juice as a model system for grape must prior to foam stability testing. The results revealed that the protein PAU5 has foam-stabilizing properties. Furthermore, the influence of heat and sulfur treatment in the presence of Botrytis cinerea was analyzed with regard to the amount of PAU5 produced by S. cerevisiae fermented in grape juice. Fermentation experiments using two different S. cerevisiae strains were performed, and the concentration of PAU5 in the samples was compared by RP-HPLC analysis. Unlike sulfur treatment, heat treatment prevented the protein degradation induced by B. cinerea and resulted in even higher amounts of PAU5 compared to the juice fermented with yeast without a previous botrytization. The two different yeast strains applied secreted PAU5 into the surrounding medium in different amounts. In further experiments, the fining process of the wine with bentonite was examined for its potential to remove PAU5 from the wine. RP-HPLC of wines processed with different fining agents revealed that bentonite treatment affected PAU5 concentrations in the final product. The extent of PAU5 removal depended on the type of bentonite applied and on the time of addition during the production process.