Host DNA depletion methods and genome-centric metagenomics of bovine hindmilk microbiome.

Bovine mastitis is a multi-etiological and complex disease, resulting in serious economic consequences for dairy farmers and industry. In recent years, the microbiological evaluation of raw milk has been investigated in-depth using next-generation sequencing approaches such as metataxonomic analysis. Despite this, host DNA is a major concern in the shotgun metagenomic sequencing of microbial communities in milk samples, and it represents a big challenge. In this study, we aimed to evaluate different methods for host DNA depletion and/or microbial DNA enrichment and assess the use of PCR-based whole genome amplification in milk samples with high somatic cell count (SCC) by using short- and long-read sequencing technologies. Our results evidenced that DNA extraction performed differently in terms of host DNA removal, impacting metagenome composition and functional profiles.. Moreover, the ratio of SCC/bacteria ultimately impacts microbial DNA yield, and samples with low SCC (SCC below 100,000 cells/mL) are the most problematic. When milk samples with high SCC (SCC above 200,000 cells/mL) underwent multiple-displacement amplification (MDA), we successfully recovered high-quality metagenome-assembled genomes (MAGs), and long-read sequencing was feasible even for samples with low DNA concentration. By associating MDA and short-read sequencing, we recovered two times more MAGs than in untreated samples, and an ongoing co-infection not reported by traditional methods was detected for mastitis pathogen. Overall, this new approach will improve the detection of mastitis-associated microorganisms and make it possible to examine host-microbiome interactions in bovine mastitis.IMPORTANCENext-generation sequencing technologies have been widely used to gain new insights into the diversity of the microbial community of milk samples and dairy products for different purposes such as microbial safety, profiling of starter cultures, and host-microbiome interactions. Milk is a complex food matrix, and additionally, the presence of host nucleic acid sequences is considered a contaminant in untargeted high-throughput sequencing studies. Therefore, genomic-centric metagenomic studies of milk samples focusing on the health-disease status in dairy cattle are still scarce, which makes it difficult to evaluate the microbial ecophysiology of bovine hindmilk. This study provides an alternative method for genome-centric metagenome studies applied to hindmilk samples with high somatic cell content, which is indispensable to examining host-microbiome interactions in bovine mastitis.

Interactions between Starmerella bacillaris and Saccharomyces cerevisiae during sequential fermentations influence the release of yeast mannoproteins and impact the protein stability of an unstable wine.

Wine protein haze formation is a problem due to grape proteins aggregation during wine storage. The cell wall components of wine yeasts, particularly high molecular weight mannoproteins, have a protective effect against haze formation, although their involvement remains poorly understood. This study aimed at characterizing glycosylated proteins released by Starmerella bacillaris and Saccharomyces cerevisiae during single and sequential fermentations in a synthetic must, and testing their impact on wine protein stability. Mannoproteins-rich extracts from sequential fermentations showed an increase in the low MW polysaccharide fraction and, when added to an unstable wine, had a greater effect on protein stability than S. cerevisiae extracts. Shotgun proteomics approaches revealed that the identified cell wall proteins exclusively found in sequential fermentations were produced by both S. bacillaris (MKC7, ENG1) and S. cerevisiae (Bgl2p). Moreover, sequential fermentations significantly increased the expression of Scw4p and 1,3-beta-glucanosyltransferase (GAS5), produced by S. cerevisiae. Finally, some of the key proteins identified might play a positive role in increasing wine protein stability.

Kombuchas from Green and Black Tea Modulate the Gut Microbiota and Improve the Intestinal Health of Wistar Rats Fed a High-Fat High-Fructose Diet.

The Western diet can negatively affect the gut microbiota and is associated with metabolic disorders. Kombucha, a tea fermented by a symbiotic culture of bacteria and yeast (SCOBY), is known for its bioactive properties and has become popular in the last years. In this study, we evaluated the effects of regular kombucha consumption on the gut microbiota and on outcomes related to the intestinal health of Wistar rats fed a high-fat high-fructose diet. After eight weeks receiving a standard diet (AIN-93M) (n = 10) or a high-fat and high-fructose diet (HFHF) (n = 30) to induce metabolic disorders, the animals were subdivided into four groups: AIN-93M (n = 10); HFHF (n = 10); GTK (HFHF + green tea kombucha (n = 10); and BTK (HFHF + black tea kombucha; n = 10) for 10 weeks. Although body composition did not differ among the groups, the HFHF diet was associated with metabolic alterations, and stimulated the growth of gram-negative bacteria such as Proteobacteria and Bacteroides. Kombucha ingestion could somewhat modulate the gut microbiota, attenuating the effects of a Western diet by increasing propionate production and favoring the growth of beneficial bacteria, such as Adlercreutzia in the GTK group. Our results suggest that regular kombucha consumption may be beneficial to intestinal health, which can be mostly attributed to its high content and diversity of phenolic compounds.

In vitro Probiotic Potential and Anti-cancer Activity of Newly Isolated Folate-Producing Streptococcus thermophilus Strains.

Most probiotic strains commercially available today are lactic acid bacteria. Within this functional group, Streptococcus thermophilus is a thermophilic species widely used as starter culture for a huge number of dairy products. Besides being rapid acidifiers, many S. thermophilus strains are able to produce and release folate during growth but, unfortunately, they are seriously impaired during passage through the human gastrointestinal tract. In this work, we studied eight S. thermophilus strains isolated from dairy environments in Italy, which already had shown good technological properties, to evaluate their possible probiotic potential and cytotoxicity against cancer cells in vitro. All strains were also evaluated for some health-related properties such as susceptibility to most common antibiotics, hemolytic activity, resistance to simulated gastrointestinal conditions, bile salts hydrolytic activity, production of folate, adhesion to HT-29 human colorectal adenocarcinoma cells and cytotoxic activity against cancer cells and production of biogenic amines. Results revealed that two fast acidifying S. thermophilus strains were found to possess in vitro probiotic properties along with anticancer activity and production of folate. These properties resulted similar and, in some cases, superior to those of Lactobacillus rhamnosus GG, a well-known commercial probiotic strain. These findings encourage further in vivo studies to evaluate the actual health benefits of these strains on the human host.

The Complete Genome Sequence of Trueperella pyogenes UFV1 Reveals a Processing System Involved in the Quorum-Sensing Signal Response.

We present here the complete genome sequence of Trueperella pyogenes UFV1. The 2.3-Mbp genome contains an extremely interesting AI-2 transporter and processing system related to the quorum-sensing signal response. This specific feature is described in this species for the first time and might be responsible for a new pathogenic behavior.

Draft Genome Sequence of the Yeast Starmerella bacillaris (syn., Candidazemplinina) FRI751 Isolated from Fermenting Must of Dried Raboso Grapes.

Starmerella bacillaris is an ascomycetous yeast commonly present in enological environments. Here, we report the first draft genome sequence of S. bacillaris FRI751, which will facilitate the study of the characteristics of this interesting enological yeast.