The chemical composition of milk can be significantly affected by different factors across the dairy supply chain, including primary production practices. Among the latter, the feeding system could drive the nutritional value and technological properties of milk and dairy products. Therefore, in this work, a combined foodomics approach based on both untargeted metabolomics and metagenomics was used to shed light onto the impact of feeding systems (i.e., hay vs. a mixed ration based on hay and fresh forage) on the chemical profile of raw milk for the production of hard cheese. In particular, ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF) was used to investigate the chemical profile of raw milk (n = 46) collected from dairy herds located in the Po River Valley (Italy) and considering different feeding systems. Overall, a total of 3320 molecular features were putatively annotated across samples, corresponding to 734 unique compound structures, with significant differences (p < 0.05) between the two feeding regimens under investigation. Additionally, supervised multivariate statistics following metabolomics-based analysis allowed us to clearly discriminate raw milk samples according to the feeding systems, also extrapolating the most discriminant metabolites. Interestingly, 10 compounds were able to strongly explain the differences as imposed by the addition of forage in the cows’ diet, being mainly glycerophospholipids (i.e., lysophosphatidylethanolamines, lysophosphatidylcholines, and phosphatidylcholines), followed by 5-(3′,4′-Dihydroxyphenyl)-gamma-valerolactone-4′-O-glucuronide, 5a-androstan-3a,17b-diol disulfuric acid, and N-stearoyl glycine. The markers identified included both feed-derived (such as phenolic metabolites) and animal-derived compounds (such as lipids and derivatives). Finally, although characterized by a lower prediction ability, the metagenomic profile was found to be significantly correlated to some milk metabolites, with Staphylococcaceae, Pseudomonadaceae, and Dermabacteraceae establishing a higher number of significant correlations with the discriminant metabolites. Therefore, taken together, our preliminary results provide a comprehensive foodomic picture of raw milk samples from different feeding regimens, thus supporting further ad hoc studies investigating the metabolomic and metagenomic changes of milk in all processing conditions.

Pseudomonas fluorescens is a psychrotrophic species associated with milk spoilage because of its lipolytic and proteolytic activities. Consequently, monitoring P. fluorescens or its antecedent activity in milk is critical to preventing quality defects of the product and minimizing food waste. Therefore, in this study, untargeted metabolomics and peptidomics were used to identify the changes in milk related to P. fluorescens activity by simulating the low-temperature conditions usually found in milk during the cold chain. Both unsupervised and supervised multivariate statistical approaches showed a clear effect caused by the P. fluorescens inoculation on milk samples. Our results showed that the levels of phosphatidylglycerophosphates and glycerophospholipids were directly related to the level of contamination. In addition, our metabolomic approach allowed us to detect lipid and protein degradation products that were directly correlated with the degradative metabolism of P. fluorescens. Peptidomics corroborated the proteolytic propensity of P. fluorescens-contaminated milk, but with lower sensitivity. The results obtained from this study provide insights into the alterations related to P. fluorescens 39 contamination, both pre and post heat treatment. This approach could represent a potential tool to retrospectively understand the actual quality of milk under cold chain storage conditions, either before or after heat treatments.

A novel Gram-stain-positive, strictly aerobic, short rod-shaped bacterium, designated 2CT, was isolated from freshly packaged microfiltered milk. This strain was able to grow within the NaCl concentration range of 0–5 % (w/v), temperature range of 8–37 °C (optimally at 30 °C) and at pH 6.0–10.0. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 2CT was closely related to species of the genus Microbacterium , with the highest sequence similarity (99.2 %) to Microbacterium lacticum DSM 20427T as well as Microbacterium flavum DSM 18909T (=YM18-098T). The phylogenetic tree based on 16S rRNA genes showed that strain 2CT clustered with M. flavum DSM 18909T. However, the phylogenetic tree based on concatenated 16S rRNA and four housekeeping genes showed that strain 2CT clustered with M. lacticum DSM 20427T. Furthermore, the phylogenomic tree showed that strain 2CT clustered with M. lacticum DSM 20427T and M. flavum DSM 18909T. The major respiratory quinones were MK-10, MK-11 and MK-12. The predominant cellular fatty acids were anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The polar lipid composition of strain 2CT consisted of diphosphatidylglycerol, phosphatidylglycerol, three unidentified glycolipids and two unidentified lipids. The cell-wall peptidoglycan type was a variant of B1α {Gly} [l-Lys] d-Glu-l-Lys, with the amino acids lysine, glycine, alanine and glutamic acid. The whole-cell sugars consisted of galactose, glucose, ribose and minor amounts of rhamnose. In addition, strain 2CT showed a glycolyl-type cell wall. The genomic DNA G+C content was 69.8mol%, while the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values with the closely related Microbacterium species were below the recognized thresholds of 95–96 % ANI and 70 % DDH for species definition. Based on the phenotypic and genotypic data, strain 2CT (=LMG 32277T=CECT 30329T) is considered to represent a new species, for which the name Microbacterium paulum sp. nov. is proposed.

This work investigated the microbiological quality and chemical profiles of two different dairy creams obtained by centrifugation vs. natural creaming separation systems. To this aim, an untargeted metabolomics approach based on UHPLC-QTOF mass spectrometry was used in combination with multivariate statistical tools to find potential marker compounds of the two different types of two dairy creams. Thereafter, we evaluated the chemical, microbiological and sensorial changes of a ricotta cheese made with a 30% milk cream (i.e., made by combining dairy creams from centrifugation and natural creaming separation) during its shelf-life period (12 days). Overall, microbiological analysis revealed no significant differences between the two types of dairy creams. On the contrary, the trend observed in the growth of degradative bacteria in ricotta during shelf-life was significant. Metabolomics revealed that triacylglycerols and phospholipids showed significant strong down-accumulation trends when comparing samples from the centrifugation and natural creaming separation methods. Additionally, 2,3-Pentanedione was among the best discriminant compounds characterising the shelf-life period of ricotta cheese (VIP score = 1.02), mainly related to sensorial descriptors, such as buttery and cheesy. Multivariate statistics showed a clear impact of the shelf-life period on the ricotta cheese, revealing 139 potential marker compounds (mainly included in amino acids and lipids). Therefore, the approach used showed the potential of a combined metabolomic, microbiological and sensory approach to discriminate ricotta cheese during the shelf-life period.

Natural whey starter cultures are undefined microbial communities mainly consisting of thermophilic lactic acid bacteria (LAB). The technological pressure that shapes the natural whey starter community before and during the back-slopping procedure can impact the amount and viability of the different thermophilic LAB. Traditional culture-dependent analytical methods are useful for evaluating natural whey cultures based on plate enumeration with various culture media and are commonly used as self-control procedures in dairy items. These methods have high variability and require days to obtain results. As the dairy industry has been searching for a solution to this problem for a long time, researchers must explore alternative methods for the technological evaluation of natural whey and assessment of the health status of the thermophilic acidifying bacteria community in the cheesemaking process. The flow cytometry approach has been considered an alternative to classical methods in this work sector. This study compared bacterial enumeration by plate counting and flow cytometry on natural whey samples. Flow cytometry results showed positive agreement with a tendency to overestimate, linearity, and correlation with plate counting. Other parameters have also been introduced for evaluating a natural whey starter, measuring the physiological state of the cells. Specifically, cell-wall damage and metabolic activity were also evaluated, allowing us to quantify the number of cells even in sub-optimal physiological conditions.

Four strains, representing two novel Bifidobacterium species, were isolated from water kefir, a fermented beverage. 16S rRNA gene analysis suggested that the novel species share high identities (98.82–98.89%) with Bifidobacterium aquikefiri LMG 28769^T. Complete genomes were assembled with a short- and long-read hybrid sequencing approach. In agreement with the 16S rRNA gene analysis, phylogenetics with 117 marker genes places the novel species closest to B. aquikefiri LMG 28769^T as well. The isolates have average nucleotide identity (ANI) scores ranging from 81.46 to 84.84% and digital DNA–DNA hybridization (dDDH) scores from 23.9 to 38.5% with the closest related species, as well as ANI scores between the proposed new species of 80.50%, indicating that the isolates represent two novel species. Matrix-assisted laser desorption/ionization-time of flight chemotaxonomic analysis supported the gene-based taxonomic placement. We propose the names Bifidobacterium fermentum sp. nov. and Bifidobacterium aquikefiricola sp. nov. for these novel species within the Bifidobacterium genus. The proposed type strain B. fermentum WK012_4_13^T (= LMG 33104^T = DSM 116073^T; GenBank accession number GCF_041080835.1) has a genome size of 2.43 Mbp, with a G+C content of 56.00 mol%. The proposed type strain for B. aquikefiricola WK041_4_12^T (= LMG 33105^T = DSM 116074^T; GenBank accession number GCF_041080795.1) has a genome size of 2.36 Mbp and a G+C content of 53.94 mol%. B. fermentum cells are Gram-positive staining, non-motile, non-spore-forming, fructose-6-phosphate phosphoketolase (F6PPK)-positive, catalase- and oxidase-negative and bacillary club shaped. B. aquikefiricola cells are Gram-positive staining, non-motile, non-spore-forming, F6PPK-positive, catalase- and oxidase-negative and square rod shaped.