Application of in-depth taxonomy, metagenomics, and cell viability assessment of bacterial communities of the dairy chain

Nella microbiologia lattiero-casearia, la ricerca è passata dallo studio del comportamento di singoli microrganismi in situazioni controllate allo studio di comunità batteriche complesse e alla loro interrelazione con il sistema circostante. In questa tesi sono stati studiati tre concetti chiave: lo studio della micro-diversità microbica, analizzando un genere specifico “Microbacterium” nel latte microfiltrato, il microbiota complesso del latte crudo e la vitalità cellulare nel siero-innesto naturale. I tre approcci utilizzati sono complementari per comprendere le dinamiche di processo e i fenomeni nelle produzioni lattiero casearie. Il genere Microbacterium è stato trovato prevalentemente nel latte fresco microfiltrato con una capacità di crescita a basse temperature. Inoltre, una nuova specie è stata scoperta e classificata con il nome Microbacterium paulum. Il profilo chimico e microbiologico misurato con tecniche omiche del latte crudo ha rivelato un'influenza del sistema di alimentazione che riguarda principalmente i metaboliti secondari e alcune unità tassonomiche significative, tra cui Pseudomonas e Staphylococcus. Infine, lo studio dello stato di salute della comunità lattica presente nel siero-innesto ha permesso la scoperta di un metodo rapido per valutare le prestazioni tecnologiche del siero-innesto sostituendosi alle tecniche basate su metodi di coltura in piastra.In dairy microbiology, research has moved from the study of the behavior of single microorganisms in milk in controlled situations to the study of complex bacterial communities and their interrelationship with the surrounding system. Three key concepts were studied in this thesis: the study of microbial micro-diversity by analyzing a specific Microbacterium genus in microfiltered milk, the complex microbiota of raw milk, and cell viability in natural whey starter. The three aims addressed were complementary to understand the process dynamics in milk and related productions. The genus Microbacterium was found predominantly in fresh microfiltered milk with an ability to grow at low temperatures. In addition, a new species was discovered more present and classified as Microbacterium paulum. The chemical and microbiological profile measured by omics techniques of raw milk revealed a feeding system influence mainly affecting secondary metabolites and some significant taxonomic units, including Pseudomonas and Staphylococcus. Finally, the study of the health status of the lactic community present in a natural whey starter has allowed the discovery of a rapid method to evaluate the technological performance of the whey by replacing the culture-dependent techniques

Case Study on the Microbiological Quality, Chemical and Sensorial Profiles of Different Dairy Creams and Ricotta Cheese during Shelf-Life

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

Application of flow cytometry for rapid bacterial enumeration and cells physiological state detection to predict acidification capacity of natural whey starters

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

A Combined Metabolomic and Metagenomic Approach to Discriminate Raw Milk for the Production of Hard Cheese

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 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

Microbacterium: adapted bacteria to microfiltrated milk

Microfiltration is a widespread technology applied to reduce the bacterial content during milk treatment. It is usually associated to pasteurization to further extend the shelf life of milk. The aim of this work was to evaluate the presence of bacteria in microfiltrated milk from the production process to the end of the shelf life. MATERIAL AND METHODS Milk samples were collected in two different plants located in Italy. Multiple time points were considered during the milk processing of the first plant, along with three different seasons. Samples from the second plant s were collected from freshly packaged at different time points. Additional samples were provided at the retail store. The following analyses were carried out for each sample: Total Bacterial Count (TBC), Bacillus cereus, Enterobacteriaceae (using enrichment medium). The colonies (about 10% of the plate) were randomly selected from the plates, purified by streaking on MPCA and then isolates were identified using the 16S rRNA gene Sanger sequencing. The proteolytic and lipolytic activity of Microbacterium isolates were evaluated using spotting technique on milk and tributyrin agar plates . Biofilm production was evaluated by streaking the strains isolated on Congo Red Agar (CRA) medium. The antibiotic resistance profile was determined according to CLSI standard methods. Moreover, bacterial growth capability in milk was also assessed at 8°C

Comparative genomics of Halobacterium salinarum strains isolated from salted foods reveals protechnological genes for food applications

Archaeal cell factories are becoming of great interest given their ability to produce a broad range of value-added compounds. Moreover, the Archaea domain often includes extremophilic microorganisms, facilitating their cultivation at the industrial level under nonsterile conditions. Halophilic archaea are studied for their ability to grow in environments with high NaCl concentrations. In this study, nine strains of Halobacterium salinarum were isolated from three different types of salted food, sausage casings, salted codfish, and bacon, and their genomes were sequenced along with the genome of the collection strain CECT 395. A comparative genomic analysis was performed on these newly sequenced genomes and the publicly available ones for a total of 19 H. salinarum strains. We elucidated the presence of unique gene clusters of the species in relation to the different ecological niches of isolation (salted foods, animal hides, and solar saltern sediments). Moreover, genome mining at the single-strain level highlighted the metabolic potential of H. salinarum UC4242, which revealed the presence of different protechnological genes (vitamins and myo-inositol biosynthetic pathways, aroma- and texture-related features, and antimicrobial compounds). Despite the presence of genes of potential concern (e.g., those involved in biogenic amine production), all the food isolates presented archaeocin-related genes (halocin-C8 and sactipeptides)