Francesca De Filippis

A Selected Core Microbiome Drives the Early Stages of Three Popular Italian Cheese Manufactures

Mozzarella (M), Grana Padano (GP) and Parmigiano Reggiano (PR) are three of the most important traditional Italian cheeses. In the three cheese manufactures the initial fermentation is carried out by adding natural whey cultures (NWCs) according to a back-slopping procedure. In this study, NWCs and the corresponding curds from M, GP and PR manufactures were analyzed by culture-independent pyrosequencing of the amplified V1–V3 regions of the 16S rRNA gene, in order to provide insights into the microbiota involved in the curd acidification. Moreover, culture-independent high-throughput sequencing of lacS gene amplicons was carried out to evaluate the biodiversity occurring within the S. thermophilus species. Beta diversity analysis showed a species-based differentiation between GP-PR and M manufactures indicating differences between the preparations. Nevertheless, all the samples shared a naturally-selected core microbiome, that is involved in the curd acidification. Type-level variability within S. thermophilus species was also found and twenty-eight lacS gene sequence types were identified. Although lacS gene did not prove variable enough within S. thermophilus species to be used for quantitative biotype monitoring, the possibility of using non rRNA targets for quantitative biotype identification in food was highlighted.

High-level adherence to a Mediterranean diet beneficially impacts the gut microbiota and associated metabolome

Objectives Habitual diet plays a major role in shaping the composition of the gut microbiota, and also determines the repertoire of microbial metabolites that can influence the host. The typical Western diet corresponds to that of an omnivore; however, the Mediterranean diet (MD), common in the Western Mediterranean culture, is to date a nutritionally recommended dietary pattern that includes high-level consumption of cereals, fruit, vegetables and legumes. To investigate the potential benefits of the MD in this cross-sectional survey, we assessed the gut microbiota and metabolome in a cohort of Italian individuals in relation to their habitual diets. Design and results We retrieved daily dietary information and assessed gut microbiota and metabolome in 153 individuals habitually following omnivore, vegetarian or vegan diets. The majority of vegan and vegetarian subjects and 30% of omnivore subjects had a high adherence to the MD. We were able to stratify individuals according to both diet type and adherence to the MD on the basis of their dietary patterns and associated microbiota. We detected significant associations between consumption of vegetable-based diets and increased levels of faecal short-chain fatty acids, Prevotella and some fibre-degrading Firmicutes, whose role in human gut warrants further research. Conversely, we detected higher urinary trimethylamine oxide levels in individuals with lower adherence to the MD. Conclusions High-level consumption of plant foodstuffs consistent with an MD is associated with beneficial microbiome-related metabolomic profiles in subjects ostensibly consuming a Western diet. Trial registration number This study was registered at clinical trials.gov as NCT02118857.

“Remake” by High-Throughput Sequencing of the Microbiota Involved in the Production of Water Buffalo Mozzarella Cheese

ABSTRACT Intermediates of production of two batches of traditional mozzarella cheese were analyzed by culture-independent pyrosequencing. The quantitative distribution of taxa within the samples suggested that thermophilic lactic acid bacteria from the natural starter were mainly responsible for the fermentation, while microorganisms found in raw milk did not develop during fermentation.

Exploring the Sources of Bacterial Spoilers in Beefsteaks by Culture-Independent High-Throughput Sequencing

Microbial growth on meat to unacceptable levels contributes significantly to change meat structure, color and flavor and to cause meat spoilage. The types of microorganisms initially present in meat depend on several factors and multiple sources of contamination can be identified. The aims of this study were to evaluate the microbial diversity in beefsteaks before and after aerobic storage at 4°C and to investigate the sources of microbial contamination by examining the microbiota of carcasses wherefrom the steaks originated and of the processing environment where the beef was handled. Carcass, environmental (processing plant) and meat samples were analyzed by culture-independent high-throughput sequencing of 16S rRNA gene amplicons. The microbiota of carcass swabs was very complex, including more than 600 operational taxonomic units (OTUs) belonging to 15 different phyla. A significant association was found between beef microbiota and specific beef cuts (P<0.01) indicating that different cuts of the same carcass can influence the microbial contamination of beef. Despite the initially high complexity of the carcass microbiota, the steaks after aerobic storage at 4°C showed a dramatic decrease in microbial complexity. Pseudomonas sp. and Brochothrix thermosphacta were the main contaminants, and Acinetobacter, Psychrobacter and Enterobacteriaceae were also found. Comparing the relative abundance of OTUs in the different samples it was shown that abundant OTUs in beefsteaks after storage occurred in the corresponding carcass. However, the abundance of these same OTUs clearly increased in environmental samples taken in the processing plant suggesting that spoilage-associated microbial species originate from carcasses, they are carried to the processing environment where the meat is handled and there they become a resident microbiota. Such microbiota is then further spread on meat when it is handled and it represents the starting microbial association wherefrom the most efficiently growing microbial species take over during storage and can cause spoilage.

Microbial Ecology Dynamics during Rye and Wheat Sourdough Preparation

ABSTRACT The bacterial ecology during rye and wheat sourdough preparation was described by 16S rRNA gene pyrosequencing. Viable plate counts of presumptive lactic acid bacteria, the ratio between lactic acid bacteria and yeasts, the rate of acidification, a permutation analysis based on biochemical and microbial features, the number of operational taxonomic units (OTUs), and diversity indices all together demonstrated the maturity of the sourdoughs during 5 to 7 days of propagation. Flours were mainly contaminated by metabolically active genera (Acinetobacter, Pantoea, Pseudomonas, Comamonas, Enterobacter, Erwinia, and Sphingomonas) belonging to the phylum Proteobacteria or Bacteroidetes (genus Chryseobacterium). Their relative abundances varied with the flour. Soon after 1 day of propagation, this population was almost completely inhibited except for the Enterobacteriaceae. Although members of the phylum Firmicutes were present at very low or intermediate relative abundances in the flours, they became dominant soon after 1 day of propagation. Lactic acid bacteria were almost exclusively representative of the Firmicutes by this time. Weissella spp. were already dominant in rye flour and stably persisted, though they were later flanked by the Lactobacillus sakei group. There was a succession of species during 10 days of propagation of wheat sourdoughs. The fluctuation between dominating and subdominating populations of L. sakei group, Leuconostoc spp., Weissella spp., and Lactococcus lactis was demonstrated. Other subdominant species such as Lactobacillus plantarum were detectable throughout propagation. As shown by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis, Saccharomyces cerevisiae dominated throughout the sourdough propagation. Notwithstanding variations due to environmental and technology determinants, the results of this study represent a clear example of how the microbial ecology evolves during sourdough preparation.

Bacteria and yeast microbiota in milk kefir grains from different Italian regions

Kefir grains are a unique symbiotic association of different microrganisms, mainly lactic acid bacteria, yeasts and occasionally acetic acid bacteria, cohabiting in a natural polysaccharide and a protein matrix. The microbial composition of kefir grains can be considered as extremely variable since it is strongly influenced by the geographical origin of the grains and by the sub-culturing method used. The aim of this study was to elucidate the bacteria and yeast species occurring in milk kefir grains collected in some Italian regions by combining the results of scanning electron microscopy analysis, viable counts on selective culture media, PCR-DGGE and pyrosequencing. The main bacterial species found was Lactobacillus kefiranofaciens while Dekkera anomala was the predominant yeast. The presence of sub-dominant species ascribed to Streptococcus thermophilus, Lactococcus lactis and Acetobacter genera was also highlighted. In addition, Lc. lactis, Enterococcus sp., Bacillus sp., Acetobacter fabarum, Acetobacter lovaniensis and Acetobacter orientalis were identified as part of the cultivable community. This work further confirms both the importance of combining culture-independent and culture-dependent approaches to study microbial diversity in food and how the combination of multiple 16S rRNA gene targets strengthens taxonomic identification using sequence-based identification approaches.

Metatranscriptomics reveals temperature-driven functional changes in microbiome impacting cheese maturation rate

Traditional cheeses harbour complex microbial consortia that play an important role in shaping typical sensorial properties. However, the microbial metabolism is considered difficult to control. Microbial community succession and the related gene expression were analysed during ripening of a traditional Italian cheese, identifying parameters that could be modified to accelerate ripening. Afterwards, we modulated ripening conditions and observed consistent changes in microbial community structure and function. We provide concrete evidence of the essential contribution of non-starter lactic acid bacteria in ripening-related activities. An increase in the ripening temperature promoted the expression of genes related to proteolysis, lipolysis and amino acid/lipid catabolism and significantly increases the cheese maturation rate. Moreover, temperature-promoted microbial metabolisms were consistent with the metabolomic profiles of proteins and volatile organic compounds in the cheese. The results clearly indicate how processing-driven microbiome responses can be modulated in order to optimize production efficiency and product quality.

NaOH-Debittering Induces Changes in Bacterial Ecology during Table Olives Fermentation

Limited information is available on the impact of the NaOH treatment on table olive fermentations, and for this reason a polyphasic approach has been adopted here to investigate its effect on the fermentation dynamics and bacterial biodiversity. The microbial counts of the main groups involved in the transformation have not shown any differences, apart from a more prompt start of the fermentation when the olives were subjected to the NaOH treatment. The data produced by culture-independent analyses highlighted that the fermentation of table olives not treated with NaOH is the result of the coexistence of two different ecosystems: the surface of the olives and the brines. A sodium hydroxide treatment not only eliminates this difference, but also affects the bacterial ecology of the olives to a great extent. As proved by high-throughput sequencing, the fermentation of the olives not treated with NaOH was characterized by the presence of halophilic bacteria, which were substituted by Lactobacillus at the later stages of the fermentation, while enterobacteria were dominant when the olives were treated with sodium hydroxide. Higher biodiversity was found for Lactobacillus plantarum isolated during untreated fermentation. Different biotypes were found on the olive surface and in the brines. When the debittering process was carried out, a decrease in the number of L. plantarum biotypes were observed and those originating from the surface of the olive did not differentiate from the ones present in the brines.

The same microbiota and a potentially discriminant metabolome in the saliva of omnivore, ovo-lacto-vegetarian and Vegan individuals.

The salivary microbiota has been linked to both oral and non-oral diseases. Scant knowledge is available on the effect of environmental factors such as long-term dietary choices on the salivary microbiota and metabolome. This study analyzed the microbial diversity and metabolomic profiles of the saliva of 161 healthy individuals who followed an omnivore or ovo-lacto-vegetarian or vegan diet. A large core microbiota was identified, including 12 bacterial genera, found in >98% of the individuals. The subjects could be stratified into three “salivary types” that differed on the basis of the relative abundance of the core genera Prevotella, Streptococcus/Gemella and Fusobacterium/Neisseria. Statistical analysis indicated no effect of dietary habit on the salivary microbiota. Phylogenetic beta-diversity analysis consistently showed no differences between omnivore, ovo-lacto-vegetarian and vegan individuals. Metabolomic profiling of saliva using 1H-NMR and GC-MS/SPME identified diet-related biomarkers that enabled a significant discrimination between the 3 groups of individuals on the basis of their diet. Formate, urea, uridine and 5-methyl-3-hexanone could discriminate samples from omnivores, whereas 1-propanol, hexanoic acid and proline were characteristic of non-omnivore diets. Although the salivary metabolome can be discriminating for diet, the microbiota has a remarkable inter-individual stability and did not vary with dietary habits. Microbial homeostasis might be perturbed with sub-standard oral hygiene or other environmental factors, but there is no current indication that a choice of an omnivore, ovo-lacto-vegetarian or vegan diet can lead to a specific composition of the oral microbiota with consequences on the oral homeostasis.

Metagenomics insights into food fermentations.

This review describes the recent advances in the study of food microbial ecology, with a focus on food fermentations. High‐throughput sequencing (HTS) technologies have been widely applied to the study of food microbial consortia and the different applications of HTS technologies were exploited in order to monitor microbial dynamics in food fermentative processes. Phylobiomics was the most explored application in the past decade. Metagenomics and metatranscriptomics, although still underexploited, promise to uncover the functionality of complex microbial consortia. The new knowledge acquired will help to understand how to make a profitable use of microbial genetic resources and modulate key activities of beneficial microbes in order to ensure process efficiency, product quality and safety.