Annalisa Rebecchi

Bacterial diversity in typical Italian salami at different ripening stages as revealed by high-throughput sequencing of 16S rRNA amplicons.

The bacterial diversity involved in food fermentations is one of the most important factors shaping the final characteristics of traditional foods. Knowledge about this diversity can be greatly improved by the application of high-throughput sequencing technologies (HTS) coupled to the PCR amplification of the 16S rRNA subunit. Here we investigated the bacterial diversity in batches of Salame Piacentino PDO (Protected Designation of Origin), a dry fermented sausage that is typical of a regional area of Northern Italy. Salami samples from 6 different local factories were analysed at 0, 21, 49 and 63 days of ripening; raw meat at time 0 and casing samples at 21 days of ripening where also analysed, and the effect of starter addition was included in the experimental set-up. Culture-based microbiological analyses and PCR-DGGE were carried out in order to be compared with HTS results. A total of 722,196 high quality sequences were obtained after trimming, paired-reads assembly and quality screening of raw reads obtained by Illumina MiSeq sequencing of the two bacterial 16S hypervariable regions V3 and V4; manual curation of 16S database allowed a correct taxonomical classification at the species for 99.5% of these reads. Results confirmed the presence of main bacterial species involved in the fermentation of salami as assessed by PCR-DGGE, but with a greater extent of resolution and quantitative assessments that are not possible by the mere analyses of gel banding patterns. Thirty-two different Staphylococcus and 33 Lactobacillus species where identified in the salami from different producers, while the whole data set obtained accounted for 13 main families and 98 rare ones, 23 of which were present in at least 10% of the investigated samples, with casings being the major sources of the observed diversity. Multivariate analyses also showed that batches from 6 local producers tend to cluster altogether after 21 days of ripening, thus indicating that HTS has the potential for fine scale differentiation of local fermented foods.

Erythromycin- and tetracycline-resistant lactobacilli in Italian fermented dry sausages

Aims:  To assess the frequency of erythromycin‐ and tetracycline‐resistant lactobacilli in Italian fermented dry sausages.

Surface microbiota analysis of Taleggio, Gorgonzola, Casera, Scimudin and Formaggio di Fossa Italian cheeses.

The composition of the bacterial consortia of the smear Italian cheeses and their role on quality and safety is still poorly understood. The objective of this study was to identify and characterize the bacterial communities present on the surface of five traditional Italian cheeses, Casera Valtellina, Scimudin, Formaggio di Fossa, Gorgonzola and Taleggio. DGGE analysis performed using total DNA obtained from cheese surfaces enabled us to identify the dominant bacterial populations. Bands showing different intensity and identified as Staphylococcus, Micrococcus, Psychrobacter, Enterococcus and Brevibacterium species were detected on the surface of cheeses. The cluster analysis showed that Gorgonzola, Taleggio and Formaggio di Fossa cheeses present high similarity in their surface bacterial composition while major differences in the DGGE profiles were observed in Scimudin and Casera. The molecular taxonomical identification among the Gram positive isolates, reveals the presence of the following bacterial genera: Staphylococcus, Micrococcus, Macrococcus, Enterococcus, Lactobacillus, Carnobacterium, Leuconostoc, Brevibacterium, Corynebacterium, Brochothrix, Bacillus. The combination of culture dependent and independent techniques allowed us to obtain information about the bacterial species covering the surface of five different traditional Italian cheeses.

Impact of cystic fibrosis disease on archaea and bacteria composition of gut microbiota

&NA; Cystic fibrosis is often associated with intestinal inflammation due to several factors, including altered gut microbiota composition. In this study, we analyzed the fecal microbiota among patients with cystic fibrosis of 10–22 years of age, and compared the findings with age‐matched healthy subjects. The participating patients included 14 homozygotes and 14 heterozygotes with the delF508 mutation, and 2 heterozygotes presenting non‐delF508 mutations. We used PCR‐DGGE and qPCR to analyze the presence of bacteria, archaea and sulfate‐reducing bacteria. Overall, our findings confirmed disruption of the cystic fibrosis gut microbiota. Principal component analysis of the qPCR data revealed no differences between homozygotes and heterozygotes, while both groups were distinct from healthy subjects who showed higher biodiversity. Archaea were under the detection limit in all homozygotes subjects, whereas methanogens were detected in 62% of both cystic fibrosis heterozygotes and healthy subjects. Our qPCR results revealed a low frequency of sulfate‐reducing bacteria in the homozygote (13%) and heterozygote (13%) patients with cystic fibrosis compared with healthy subjects (87.5%). This is a pioneer study showing that patients with cystic fibrosis exhibit significant reduction of H2‐consuming microorganisms, which could increase hydrogen accumulation in the colon and the expulsion of this gas through non‐microbial routes.

Microbial ecology involved in the ripening of naturally fermented llama meat sausages. A focus on lactobacilli diversity

Llama represents for the Andean regions a valid alternative to bovine and pork meat and thanks to the high proteins and low fat content; it can constitute a good product for the novel food market. In this study, culture-dependent and independent methods were applied to investigate the microbial ecology of naturally fermented llama sausages produced in Northwest Argentina. Two different production technologies of llama sausage were investigated: a pilot-plant scale (P) and an artisanal one (A). Results obtained by High-Throughput Sequencing (HTS) of 16S rRNA amplicons showed that the production technologies influenced the development of microbial communities with a different composition throughout the entire fermentation process. Both sequencing and microbiological counts demonstrated that Lactic Acid Bacteria (LAB) contributed largely to the dominant microbiota. When a total of 230 isolates were approached by RAPD-PCR, presumptive LAB strains from P production exhibited an initial variability in RAPD fingerprints switching to a single profile at the final of ripening, while A production revealed a more heterogeneous RAPD pattern during the whole fermentation process. The constant presence of Lactobacillus sakei along the fermentation in both productions was revealed by HTS and confirmed by species-specific PCR from isolated strains. The technological characterization of Lb. sakei isolates evidenced their ability to grow at 15°C, pH4.5 and 5% NaCl (95%). Most strains hydrolyzed myofibrillar and sarcoplasmic proteins. Bacteriocins encoding genes and antimicrobial resistance were found in 35% and 42.5% of the strains, respectively. An appropriate choice of a combination of autochthonous strains in a starter formulation is fundamental to improve and standardize llama sausages safety and quality.

High-throughput assessment of bacterial ecology in hog, cow and ovine casings used in sausages production

Natural casings derived from different intestine portions have been used for centuries in the production of fresh and dry-fermented sausages. Here we analysed by means of culture-dependent methods and Illumina high-throughput sequencing of 16S rRNA amplicons the bacterial ecology of hog, cow and ovine casings at different stages of their preparation for sausages production. Several strains of Staphylococcus, Lactobacillus, Bifidobacterium, Vagococcus and Clostridium were counted, isolated and characterised at phylogenetic level. High-throughput sequencing analyses revealed a high bacterial diversity, which differed strongly between casings of different animal species. The technological processes involved in the preparation for casing had also a strong impact on the casings bacterial ecology, with a significant reduction of undesired microorganisms, and an increase in the proportion of lactobacilli and staphylococci. Natural casings were demonstrated to be complex ecological environments, whose role as microbiological inoculants in the production of sausages should not be underestimated.

Archaeal microbiota population in piglet feces shifts in response to weaning: Methanobrevibacter smithii is replaced with Methanobrevibacter boviskoreani

Methanogens commonly inhabit swine intestine. We analyzed the gut archaeal population by extracting DNA from the feces of nine piglets. We performed PCR to target the V6-V8 region of the 16S rRNA gene. Subsequent denaturing gradient gel electrophoresis (DGGE) revealed the presence of Methanobrevibacter boviskoreani, which has not previously been identified in pigs. We confirmed these data with a PCR-DGGE analysis of the mcrA gene, and subsequent sequencing. At 63 days old, the only band in fecal samples corresponded to M. boviskoreani. The DGGE analysis also showed that Methanobrevibacter smithii, which was abundant at 28 days, was dramatically reduced at 42 days, and it completely disappeared at 63 days. To confirm these data, we quantified M. smithii and the total archaeal population by quantitative PCR (qPCR); moreover, we designed a new set of species-specific primers based on the 16S rRNA gene of M. boviskoreani. The qPCR results confirmed the reduction in M. smithii over time and a simultaneous increase in M. boviskoreani. At 63 days, the total numbers of archaea and M. boviskoreani genomes were comparable, which suggested that M. boviskoreani represented the dominant archaea. This work showed that the archaeal population shifted during weaning, and M. boviskoreani replaced M. smithii.

Impact of cooking and fermentation by lactic acid bacteria on phenolic profile of quinoa and buckwheat seeds

In this work, quinoa and buckwheat cooked seeds were fermented by two autochthonous strains of lactic acid bacteria isolated from the corresponding seeds, namely Lactobacillus paracasei A1 2.6 and Pediococcus pentosaceus GS·B, with lactic acid chemically acidified seeds as control. The impact of cooking and fermentation on the comprehensive phenolic profile of quinoa and buckwheat seeds was evaluated through untargeted ultra-high-pressure liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF-MS). Samples were analyzed also for in vitro antioxidant capacity (as FRAP and ORAC assays) and total phenolic content (TPC). The in vitro spectrophotometric assays highlighted that the microbial fermentation was more efficient in increasing (p < .05) the TPC and in vitro antioxidant potential in quinoa cooked seeds. However, an increase (p < .05) in TPC and ORAC radical scavenging was observed in both pseudocereals after the different cooking processes (i.e., boiling or toasting). The untargeted phenolic profiling depicted the comprehensive phenolic composition in these matrices. Raw seeds of both pseudocereals possessed a similar phenolic content (4.4 g kg-1 equivalents; considering free and bound fractions). Besides, the metabolomics-based approach showed that all treatments (i.e., cooking and fermentation) induced the release of specific classes, namely phenolic acids and tyrosols. The PLS-DA multivariate approach identified in flavonoids the best markers allowing to discriminate the different treatments considered (i.e., cooking, chemical acidification and microbial fermentation). These findings support the use of cooking and microbial fermentation to ensure the health-promoting properties of non-wheat grains, such as buckwheat and quinoa.