Diversity within Italian Cheesemaking Brine-Associated Bacterial Communities Evidenced by Massive Parallel 16S rRNA Gene Tag Sequencing

[EN]This study explored the bacterial diversity of brines used for cheesemaking in Italy, as well as their physicochemical characteristics. In this context, 19 brines used to salt soft, semi-hard, and hard Italian cheeses were collected in 14 commercial cheese plants and analyzed using a culture-independent amplicon sequencing approach in order to describe their bacterial microbiota. Large NaCl concentration variations were observed among the selected brines, with hard cheese brines exhibiting the highest values. Acidity values showed a great variability too, probably in relation to the brine use prior to sampling. Despite their high salt content, brine microbial loads ranged from 2.11 to 6.51 log CFU/mL for the total mesophilic count. Microbial community profiling assessed by 16S rRNA gene sequencing showed that these ecosystems were dominated by Firmicutes and Proteobacteria, followed by Actinobacteria and Bacteroidetes. Cheese type and brine salinity seem to be the main parameters accountable for brine microbial diversity. On the contrary, brine pH, acidity and protein concentration, correlated to cheese brine age, did not have any selective effect on the microbiota composition. Nine major genera were present in all analyzed brines, indicating that they might compose the core microbiome of cheese brines. Staphylococcus aureus was occasionally detected in brines using selective culture media. Interestingly, bacterial genera associated with a functional and technological use were frequently detected. Indeed Bifidobacteriaceae, which might be valuable probiotic candidates, and specific microbial genera such as Tetragenococcus, Corynebacterium and non-pathogenic Staphylococcus, which can contribute to sensorial properties of ripened cheeses, were widespread within brines.S

Discriminative power of DNA-based, volatilome, near infrared spectroscopy, elements and stable isotopes methods for the origin authentication of typical Italian mountain cheese using sPLS-DA modeling

Origin authentication methods are pivotal in counteracting frauds and provide evidence for certification systems. For these reasons, geographical origin authentication methods are used to ensure product origin. This study focused on the origin authentication (i.e. at the producer level) of a typical mountain cheese origin using various approaches, including shotgun metagenomics, volatilome, near infrared spectroscopy, stable isotopes, and elemental analyses. DNA-based analysis revealed that viral communities achieved a higher classification accu racy rate (97.4 ± 2.6 %) than bacterial communities (96.1 ± 4.0 %). Non-starter lactic acid bacteria and phages specific to each origin were identified. Volatile organic compounds exhibited potential clusters according to cheese origin, with a classification accuracy rate of 90.0 ± 11.1 %. Near-infrared spectroscopy showed lower discriminative power for cheese authentication, yielding only a 76.0 ± 31.6 % classification accuracy rate. Model performances were influenced by specific regions of the infrared spectrum, possibly associated with fat content, lipid profile and protein characteristics. Furthermore, we analyzed the elemental composition of mountain Caciotta cheese and identified significant differences in elements related to dairy equipment, macronutrients, and rare earth elements among different origins. The combination of elements and isotopes showed a decrease in authentication performance (97.0 ± 3.1 %) compared to the original element models, which were found to achieve the best classification accuracy rate (99.0 ± 0.01 %). Overall, our findings emphasize the potential of multi-omics techniques in cheese origin authentication and highlight the complexity of factors influencing cheese composition and hence typicity

BMC Genomics

Oenococcus oeni is a lactic acid bacteria species adapted to the low pH, ethanol-rich environments of wine and cider fermentation, where it performs the crucial role of malolactic fermentation. It has a small genome and has lost the mutS-mutL DNA mismatch repair genes, making it a hypermutable and highly specialized species. Two main lineages of strains, named groups A and B, have been described to date, as well as other subgroups correlated to different types of wines or regions. A third group "C" has also been hypothesized based on sequence analysis, but it remains controversial. In this study we have elucidated the species population structure by sequencing 14 genomes of new strains isolated from cider and kombucha and performing comparative genomics analyses. Sequence-based phylogenetic trees confirmed a population structure of 4 clades: The previously identified A and B, a third group "C" consisting of the new cider strains and a small subgroup of wine strains previously attributed to group B, and a fourth group "D" exclusively represented by kombucha strains. A pair of complete genomes from group C and D were compared to the circularized O. oeni PSU-1 strain reference genome and no genomic rearrangements were found. Phylogenetic trees, K-means clustering and pangenome gene clusters evidenced the existence of smaller, specialized subgroups of strains. Using the pangenome, genomic differences in stress resistance and biosynthetic pathways were found to uniquely distinguish the C and D clades. The obtained results, including the additional cider and kombucha strains, firmly established the O. oeni population structure. Group C does not appear as fully domesticated as group A to wine, but showed several unique patterns which may be due to ongoing specialization to the cider environment. Group D was shown to be the most divergent member of O. oeni to date, appearing as the closest to a pre-domestication state of the species.MICROWINE - Microbial metagenomics and the modern wine industr

Diversity within Italian Cheesemaking Brine-Associated Bacterial Communities Evidenced by Massive Parallel 16S rRNA Gene Tag Sequencing

This study explored the bacterial diversity of brines used for cheesemaking in Italy, as well as their physicochemical characteristics. In this context, 19 brines used to salt soft, semi-hard, and hard Italian cheeses were collected in 14 commercial cheese plants and analyzed using a culture-independent amplicon sequencing approach in order to describe their bacterial microbiota. Large NaCl concentration variations were observed among the selected brines, with hard cheese brines exhibiting the highest values. Acidity values showed a great variability too, probably in relation to the brine use prior to sampling. Despite their high salt content, brine microbial loads ranged from 2.11 to 6.51 log CFU/mL for the total mesophilic count. Microbial community profiling assessed by 16S rRNA gene sequencing showed that these ecosystems were dominated by Firmicutes and Proteobacteria, followed by Actinobacteria and Bacteroidetes. Cheese type and brine salinity seem to be the main parameters accountable for brine microbial diversity. On the contrary, brine pH, acidity and protein concentration, correlated to cheese brine age, did not have any selective effect on the microbiota composition. Nine major genera were present in all analyzed brines, indicating that they might compose the core microbiome of cheese brines. Staphylococcus aureus was occasionally detected in brines using selective culture media. Interestingly, bacterial genera associated with a functional and technological use were frequently detected. Indeed Bifidobacteriaceae, which might be valuable probiotic candidates, and specific microbial genera such as Tetragenococcus, Corynebacterium and non-pathogenic Staphylococcus, which can contribute to sensorial properties of ripened cheeses, were widespread within brines. \ua9 2017 Marino, Innocente, Maifreni, Mounier, Cobo-D\uedaz, Coton, Carraro and Cardazzo

Microbial Ecology of French Dry Fermented Sausages and Mycotoxin Risk Evaluation During Storage

Dry fermented sausages are produced worldwide by well-controlled fermentationprocesses involving complex microbiota including many bacterial and fungal species with key technological roles. However, to date, fungal diversity on sausage casings during storage has not been fully described. In this context, we studied the microbial communities from dry fermented sausages naturally colonized or voluntarily surface inoculated with molds during storage using both culture-dependent and metabarcoding methods. Staphylococci and lactic acid bacteria largely dominated in samples, although some halotolerant genera (e.g., Halomonas, Tetragenococcus, and Celerinatantimonas spp.) were also frequently observed. Fungal populations varied from 7.2 to 9.8 log TFU/cm2 sausage casing during storage, suggesting relatively low count variability among products. Fungal diversity identified on voluntarily inoculated casings was lower (dominated by Penicillium nalgiovense and Debaryomyces hansenii) than naturally environment-inoculated fermented sausages (colonized by P. nalgiovense, Penicillium nordicum, and other Penicillium spp. and sporadically by Scopulariopsis sp., D. hansenii, and Candida zeylanoïdes). P. nalgiovense and D. hansenii were systematically identified, highlighting their key technological role. The mycotoxin risk was then evaluated, and in situ mycotoxin production of selected mold isolates was determined during pilot-scale sausage productions. Among the identified fungal species, P. nalgiovense was confirmed not to produce mycotoxins. However, some P. nordicum, Penicillium chrysogenum, Penicillium bialowienzense, Penicillium brevicompactum, and Penicillium citreonigrum isolates produced one or more mycotoxins in vitro. P. nordicum also produced ochratoxin A during pilotscale sausage productions using “worst-case” conditions in the absence of biotic competition. These data provide new knowledge on fermented sausage microbiota and the potential mycotoxin risk during storage

Unraveling microbial ecology of industrial-scale Kombucha fermentations by metabarcoding and culture-based methods.

Kombucha, historically an Asian tea-based fermented drink, has recently become trendy in Western countries. Producers claim it bears health-enhancing properties that may come from the tea or metabolites produced by its microbiome. Despite its long history of production, microbial richness and dynamics have not been fully unraveled, especially at an industrial scale. Moreover, the impact of tea type (green or black) on microbial ecology was not studied. Here, we compared microbial communities from industrial-scale black and green tea fermentations, still traditionally carried out by a microbial biofilm, using culture-dependent and metabarcoding approaches. Dominant bacterial species belonged to Acetobacteraceae and to a lesser extent Lactobacteriaceae, while the main identified yeasts corresponded to Dekkera, Hanseniaspora and Zygosaccharomyces during all fermentations. Species richness decreased over the 8-day fermentation. Among acetic acid bacteria, Gluconacetobacter europaeus, Gluconobacter oxydans, G. saccharivorans and Acetobacter peroxydans emerged as dominant species. The main lactic acid bacteria, Oenococcus oeni, was strongly associated with green tea fermentations. Tea type did not influence yeast community, with Dekkera bruxellensis, D. anomala, Zygosaccharomyces bailii and Hanseniaspora valbyensis as most dominant. This study unraveled a distinctive core microbial community which is essential for fermentation control and could lead to Kombucha quality standardization

Cider (Cyder; Hard Cider): The Product and Its Manufacture

International audienceCider, although the term ‘ciders’ is more suitable to represent the diversity of this product, is a fermented beverage produced and consumed worldwide. In this article, its historical and geographical origins, as well as definition from a regulatory point of view, are presented. Cidermaking is then discussed by describing two main processes (in France and the UK) that are radically different, although both lead to products named ciders. The importance of microorganisms and their metabolism during production in terms of quality or spoilage is reviewed. Finally, product composition and potential impacts on health are presented

Penicillium roqueforti

International audiencePenicillium roqueforti is used as a fungal adjunct culture for the production of blue-veined cheeses worldwide. The physiological traits of this fungus explain its adaptation to the cheese matrix and thus its ability to develop in the cheese-making environment. The various metabolic activities of this fungus, including proteolysis and lipolysis, are largely involved in cheese ripening and provide the typical organoleptic properties (visual aspect, color, texture and aroma) to the final product. However, this species is also known to be a common spoiler in various dairy products as well as in other food (e.g., bread) and feed (especially silage) products

Evaluation of enniatins and beauvericin toxicity by innovative in vitro model approaches

International audienceMycotoxins contaminate more than 70% of cereal crops worldwide and therefore constitute a public health problem [1]. Among mycotoxigenic molds, Fusarium spp. are the most recurring and are linked to enniatins (ENNs) contamination [2]. ENNs are considered "emerging" mycotoxins, a term referring to poorly characterized or only recently identified as secondary metabolites. However, their occurrence on cereals is particularly high. A few in vitro studies, mainly on ENNs B and B1, have shown various effects including DNA alteration, apoptosis, mitochondrial damage and production of reactive oxygen species. Yet, the lack of in vitro and in vivo ENN toxicity data prevents robust human health risk assessment [3]–[6]. It is therefore important to address these issues by mimicking human exposure conditions as closely as possible. In order to take into account the mycotoxin exposure process (i.e. through food consumption), we evaluated ENNs toxicity on liver cells as they are responsible for xenobiotic detoxification. Acute toxicity of enniatins A, A1, B, B1, and beauvericin was evaluated by cell viability tests on 2D and 3D hepatic cell line models (HepaRG). To further understand the impact of ENN B and ENN B1 (IC0, IC10 and IC30) toxicity, RNA-seq analyses on HepaRG spheroids, a more relevant hepatic model, were also performed and over- and under-expressed genes were identified. These key genes pinpointed the main pathways involved after acute exposure. The obtained data provides novel knowledge on ENNs toxicity after acute exposure and the impact on cell gene expression.[1]G. Schatzmayr et E. Streit, « Global occurrence of mycotoxins in the food and feed chain: facts and figures », World Mycotoxin J., vol. 6, no 3, p. 213‑222, août 2013, doi: 10.3920/WMJ2013.1572.[2]D. Ferrigo, A. Raiola, et R. Causin, « Fusarium Toxins in Cereals: Occurrence, Legislation, Factors Promoting the Appearance and Their Management », Molecules, vol. 21, no 5, p. 627, mai 2016, doi: 10.3390/molecules21050627.[3]A. Juan-García, L. Manyes, M.-J. Ruiz, et G. Font, « Involvement of enniatins-induced cytotoxicity in human HepG2 cells », Toxicol. Lett., vol. 218, no 2, p. 166‑173, avr. 2013, doi: 10.1016/j.toxlet.2013.01.014.[4]A. Prosperini, A. Juan-García, G. Font, et M. J. Ruiz, « Reactive oxygen species involvement in apoptosis and mitochondrial damage in Caco-2 cells induced by enniatins A, A1, B and B1 », Toxicol. Lett., vol. 222, no 1, p. 36‑44, sept. 2013, doi: 10.1016/j.toxlet.2013.07.009.[5]G. Meca, J. Mañes, G. Font, et M. J. Ruiz, « Study of the potential toxicity of enniatins A, A1, B, B1 by evaluation of duodenal and colonic bioavailability applying an in vitro method by Caco-2 cells », Toxicon, vol. 59, no 1, p. 1‑11, janv. 2012, doi: 10.1016/j.toxicon.2011.10.004.[6]G. Meca, G. Font, et M. J. Ruiz, « Comparative cytotoxicity study of enniatins A, A1, A2, B, B1, B4 and J3 on Caco-2 cells, Hep-G2 and HT-29 », Food Chem. Toxicol., vol. 49, no 9, p. 2464‑2469, sept. 2011, doi: 10.1016/j.fct.2011.05.020