Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments

[EN] Deep investigation of the microbiome of food-production and foodprocessing environments through whole-metagenome sequencing (WMS) can provide detailed information on the taxonomic composition and functional potential of the microbial communities that inhabit them, with huge potential benefits for environmental monitoring programs. However, certain technical challenges jeopardize the application of WMS technologies with this aim, with the most relevant one being the recovery of a sufficient amount of DNA from the frequently low-biomass samples collected from the equipment, tools and surfaces of food-processing plants. Here, we present the first complete workflow, with optimized DNA-purification methodology, to obtain high-quality WMS sequencing results from samples taken from food-production and food-processing environments and reconstruct metagenome assembled genomes (MAGs). The protocol can yield DNA loads >10 ng in >98% of samples and >500 ng in 57.1% of samples and allows the collection of, on average, 12.2 MAGs per sample (with up to 62 MAGs in a single sample) in ~1 week, including both laboratory and computational work. This markedly improves on results previously obtained in studies performing WMS of processing environments and using other protocols not specifically developed to sequence these types of sample, in which <2 MAGs per sample were obtained. The full protocol has been developed and applied in the framework of the European Union project MASTER (Microbiome applications for sustainable food systems through technologies and enterprise) in 114 food-processing facilities from different production sectors.SIThis work was funded by the European Commission under the European Union’s Horizon 2020 research and innovation program under grant agreement no. 818368 (MASTER). C.B. is grateful to Junta de Castilla y León and the European Social Fund for awarding her a pre-doctoral grant (BOCYL-D-07072020-6). A.P. is grateful to Ministerio de Ciencia e Innovación for awarding her a pre-doctoral grant (PRE2021-098910). N.M.Q. is currently funded by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 101034371. We thank AV Star Systems for their role in creating the Supplementary Video, and M. Coakley and S. Mortensen for their help in its preparation

Fecal microbiota cooperative metabolism of pectins derived from apple pomace: A functional metagenomic study

Complex polysaccharides such as pectin are receiving increasing attention as emergent prebiotics which could be of interest for human health. Pectin comprises a wide array of highly complex and diverse polysaccharide structures, which are abundant in agricultural by-products. Pectin metabolization by the gut microbiota requires cooperative metabolism and cross-feeding networks among microbial species. Herein we have deepened into the gut modulatory properties of apple pomaces and pectins derived from this by product, through a shotgun metagenomics approach coupled to advanced functional annotation and prediction of cooperating networks of metagenome-assembled genomes (MAGs). For this purpose, we have used samples from batch in vitro fecal fermentations performed with samples from healthy donors and Crohn's disease patients, in the presence of apple pomace and pectins exhibiting different structural properties. Our results confirm the capacity of apple pomace/pectins to promote key taxa generally underrepresented in Crohn's disease patients but that have been attributed anti-inflammatory properties, Faecalibacterium, Ruminococcaceae members and Akkermansia being very remarkable. Besides, some Ruminococcus and Akkermansia species have been identified as key symbionts, exhibiting complementary metabolic traits to fully metabolise pectins from apple pomace. These results will aid developing microbiome-targeted prebiotic and/or synbiotic strategies for specific population groups.The research performed was funded by the European Union's Horizon2020 Research and Innovation Programme under grant agreement No 818368 (MASTER), and the grants from the Spanish State Research Agency RTI2018-095021-J-I00. CS is a postdoctoral researcher supported by the Juan de la Cierva-Formación Postdoctoral Trainee Program from MICINN, the Spanish Ministry of Science and Innovation (FJC2019-042125-I). Research in our group is also supported by Grant AYUD-2021-50910 from the autonomic Government of Principado de Asturias (FICYT, supported by FEDER)

Supplementary data Fecal microbiota cooperative metabolism of pectins derived from apple pomace: a functional metagenomic study

Multimedia component 1.Peer reviewe

Arabinoxylan and Pectin Metabolism in Crohn’s Disease Microbiota: An In Silico Study

Inflammatory bowel disease is a chronic disorder including ulcerative colitis and Crohn’s disease (CD). Gut dysbiosis is often associated with CD, and metagenomics allows a better understanding of the microbial communities involved. The objective of this study was to reconstruct in silico carbohydrate metabolic capabilities from metagenome-assembled genomes (MAGs) obtained from healthy and CD individuals. This computational method was developed as a mean to aid rationally designed prebiotic interventions to rebalance CD dysbiosis, with a focus on metabolism of emergent prebiotics derived from arabinoxylan and pectin. Up to 1196 and 1577 MAGs were recovered from CD and healthy people, respectively. MAGs of Akkermansia muciniphila, Barnesiella viscericola DSM 18177 and Paraprevotella xylaniphila YIT 11841 showed a wide range of unique and specific enzymes acting on arabinoxylan and pectin. These glycosidases were also found in MAGs recovered from CD patients. Interestingly, these arabinoxylan and pectin degraders are predicted to exhibit metabolic interactions with other gut microbes reduced in CD. Thus, administration of arabinoxylan and pectin may ameliorate dysbiosis in CD by promoting species with key metabolic functions, capable of cross-feeding other beneficial species. These computational methods may be of special interest for the rational design of prebiotic ingredients targeting at CDThe work in our research groups was funded by the grants RTI2018-095021-J-I00 (funded by (MCIU/AEI/FEDER, UE) and AGL2016-78311-R (funded by (MINECO/AEI/FEDER, UE). Acknowledgments: Carlos Sabater acknowledges his Postdoctoral research contract funded by the Instituto de Investigación Sanitaria del Principado de Asturias (ISPA) and Postdoctoral research contract Juan de la Cierva-Formación from Spanish Ministry of Science and Innovation (FJC2019-042125-I)

Supplementary Materials Arabinoxylan and Pectin Metabolism in Crohn’s Disease Microbiota: An In Silico Study

Peer reviewe

Pectinas de bagazos de manzana modulan de forma diferencial grupos clave en la microbiota intestinal

Trabajo presentado al XIII Workshop Sociedad Española de Microbiota, Probióticos y Prebióticos, celebrado en Valencia (España), del 7 la 9 de junio de 2022

Estudio del metabolismo de carbohidratos de subespecies de Bifidobacterium longum para su aislamiento diferencial

Resumen trabajo presentado en el XII Workshop Sociedad Española de Microbiota, Probióticos y Prebióticos (SEMiPyP) y I Congreso Sociedad Iberoamericana de Microbiota, Probióticos y Prebióticos (SIAMPYP), celebrado de forma virtual del 15 al 18 de septiembre de 2021Introducción/objetivos: Bifidobacterium longum es una de las especies beneficiosas más abundantes en la microbiota intestinal, que posee diferentes subespecies como B. longum subsp. longum y B. longum subsp. infantis cuyo metabolismo de carbohidratos les otorga una ventaja a la hora de colonizar el intestino humano. En este sentido, B. longum subsp. longum es capaz de metabolizar carbohidratos vegetales complejos. Por tanto, el objetivo de este trabajo fue estudiar las actividades glicosidasas codificadas en genomas de B. longum para diseñar un medio de cultivo específico que permita diferenciar estas dos subespecies. Metodología: en primer lugar, se realizó un estudio in silico donde se construyeron mapas metabólicos con 707 genomas de bifidobacterias incluidas en la lista QPS de la EFSA (B. adolescentis, B. animalis, B. bifidum, B. breve, y B. longum (subespecies infantis y longum), además de 320 genomas de otras especies presentes en la microbiota intestinal humana (B. angulatum, B. catenulatum, B. dentium, B. faecale, B. gallicum, B. pseudocatenulatum, B. scardovii). Las secuencias se compararon con la base de datos CAZy. Posteriormente, se realizó un estudio in vitro donde se prepararon medios de cultivo con arabinoxilanos y arabinoxilo-oligosacáridos como única fuente de carbono, y se estudió el crecimiento diferencial de varias cepas previamente clasificadas como B. longum subsp. infantis o B. longum subsp. longum. Resultados: la mayoría de las especies QPS presentaron similitudes en su metabolismo de carbohidratos. La presencia de dominios de glicosidasas que actúan sobre xilanos y arabinoxilanos fue mayor en B. longum subsp. longum que en B. longum subsp. infantis. Estos resultados se verificaron experimentalmente, observándose que la presencia de arabinoxilanos y arabinoxilo-oligosacáridos posibilita el crecimiento diferencial de B. longum subsp. longum. Conclusiones: el uso de medios suplementados con arabinoxilanos y arabinoxilo-oligosacáridos permite aislar diferencialmente las subespecies B. longum subsp. longum y B. longum subsp. infantis

Structural properties of apple pomace and pectins derived therefrom influence its prebiotic potential and modulatory effects on key target commensal microbial populations

Resumen del trabajo presentado en la FoodMicro2022 Conference, celebrada en Atenas (Grecia); del 28 al 31 de agosto de 2022Pectin is a group of structurally diverse dietary fibers, very abundant in agri-food waste and by-products such as those generated during apple cider manufacturing. In recent years, pectin and pectinoligosaccharides have demonstrated good fermentation properties, as well as prominent health promoting traits particularly ameliorating certain inflammatory conditions. In previous investigations apple pomace derived from production of monovarietal Asturian ciders was demonstrated to represent a good source of pectin with varied structural characteristics. In this work we investigated in vitro the modulatory effect of pectin and pomace fractions derived from the production of selected monovarietal Asturian ciders on human microbiota from healthy subjects and inflammatory bowel disease patients through fecal batch fermentations and 16S sequencing. Overall, these fractions selectively promoted the growth of Akkermansia, Lachnospiraceae UCG-010, Prevotella, Sucinivibrio and Turicibacter on samples from healthy donors, while Blautia, Rumicoccaceae CAG-56, Dialister, E. eligens and Intestinimonas were stimulated in fermentations from IBD patients. The growth of Akkermansia, Blautia, E. eligens group, Intestinimonas and Succinivibrio was exclusively associated to pomace and pectin derived from the tested by-products, and did not occur with other non-pectic prebiotics/substrates. Gal content and (Ara+Gal)/Rha ratio was positively associated to the promotion of most of these genera. This work provides a comprehensive characterisation of gut microbiota modulation effects of apple pectin and pomace fractions derived from cider by-products, demonstrating diverse gut microbiota modulatory capacity of structurally distinct pectin and apple pomace fractions, diversifying the opportunities to achieve cider by-products valorisation through formulation of novel prebiotics for particular population groups