Systematic optimization of fermentation conditions for in vitro fermentations with fecal inocula
In vitro fermentation strategies with fecal inocula are considered cost-effective methods to gain mechanistic insights into fecal microbiota community dynamics. However, all in vitro approaches have their limitations due to inherent differences with respect to the in vivo situation mimicked, introdu...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-07-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1198903/full |
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| author | Jonas Poppe Sara Vieira-Silva Sara Vieira-Silva Jeroen Raes Jeroen Raes Kristin Verbeke Kristin Verbeke Gwen Falony Gwen Falony Gwen Falony |
| author_facet | Jonas Poppe Sara Vieira-Silva Sara Vieira-Silva Jeroen Raes Jeroen Raes Kristin Verbeke Kristin Verbeke Gwen Falony Gwen Falony Gwen Falony |
| author_sort | Jonas Poppe |
| collection | DOAJ |
| description | In vitro fermentation strategies with fecal inocula are considered cost-effective methods to gain mechanistic insights into fecal microbiota community dynamics. However, all in vitro approaches have their limitations due to inherent differences with respect to the in vivo situation mimicked, introducing possible biases into the results obtained. Here, we aimed to systematically optimize in vitro fermentation conditions to minimize drift from the initial inoculum, limit growth of opportunistic colonizers, and maximize the effect of added fiber products (here pectin) when compared to basal medium fermentations. We evaluated the impact of varying starting cell density and medium nutrient concentration on these three outcomes, as well as the effect of inoculation with fresh vs. stored fecal samples. By combining GC–MS metabolite profiling and 16 s rRNA gene-based amplicon sequencing, we established that starting cell densities below 1010 cells/ml opened up growth opportunities for members the Enterobacteriaceae family. This effect was exacerbated when using fecal samples that were stored frozen at −80°C. Overgrowth of Enterobacteriaceae resulted in lowered alpha-diversity and larger community drift, possibly confounding results obtained from fermentations in such conditions. Higher medium nutrient concentrations were identified as an additional factor contributing to inoculum community preservation, although the use of a less nutrient dense medium increased the impact of fiber product addition on the obtained metabolite profiles. Overall, our microbiome observations indicated that starting cell densities of 1010 cells/ml limited opportunities for exponential growth, suppressing in vitro community biases, whilst metabolome incubations should preferably be carried out in a diluted medium to maximize the impact of fermentable substrates. |
| first_indexed | 2024-03-12T22:08:17Z |
| format | Article |
| id | doaj.art-a55992f8748e487c93d45f1376e8da20 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-03-12T22:08:17Z |
| publishDate | 2023-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-a55992f8748e487c93d45f1376e8da202023-07-24T09:54:42ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-07-011410.3389/fmicb.2023.11989031198903Systematic optimization of fermentation conditions for in vitro fermentations with fecal inoculaJonas Poppe0Sara Vieira-Silva1Sara Vieira-Silva2Jeroen Raes3Jeroen Raes4Kristin Verbeke5Kristin Verbeke6Gwen Falony7Gwen Falony8Gwen Falony9Translational Research in Gastrointestinal Disorders (TARGID), Department of Chronic Diseases and Metabolism, KU Leuven, Leuven, BelgiumInstitute of Medical Microbiology and Hygiene and Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, GermanyInstitute of Molecular Biology (IMB), Mainz, GermanyLaboratory of Molecular Bacteriology, Department of Microbiology and Immunology, KU Leuven, Leuven, BelgiumCenter for Microbiology, VIB-KU Leuven, Leuven, BelgiumTranslational Research in Gastrointestinal Disorders (TARGID), Department of Chronic Diseases and Metabolism, KU Leuven, Leuven, BelgiumLeuven Food Science and Nutrition Research Center (LFoRCe), KU Leuven, Leuven, BelgiumInstitute of Medical Microbiology and Hygiene and Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, GermanyLaboratory of Molecular Bacteriology, Department of Microbiology and Immunology, KU Leuven, Leuven, BelgiumCenter for Microbiology, VIB-KU Leuven, Leuven, BelgiumIn vitro fermentation strategies with fecal inocula are considered cost-effective methods to gain mechanistic insights into fecal microbiota community dynamics. However, all in vitro approaches have their limitations due to inherent differences with respect to the in vivo situation mimicked, introducing possible biases into the results obtained. Here, we aimed to systematically optimize in vitro fermentation conditions to minimize drift from the initial inoculum, limit growth of opportunistic colonizers, and maximize the effect of added fiber products (here pectin) when compared to basal medium fermentations. We evaluated the impact of varying starting cell density and medium nutrient concentration on these three outcomes, as well as the effect of inoculation with fresh vs. stored fecal samples. By combining GC–MS metabolite profiling and 16 s rRNA gene-based amplicon sequencing, we established that starting cell densities below 1010 cells/ml opened up growth opportunities for members the Enterobacteriaceae family. This effect was exacerbated when using fecal samples that were stored frozen at −80°C. Overgrowth of Enterobacteriaceae resulted in lowered alpha-diversity and larger community drift, possibly confounding results obtained from fermentations in such conditions. Higher medium nutrient concentrations were identified as an additional factor contributing to inoculum community preservation, although the use of a less nutrient dense medium increased the impact of fiber product addition on the obtained metabolite profiles. Overall, our microbiome observations indicated that starting cell densities of 1010 cells/ml limited opportunities for exponential growth, suppressing in vitro community biases, whilst metabolome incubations should preferably be carried out in a diluted medium to maximize the impact of fermentable substrates.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1198903/fullfermentationin vitromicrobiomeoptimizationprebiotic |
| spellingShingle | Jonas Poppe Sara Vieira-Silva Sara Vieira-Silva Jeroen Raes Jeroen Raes Kristin Verbeke Kristin Verbeke Gwen Falony Gwen Falony Gwen Falony Systematic optimization of fermentation conditions for in vitro fermentations with fecal inocula Frontiers in Microbiology fermentation in vitro microbiome optimization prebiotic |
| title | Systematic optimization of fermentation conditions for in vitro fermentations with fecal inocula |
| title_full | Systematic optimization of fermentation conditions for in vitro fermentations with fecal inocula |
| title_fullStr | Systematic optimization of fermentation conditions for in vitro fermentations with fecal inocula |
| title_full_unstemmed | Systematic optimization of fermentation conditions for in vitro fermentations with fecal inocula |
| title_short | Systematic optimization of fermentation conditions for in vitro fermentations with fecal inocula |
| title_sort | systematic optimization of fermentation conditions for in vitro fermentations with fecal inocula |
| topic | fermentation in vitro microbiome optimization prebiotic |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1198903/full |
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