ena-STUDY-UNIVERSITY OF COPENHAGEN, SECTION OF MICROBIOLOGY-04-04-2019-10:22:38:389-51
Modulation of microbiota by fermentation of potato fiber FiberBind 400 in TIM-2 colon model
This study investigated the effect of potato fiber FiberBind 400 on composition of the gut microbiota using the TIM-2 colon model. Native starch and cross-linked resistant starch were used as reference substrates. FiberBind 400 and starches were commercial products from a potato variety Solanum tuberosum produced at KMC (Brande, Denmark). The TIM-2 model provides fully anaerobic conditions, peristaltic movements and removal of metabolites during fermentation. Inoculum consisted of pooled fecal microbiota collected from 8 healthy Caucasian adults (male: n = 4, female: n = 4; age of 25 – 42 years). TIM-2 fermentations were performed at 37 °C and pH 5.8. The units of the system were fed with the basal simulated ileal efflux medium (SIEM) containing 7.5 g per day of FiberBind 400, native starch or resistant starch as the only carbohydrate source. Microbiota samples were removed at the start of fermentation (0 h) and after 24, 48, 56 and 72 h fermentation for analyses of microbiota. TIM-2 fermentation of each substrate was performed as independent duplicates. The total bacterial DNA was isolated from the microbiota samples using the PowerLyzer@PowerSoil DNA Isolation Kit (Qiagen). To prepare the DNA library, the V3 region of the 16S rRNA gene was PCR amplified using AccuPrime SuperMix II and primers comparable with Nextera Index Kit, NXt_388F and NXt_518R (Illumina). The second PCR step was performed using Phusion High-Fidelity PCR Master and primers P5 and P7 (Nextera Index Kit) to incorporate adapters and tags in the PCR product. Sequencing was conducted using 2x150 cycles, MID output, V2 chemistry on the Illumina NextSeq 500 platform (Illumina, CA, USA). The raw dataset containing pair-ended reads were merged, trimmed and clustered (operational taxonomic units, OTUs, with 97% similarity) using the UPARSE pipeline. Sequencing data were analyzed using QIIME software and the Greengenes database for taxonomy annotation.
This study investigated the effect of potato fiber FiberBind 400 on composition of the gut microbiota using the TIM-2 colon model. Native starch and cross-linked resistant starch were used as reference substrates. FiberBind 400 and starches were commercial products from a potato variety Solanum tuberosum produced at KMC (Brande, Denmark). The TIM-2 model provides fully anaerobic conditions, peristaltic movements and removal of metabolites during fermentation. Inoculum consisted of pooled fecal microbiota collected from 8 healthy Caucasian adults (male: n = 4, female: n = 4; age of 25 – 42 years). TIM-2 fermentations were performed at 37 °C and pH 5.8. The units of the system were fed with the basal simulated ileal efflux medium (SIEM) containing 7.5 g per day of FiberBind 400, native starch or resistant starch as the only carbohydrate source. Microbiota samples were removed at the start of fermentation (0 h) and after 24, 48, 56 and 72 h fermentation for analyses of microbiota. TIM-2 fermentation of each substrate was performed as independent duplicates. The total bacterial DNA was isolated from the microbiota samples using the PowerLyzer@PowerSoil DNA Isolation Kit (Qiagen). To prepare the DNA library, the V3 region of the 16S rRNA gene was PCR amplified using AccuPrime SuperMix II and primers comparable with Nextera Index Kit, NXt_388F and NXt_518R (Illumina). The second PCR step was performed using Phusion High-Fidelity PCR Master and primers P5 and P7 (Nextera Index Kit) to incorporate adapters and tags in the PCR product. Sequencing was conducted using 2x150 cycles, MID output, V2 chemistry on the Illumina NextSeq 500 platform (Illumina, CA, USA). The raw dataset containing pair-ended reads were merged, trimmed and clustered (operational taxonomic units, OTUs, with 97% similarity) using the UPARSE pipeline. Sequencing data were analyzed using QIIME software and the Greengenes database for taxonomy annotation.