Location: Functional Foods Research
Project Number: 5010-41000-182-001-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2020
End Date: Sep 30, 2022
Objective:
The objective is to determine the role of microbiome composition in contributing to gas production during fermentation of pulses and the effect of dual-processing operation on the reduction of oligosaccharide concentration and increase in the accessibility of dietary fibers to fermentation by the gut microbiota.
Approach:
In vitro digestion and fermentation: In vitro digestion of processed dry bean products will be performed using a simulated gastric digestion followed by a small intestinal phase. Following digestion, materials will be dialyzed to simulate absorption of food components. Dialysis tubing with a molecular weight cut off of 1000 Da will be used to remove digested sugars and amino acids while retaining any remaining non-digestible oligosaccharides.
In vitro fermentation will be performed after in vitro digestion by suspending the digested material in a sterile nutrient basal medium and then inoculating with a human fecal microbiota slurry. Stool samples containing and not containing Megasphaera Elsdenii, a lactate metabolizer, will be compared for gas production, carbohydrate fermentation, and short chain fatty acid production. In subsequent experiments, communities containing an enrichment of M. elsdenii will be mixed with microbiomes devoid of M. elsdenii to examine the effects on gas production. All procedures involving human subjects will be conducted in accordance with guidelines approved by the cooperator’s internal review policies.
Fermentation analyses: Gas produced during in vitro fermentation will be monitored in gas tight syringes affixed above the fermentation vessels.
Dietary fiber fermentation will be quantified as the percentage of non-digestible carbohydrates utilized by the microbiota during in vitro fermentation. This will be determined by quantifying the non-digestible carbohydrates at the beginning and end of fermentation.
Short chain fatty acids, including butyrate, will be quantified in the fermentation media using gas chromatography.
Microbiota analysis will be performed by first separating the cells from the fermentation slurry by centrifugation (8000g). DNA will be isolated from the pellets after mechanical and enzymatic bacterial cell lysis using a commercially available kit. Microbiota characterization will be performed by amplicon sequencing of the V4 region (tentatively) of the 16S rRNA gene with a high-throughput sequencing platform. Sequenced reads will be processed, and will undergo quality control and trimming and denoising using publicly available software. Taxonomy will then be assigned using a commercially available database.
Subject dietary pattern: Subjects that provide stool samples will complete an online diet history questionnaire through the NIH, National Cancer Institute website. Dietary fiber intake (and intake of other nutrients) as well as pulse intake will be calculated using the online tools available on the NIH NCI website.
