Microbiota Diversity and Composition and Polyphenol Bioavailability

Investigators propose conducting a randomized, controlled feeding, crossover trial in 10 adults who have a regular bowel movement (≥5 times/wk) and are willing to consume a diet free of plant foods. The trial will include two 5-d intervention phases with a 2-wk washout period between crossovers. All meals will be provided to subjects during the intervention phases. Subjects will be randomly assigned to receive either Con or FWCP diet containing 30 gram matched placebo or FWCP. The dose is selected in order to provide sufficient amounts of flavonoid and PAC that can be comparable to the average flavonoid and PAC intake at 157 mg and 95 mg/d, respectively in US. The plant food free diet will be formulated to contain only animal based ingredients. All meals will be designed and overseen by Helen Rasmussen, PhD, RD, and be prepared in the kitchen of the Metabolic Research Unit. Packed frozen meals will be provided to subjects to consume at home or work. Coffee, tea, and other plant-based beverages will not be allowed during the intervention phases.

All subjects will be required to provide their informed consent prior to enrolling in the study. Individuals will be screened for study eligibility according to the exclusion criteria, as followed: 1) no antibiotic medications or drugs known to influence fecal microbiota were taken 3 mo before the study; 2) active treatment for doctor diagnosed diseases, such as cancer, cardiovascular diseases, gastrointestinal diseases; 3) regular use of any dietary supplements; 4) colonoscopy 2 mo prior to their enrollment or scheduled during the study; 5) values of standard blood biochemistries are critically abnormal based on study physician's discretion; and 6) use of ≥14/wk serving of alcohol. Subjects who meet all other eligibility criteria and have values of blood biochemistries within normal ranges will be asked to participate in the study.

During the trial, eligible subjects will be required to attend 1 screening visit and 9 study visits over 5-7 wks. At the end of Visits 2 and 6, subjects will receive the randomly assigned frozen meals and begin consuming the study meals for 5 d. Subjects may consume the study meals ≥5 da until the post-intervention stool sample is collected. The whole stool sample will be placed in a sealed bag and then in a cooler with ice packs and transported to the MRU within 24 h. Morning spot urine and 30 mL fasted blood as well as all other clinical data will be collected before and after each intervention phase. All collected samples will be stored at -80 degree C until uses.

The sample size of the human trial was estimated based on the effect of FWCP on the plant food free diet induced increase in bile tolerant bacteria and on the increase in Enterococcus, Bifidobacterium, Eggerthella lenta, and Blautia coccoides-Eubacterium rectale. Since there is no data in the literature illustrating the effect of FWCP on bile tolerant bacteria for power calculation, investigators will employ in the proposed crossover controlled feeding trial 10 subjects to test our hypothesis.

Fecal DNA will be extracted by enzymatic digestion and bead-beating steps followed by the use of QIAamp Stool DNA Mini Kit (Qiagen). 16S rRNA gene amplicons will be generated from the extracted DNA using PCR with a barcoded primer set targeted to the V4 variable region. Amplicons will be sequenced on an Illumina ® MiSeq Sequencer at the Tufts University Core Facility which is directed by Dr. Anne Kane. Microbiota Analysis will be performed using Qiime v 1.7. Operational taxonomic units will be generated using UClust. Alpha and beta diversity parameters will be calculated using Qiime. Urinary flavonoids and phenolic acids will be determined by our routine HPLC-electrochemical detection method. Anthocyanins and their glucuronidate metabolites in urine will be quantified by a LC-MS/MS assay investigators use routinely. Fecal short chain fatty acids will be determined using a GC-MS method that has been established in our laboratory. The pH value in the fecal water of the feces will be determined using a pH meter after centrifugation. Individual bile acids in feces will be determined using a LC-QTOF-MS method. TMA in feces will be determined using a spectrophotometric assay.