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.

Inclusion Criteria:

1. Age between 20-55 y

2. BMI between 18.5-29.9 kg/m2

3. Bowel movement ≥3 times/wk

4. Consume ≥3 servings of fruits and vegetables daily on average

5. Willing to consume animal based diets for ≥10 days

6. Willing to consume freeze-dried whole cranberry powder

7. Willing to not take prebiotics and probiotics during the trial

8. Do not have allergic reaction to cranberries

Exclusion Criteria:

1. Colonoscopy 2 mo prior to their enrollment or scheduled during the study

2. No antibiotic medications or drugs known to influence fecal microbiota were taken or
used 3 mo before the study

3. History of a bilateral mastectomy

4. History of autoimmune disorders (rheumatoid arthritis, lupus, multiple sclerosis,
vitiligo, psoriasis)

5. Consume <3 servings of fruits and vegetables daily one average

6. Gastrointestinal diseases, conditions, or medications influencing gastrointestinal
absorption including active peptic ulcer disease or inflammatory bowel disease which
will be found based on the self-report during the screening visit

7. Regular use of any acid-lowering medications (≥3 times/week)

8. Use of ≥14/wk serving of alcohol (168 oz beer, 56 oz wine, 14 oz hard liquor)

9. Intend to be pregnant, pregnancy, and breastfeeding

10. Infrequent (<3/wk) or excessive (>3/d) number of regular bowel movements

11. Active treatment for cancer (except non-melanom skin cancer) and cardiovascular
disease of any type >1 y

12. Having diabetes and/or receiving medications for diabetic condition, which will be
found based on the self-report during the screening visit

13. Thyroid disease unstable or medication adjustments in past 6 months, which will be
found based on the self-report during the screening visit

14. Values of standard blood biochemistries are unacceptable for the study based on study
physician's discretion

15. vegetarians and vegans, unwillingness or inability to consume animal-based foods
including chicken, turkey, beef, eggs, cheese, other milk products, etc. Allergy to
eggs or milk/dairy products.

16. Use anticoagulants, such as heparin, warfarin (coumadin) in past 6 months

17. On or planning to be on a weight loss regimen

18. Regular use of any dietary supplements containing vitamins, minerals, herbal or other
plant-based preparations, fish oil supplements (including cod liver oil) or
homeopathic remedies; however, subjects who are willing to refrain from the use of
these supplements at enrollment and throughout the entire study may be considered
eligible

19. Use of monoamine oxidase inhibitors (MAOIs)

20. Glomerular filtration rate (GFR) <60 mL/min/1.73 m2

21. Total cholesterol (TC) >250 mg/dL

22. Total triglycerides (TG) >300 mg/dL