Microbiota Diversity and Composition and Polyphenol Bioavailability
Microbiota Diversity and Composition and Polyphenol Bioavailability
Composition and diversity of intestinal microbiota that are subject to the influence of diet
 have a significant impact on health in the gut and whole body via complex interactions
 between food constituents, bacteria, and host. Polyphenols are poorly absorbed in the upper
 gastrointestinal tract and reach the colon, where they may have a reciprocal relationship
 with microbiota. Because how polyphenols mediate gut health and reduce risk for other
 pathogenesis remains to be elucidated, we propose to conduct a pilot controlled feeding study
 to examine the impact of polyphenols and other constituents in the free dried whole cranberry
 powder (FWCP) on composition and diversity of gut microbiota, as well as to substantiate
 bioavailability of cranberry polyphenols. The central hypothesis underlying the proposed
 research is that FWCP polyphenols will diminish the magnitude of a plant food free diet
 induced increase in Alistipes, Bilophila and Bacteroides and will increase Enterococcus,
 Bifidobacterium, Eggerthella lenta, and Blautia coccoides-Eubacterium rectale groups. The
 proposed research is novel as no study has examined the specific impact of FWCP polyphenols
 incorporated into a plant food free diet on gut microbiota. This proposed study is innovative
 because the positive results will demonstrate with a high degree of confidence that the
 impact of FWCP constituents on production of beneficial short chain fatty acids, carcinogenic
 bile acids, and atherogenic trimethylamine. The results generated from the study using a
 plant food free dietary regimen as the background diet will provide definite proof on
 microbial modulating actions of FWCP, inform mechanism of actions in urinary tract infection
 (UTI), and be used to formulate messages in consumer communications for gut health.
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