1. Whole-grain wheat breakfast cereal has a prebiotic effect on the human gut microbiota: a double-blind, placebo-controlled, crossover study By: Tess Soper

2. Whole grain: cereal grain that contains germ, endosperm, bran Ex) wheat Whole grain: cereal grain that contains germ, endosperm, bran Ex) wheat

3. Introduction: Whole Grains  Rich in fermentable carbs (dietary fiber, resistant starch, etc)  Protective against cancer, diabetes, obesity, and CVD  Protective mechanicsm=effect on human gut microbiota dominant members of gut microbiota are mediators of health promoting activites benefical to health: bifidobacterium, lactobacillus, Eubacterium spp., atopobium spp., and certain bacteroides spp.

4. Introduction: Prebiotics  Prebiotics : a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the GI microbiota that confers benefits upon host well-being and health  Health benefits: on bowel habit and constipation, increased minimum absorption, and reducing risk for some chronic diseases

5. Rationale  Purpose: to id differences in the population levels of intestinal bacteria and their metabolic activities upon ingestion of WG vs. WB breakfast cereal and to assess possible health effects by measuring biomarkers of gut health (bowel habit and faecal water genotoxicity) and CVD (blood lipid parameters)  Hypothesis: WGs can influence bacterial populations and their activities (observed protection- impact on gut microbiota)

6. Methods: Subjects Subjects  32 healthy volunteers (20-42 years old, average age of 25 years old)  16 females, 15 males  Healthy: BMI between 20 and 30 kg/m^2, good general health determined by medical questionare,  Eat usual diet and fluid intake during trial periods  Prohibited: Prebiotics, probiotics, high bran or whole grain breakfast cereals, drugs active on GI motility, antibiotics, or laxatives prohibited

7. Methods: Study Design  Double blind, randomized, placebo-controlled crossover dietary intervention  2 week period prior to dietary intervention- followed restricted diet  WG wheat breakfast cereal vs. WB breakfast cereal (Placebo)  Double blinded: Investigators and volunteers were not aware of the breakfast cereal given (WB-placebo or WG) Group 1: 16 volunteers 100% WG breakfast cereal (48g/d) for 3 weeks 2 week washout 100% WB breakfast cereal (placebo) (48g/d) for 3 weeks Group 1: 16 volunteers 100% WG breakfast cereal (48g/d) for 3 weeks 2 week washout 100% WB breakfast cereal (placebo) (48g/d) for 3 weeks Group 2: 15 volunteers 100% WB breakfast cereal (placebo) (48g/d) for 3 weeks 2 week washout 100% WG breakfast cereal (48g/d) for 3 weeks Group 2: 15 volunteers 100% WB breakfast cereal (placebo) (48g/d) for 3 weeks 2 week washout 100% WG breakfast cereal (48g/d) for 3 weeks

8. Methods- Study Design Fecal samples, 24h urine, and 30ml fasting venous blood collected from each volunteer at 5 different time points before and after each treatment arm and 14d after the second treatment arm within 48 h (0,21,36,58,73 d) Diaries- stool frequency, consistency, and intestinal bloating

9. Methods: Study Design  Composition of breakfast cereals:  Energy intake WB (1184kJ/100g) and WG (1442kJ/100g) different  WG: more carbs  WB: more sugars, higher fiber (27g/100g) vs WG (11.8g/100g)  Fat and protein similar

10. Methods  Faecal Microbial populations by fluorescence in situ hybridization:  Population levels of the dominant members of human gut microbiota were determined using flourescence in situ hydridisation and expressed as cells/g feces  Dominant members: Bacteroides spp., bifidobacterium spp., eubacterium rectale group, clostridium histolyticum group, and the lactobacilli/enterococci  Colonic Metabolic Output: (SCFA and Ferulic Acid concentrations)  13 phenolic acids and their metabolites were quantified by LC–MS/MS in urine and plasma after treatment  Plasma ferulic acid: main phenolic compound in wheat

11. Biomarkers of Gut Health  Diaries- record stool frequency and consistency (constipation, hard, formed, soft or diarrhea), intestinal bloating (none mild moderate severe and flatulence(none, mild, moderate, or severe) Fecal Water Genotoxicity  Fecal samples diluted in a stomacher bag and homogenized until uniform consistency, fecal water removed, used for single gel electrophoresis Blood Lipid Parameters  TC, HDL-cholesterol, glucose and insulin, and NEFA measured in blood samples taken at baseline and following ingestion of WB and WG

12. Results: Faecal Bacteria Bifidobacteria: higher in WG (9.3) vs. WB (8.8) ingestion Bifiodobacteria: higher in WG (9.3) vs. Pre-WG (8.5) Bifidobacteria: no changes in pre-WB and WB Lactobacilli/ enterococci: higher WG(9.3) and WB(8.8) vs. Pre-WG(8.5) and Pre-WB(8.9) respectively No significant changes in # of total bacteria, Bacteroides spp., Atopobium spp. and Eubacterium rectale group

13. Results: Colonic metabolic output. increased w/intake WG or WB WB conc. > WG average conc: 2·28 m g/l @ baseline to 5·70 m g/l after WG and 6·22 m g/l after WB consumption

14. Results: Colonic metabolic output No significant changes in fecal concentrations of SCFAs neither between pre- WG and WG and pre-WB and WB or when placebo (WB) and treatment (WG) were compared Acetic Acid Butyric Acid Caproic Acid Propionic Acid

15. Results: Fecal water genotoxicity varied greatly between volunteers at the beg of the: 6·3% tail intensity to 66·1% tail intensity Baseline, WG, WB similar :means no difference between cereal treatments. 66.1% 6.3%

16. Results: Blood lipid/Metabolic parameters Fasting blood samples after intervention WB or WG did not impact blood lipid/metabolic parameters during the short 3-week feeding period No significant differences were observed in these parameters between WG and WB feeding periods Significant reduction in TC shown: for both WG (4.216) and WB (4.385) from highest starting TC conc. (top quartile) TAG TC HDL Glucose Insulin NEFA

17. Biomarkers of gut health: digestive tolerance and stool consistency Stool frequency: WB (37.84) higher vs. WG (26.97) Changes in digestive tolerance: severity and frequency varied greatly, neither resulting in adverse symptomology

18. Discussion  Implications of the results:  different effects of WG and WB cereals on gut microbiota may be due to the different composition  WG: higher non-sugar carbs- might represent fermentable carbohydrates (fiber)  May act as prebiotic  no differences in faecal SCFA after ingestion of WG cereals  SCFA are products of dietary fiber fermentation

19. Discussion  Implications of the results:  significant increase of the ferulic acid plasma concentrations was observed upon ingestion of both WG and WB, without significant differences between the two types of breakfast cereals.  for the first time that in human subjects the regular consumption of WG and WB is followed by a slow and continuous release of phenolic acids into the bloodstream-continously prescence of antioxidants- preserve oxidation targets LDL and TAG- seen in association between WG consumption and prevention of CVD.

20. Discussion  Implications of Results:  Improvement in bowel health for WG and WB  No significant differences in blood lipid or metabolic (glucose or insulin) parameters  Not surprising for a short-term feeding study in healthy individuals  Significant reduction in TC for both breakfast cereals in high TC individuals  No significant change in HDL, indicating that this reduction on TC may be viewed as beneficial in terms of CVD risk  Intake of either breakfast cereal may have a beneficial impact on blood lipid profiles  No difference in faecal water genotoxicity after intervention due to high inter-individual variation (ate open diet besides restrictions)  No differences: WB and WG similar (many of the complex polyphenolic compounds in WB are also in WG wheat)

21. Important Results  Improvement in bowel habit of both WG and WB  Reduced water genotoxicity with both WG and WB  Reduction in TC observed for both WG and WB  WG results in higher numbers of Bifidobacterium spp. in stool samples  WG results in higher numbers of lactobacilli

22. Possible Future Research  longer-term trials in high-risk populations to investigate the efficacy of WG and WB breakfast cereals in impacting on biomarkers of CVD

23. Conclusion  In conclusion…  this study has for the first time demonstrated the differential impact of WG wheat and WB on the microbrial ecology of the human gut and established a prebiotic mode of activity for the WG breakfst cereal investigated with increase pops of bifidobacteria and lactobacilli compared to starting levels of WB.  Increase of the ferulic acid concentration in fasting plasma suggests that the WG intake caused a continuous release of antioxidant in the bloodstream.  This study has established a prebiotic mode of action for WG cereal, which together with antioxidant activities may contribute to the underlying mechanisms of protective health effects of WG wheat.