1. PREBIOTIC EFFECTS OF ALMONDS AND ALMOND SKINS ON INTESTINAL MICROBIOTA IN HEALTHY ADULT HUMANS By: Tess Soper

2. Introduction: Intestinal Microbiota  Intestinal microbiota play an essential role in influencing the health of the host  Good bacteria: Bifidobacterium spp. and Lactobacillus spp.  Bad Bacteria: E Coli and C. perfringens  Intestinal microbiota play an essential role in influencing the health of the host  Good bacteria: Bifidobacterium spp. and Lactobacillus spp.  Bad Bacteria: E Coli and C. perfringens

3. Introduction: Prebiotics  Use as functional food ingredients to manipulate the composition of colonic microbiota in order to improve health  Prebiotic properties: food oligosaccharides and polysaccharides (including dietary fiber)

4. Introduction: Almonds  Almond or almond skin can serve as a candidate food for potential prebiotic effects  Almonds: good source of nutrients and amounts of potential prebiotic indigestible carbohydrates  Almond Skins:  4% of the total almond weight  Flavonoids in skin: may contribute to the health benefits associated with almond consumption  Almond or almond skin can serve as a candidate food for potential prebiotic effects  Almonds: good source of nutrients and amounts of potential prebiotic indigestible carbohydrates  Almond Skins:  4% of the total almond weight  Flavonoids in skin: may contribute to the health benefits associated with almond consumption

5. Rationale  Purpose: to investigate the effects of daily consumption of either almonds or almond skins on the composition of the fecal microbiota and on selected indicators of microbial activity in healthy adult volunteers  Hypothesis: almonds and almond skins possess potential prebiotic properties  Purpose: to investigate the effects of daily consumption of either almonds or almond skins on the composition of the fecal microbiota and on selected indicators of microbial activity in healthy adult volunteers  Hypothesis: almonds and almond skins possess potential prebiotic properties

6. Methods: Subjects 48 subjects (24 male and 24 females) 18-22 years old Similar living environment in dorms Criteria: good health, nonsmokers, stable weight No antibiotics/meds 3 months prior to study No yogurt or products containing bifidobacteria, lactobacilli, or prebiotics in the 3 weeks prior to the study 48 subjects (24 male and 24 females) 18-22 years old Similar living environment in dorms Criteria: good health, nonsmokers, stable weight No antibiotics/meds 3 months prior to study No yogurt or products containing bifidobacteria, lactobacilli, or prebiotics in the 3 weeks prior to the study

7. Methods: Study Design  2 week run-in period (dietary restrictions- no nuts), 6 week treatment period (3 groups), 2 week washout period (return to normal diet) Group 1 (The Control Group): commercial fructooligosaccharides (FOS), 8 g/d (4 g/serving for lunch and supper, which was dissolved in 100 mL drinking water) Group 1 (The Control Group): commercial fructooligosaccharides (FOS), 8 g/d (4 g/serving for lunch and supper, which was dissolved in 100 mL drinking water) Group 2 (The Almond Skin Group): almond skin powder, 10 g/d (5 g/serving for lunch and supper, which was consumed after mix into the basic diet) Group 2 (The Almond Skin Group): almond skin powder, 10 g/d (5 g/serving for lunch and supper, which was consumed after mix into the basic diet) Group 3 (The Almond Group): roasted, unsalted whole almonds, 56 g/d (28 g/serving for lunch and supper, consumed directly), respectively Group 3 (The Almond Group): roasted, unsalted whole almonds, 56 g/d (28 g/serving for lunch and supper, consumed directly), respectively

8. Methods: Fecal Sample Collection  Subjects collected stool samples (10g) in seal specimen cup,  1 specimen cup: weighted into 3 samples (1g), for measurement of fecal pH and water content, enumeration of fecal bacteria, and bacterial enzyme assays  Subjects collected stool samples (10g) in seal specimen cup,  1 specimen cup: weighted into 3 samples (1g), for measurement of fecal pH and water content, enumeration of fecal bacteria, and bacterial enzyme assays Baseline Samples Week 0: at end of Run-in Period Ingestion Samples Week 1, 3,6: during treatment Evacuation Sample Week 8: at the end of wash out period

9. Methods: Measurement of Fecal pH and Water Content Fecal Water Content 1g fecal samples weighed before and after drying Vaccum oven 105 degrees by infrared moisture gauge Fecal pH Measurement 1g feces diluted w/DI Dispersed by vortexing pH measured: lab pH meter with a protein- resistant electrode (at room temp)

10. Methods: Enumeration of Fecal Bacteria 1. Raffinose- Bifidobacterium (RB) agar For enumeration of Bifidobacterium spp. 1. Raffinose- Bifidobacterium (RB) agar For enumeration of Bifidobacterium spp. 2. lactobacilli select (LBS) agar For enumeration of Lactobacillus spp., 2. lactobacilli select (LBS) agar For enumeration of Lactobacillus spp., 3. alizarin- β - galactosidase (ALIZ-GAL) agar For enumeration of E. coli 3. alizarin- β - galactosidase (ALIZ-GAL) agar For enumeration of E. coli 4. sulfite– polymyxin– sulfadiazine (SPS agar ) for enumeration of Clostridum perfringens From each fecal sample: 1 g feces mixed with 99 mL sterile H2O, 10-fold dilutions prepped, 0.1-mL aliquot of each dilution was used to inoculate plates of 4 selective media After incubation: plates examined for bacterial colonies (colony forming units CFU) per g wet feces

11. Methods: Enzyme Assays  Baseline Week 0 and Ingestion Week 6 Samples used for determinations of β -galactosidase, β -glucuronidase, nitroreductase and azoreductase enzyme activities  Samples for Enzyme Assay: 1 g of feces diluted 10-fold in 0.1 M potassium phosphate buffer, homogenized in a blender, sonicated for 15 min, and centrifuged at 12,000 rpm for 10 min at 4 °C.  Measuring the bacterial enzyme activities: expressed as units per gram (U/g) of wet feces  Baseline Week 0 and Ingestion Week 6 Samples used for determinations of β -galactosidase, β -glucuronidase, nitroreductase and azoreductase enzyme activities  Samples for Enzyme Assay: 1 g of feces diluted 10-fold in 0.1 M potassium phosphate buffer, homogenized in a blender, sonicated for 15 min, and centrifuged at 12,000 rpm for 10 min at 4 °C.  Measuring the bacterial enzyme activities: expressed as units per gram (U/g) of wet feces

12. Results

13. Study Subjects: Body Composition, Water Intake, Defecation- no significant differences

14. Control Group: significant decrease in fecal pH at Week 3 Week 1: fecal samples moister & 4 subjects reported mild diarrhea Almond Group: water content decreased at week 3 and 6 Changes in Fecal Moisture and pH during treatment period for all subjects: no significant differences except in control group

15. Microbial Pops in Wet Feces

16. Bifidobacteria: increased significantly after 6 Weeks of ingesting ALL groups Ingestion Period: (Week 1,3,6) High counts throughout for FOS Control and Almond Skin Evacuation Period: (Week 8) pops higher than at baseline for ALL groups Almond Group: no significant change until week 6 onward (sign increase observed) Bifidobacteria: increased significantly after 6 Weeks of ingesting ALL groups Ingestion Period: (Week 1,3,6) High counts throughout for FOS Control and Almond Skin Evacuation Period: (Week 8) pops higher than at baseline for ALL groups Almond Group: no significant change until week 6 onward (sign increase observed) Microbial Pops in Wet Feces: Bifidobacteria

17. Lactobacillus: increased significantly after 6 Weeks of ingesting ALL groups FOS and Almond Group: no significant change until week 6 (significant increase observed) Ingestion Period: Almond Skin group maintined higher levels throughout Evacuation Period: remained high for FOS and Almond Skin, but Almond group levels decreased to initial level Lactobacillus: increased significantly after 6 Weeks of ingesting ALL groups FOS and Almond Group: no significant change until week 6 (significant increase observed) Ingestion Period: Almond Skin group maintined higher levels throughout Evacuation Period: remained high for FOS and Almond Skin, but Almond group levels decreased to initial level Microbial Pops in Wet Feces: Lactobacillus

18. No significant differences during intake of any Group Microbial Pops in Wet Feces: E Coli

19. At Week 6, all groups decreased Evacuation period: the clostridia populations raised to the baseline level for all groups At Week 6, all groups decreased Evacuation period: the clostridia populations raised to the baseline level for all groups Microbial Pops in Wet Feces: C. perfringens

20. Fecal Bacterial Enzymes

21. FOS and Almond Skin : resulted in a significant increase, Almond: increased trend observed FOS and Almond Skin : resulted in a significant increase, Almond: increased trend observed Fecal B-Galactosidase Activities

22.  FOS and Almond: decreased trend in activity  Almond skin: activity decreased significantly  FOS and Almond: decreased trend in activity  Almond skin: activity decreased significantly Fecal B-Glucuronidase Activities

23. All groups: significant decrease of activity FOS Week 6: activity was lower than after almond skin intake or almond intake Fecal Nitroreductase Activities

24.  All groups: activity tended to decrease but no statistically significant differences observed Fecal Azoreductase Activities

25. Discussion: Implication of Results  the fecal pH and water content did not change significantly: fecal pH may not accurately reflect the pH in the colon and depends on absorption of SCFAs and bicarbonate secretion  in the short term, almond and almond skin bring no short term significant changes in body composition characteristics

26. Discussion: Implication of Results  almond skins and almonds possess bifidobacteria and lactobacili stimulation effects  stimulation effects of almond skin and almond intake on bifidobacteria and lactobacilli were different  the stimulation effect of almond intake was not obvious until the end of 6 weeks  eventually (week 6) the pops of bifidobacteria or lactobacilli in both groups reached a similar level (no significant difference)  almond skins and almonds possess bifidobacteria and lactobacili stimulation effects  stimulation effects of almond skin and almond intake on bifidobacteria and lactobacilli were different  the stimulation effect of almond intake was not obvious until the end of 6 weeks  eventually (week 6) the pops of bifidobacteria or lactobacilli in both groups reached a similar level (no significant difference)

27. Discussion: Implication of Results  Almond skin or almond ingestion for 6 weeks also induced a decreasing trend of the viable counts of C. perfringens  an organism known for causing histotoxin and gastrointestinal diseases in humans

28. Discussion: Implication of Results  Indicate the stimulation effects of almond skin and almond intake were typical prebiotic effects

29. Discussion: Implication of Results

30. Important Results  After six weeks of almond skin or almond ingestion, the populations of Bifidobacterium spp. and Lactobacillus spp. increased significantly  Almond skin or almond ingestion for 6 weeks also induced a decreasing trend of the viable counts of C. perfringens  the activity of β -galactosidase increased and the activities of β - glucuronidase, nitroreductase and azoreductase decreased

31. Future Implications  The abundance of dietary fiber and polyphenols may be associated with the prebiotic effects observed upon ingestion of almond skins and almonds.  Further study: explore the specific prebiotic components in almonds and almond skins

32. Conclusion almond skin and almond ingestion lead to an improvement of the intestinal microbiota profile and modify the intestinal bacterial activities induces the promotion of health beneficial factors and inhibition of harmful factors almond skins and almonds possess potential prebiotic properties

33. Questions/Comments