Tyler Culpepper

The Metabolizable Energy of Dietary Resistant Maltodextrin Is Variable and Alters Fecal Microbiota Composition in Adult Men

Resistant maltodextrin (RM) is a novel soluble, nonviscous dietary fiber. Its metabolizable energy (ME) and net energy (NE) values derived from nutrient balance studies are unknown, as is the effect of RM on fecal microbiota. A randomized, placebo-controlled, double-blind crossover study was conducted (n = 14 men) to determine the ME and NE of RM and its influence on fecal excretion of macronutrients and microbiota. Participants were assigned to a sequence consisting of 3 treatment periods [24 d each: 0 g/d RM + 50 g/d maltodextrin and 2 amounts of dietary RM (25 g/d RM + 25 g of maltodextrin/d and 50 g/d RM + 0 g/d maltodextrin)] and were provided all the foods they were to consume to maintain their body weight. After an adaptation period, excreta were collected during a 7-d period. After the collection period, 24-h energy expenditure was measured. Fluorescence in situ hybridization, quantitative polymerase chain reaction, and 454 titanium technology-based 16S rRNA sequencing were used to analyze fecal microbiota composition. Fecal amounts of energy (544, 662, 737 kJ/d), nitrogen (1.5, 1.8, 2.1 g/d), RM (0.3, 0.6, 1.2 g/d), and total carbohydrate (11.1, 14.2, 16.2 g/d) increased with increasing dose (0, 25, 50 g) of RM (P < 0.0001). Fat excretion did not differ among treatments. The ME value of RM was 8.2 and 10.4 kJ/g, and the NE value of RM was -8.2 and 2.0 kJ/g for the 25 and 50 g/d RM doses, respectively. Both doses of RM increased fecal wet weight (118, 148, 161 g/d; P < 0.0001) and fecal dry weight (26.5, 32.0, 35.8 g/d; P < 0.0001) compared with the maltodextrin placebo. Total counts of fecal bacteria increased by 12% for the 25 g/d RM dose (P = 0.17) and 18% for the 50 g/d RM dose (P = 0.019). RM intake was associated with statistically significant increases (P < 0.001) in various operational taxonomic units matching closest to ruminococcus, eubacterium, lachnospiraceae, bacteroides, holdemania, and faecalibacterium, implicating RM in their growth in the gut. Our findings provide empirical data important for food labeling regulations related to the energy value of RM and suggest that RM increases fecal bulk by enhancing the excretion of nitrogen and carbohydrate and the growth of specific microbial populations.

Galactooligosaccharide supplementation reduces stress-induced gastrointestinal dysfunction and days of cold or flu: a randomized, double-blind, controlled trial in healthy university students

BACKGROUND Acute psychological stress induced by academic exams is associated with dysregulated gastrointestinal and immune function. OBJECTIVE We examined whether supplementation with galactooligosaccharides reduced gastrointestinal dysfunction and the percentage of days with cold or flu in academically stressed undergraduate students. DESIGN In a randomized, double-blind study, subjects (n = 427) received 0, 2.5, or 5.0 g galactooligosaccharides for 8 wk around the time of fall final exams. Levels of stress and cold or flu symptom intensity (SI; 0 = not experiencing to 3 = severe) were recorded daily. The SI from 9 cold or flu symptoms was summed with 1 d of cold or flu defined as a sum >6. The Gastrointestinal Symptom Response Scale was completed weekly. RESULTS Stress was positively related to diarrhea, indigestion, and reflux syndromes and with abdominal pain, average daily cold or flu SI score, and the percentage of days with cold or flu. Gastrointestinal symptom scores for diarrhea (P = 0.0298), constipation (P = 0.0342), abdominal pain (P = 0.0058), and indigestion (P = 0.0003) syndromes were lower after galactooligosaccharide supplementation. The cold or flu SI score was affected by galactooligosaccharides and stress (P < 0.0001); 2.5 g was associated with a lower SI score across all levels of stress, but 5.0 g was protective only at lower levels of stress. The percentage of days with cold or flu was associated with galactooligosaccharides within different body mass index categories (P = 0.0002), wherein a 40% reduction in the percentage of days with cold or flu was observed in normal-weight individuals with 5.0 g galactooligosaccharides. This effect was not observed in overweight or obese individuals. CONCLUSIONS Acute psychological stress was directly related to symptoms of gastrointestinal dysfunction and cold or flu. Galactooligosaccharide supplementation reduced these symptoms and the number of days with cold or flu. This trial was registered at clinicaltrials.gov as NCT01137760.

Fecal Lactic Acid Bacteria Increased in Adolescents Randomized to Whole-Grain but Not Refined-Grain Foods, whereas Inflammatory Cytokine Production Decreased Equally with Both Interventions

The intake of whole-grain (WG) foods by adolescents is reported to be approximately one-third the recommended intake of 48 g/d. This 6-wk randomized interventional study determined the effect of replacing grains within the diet with refined-grain (RG; n = 42) or WG (n = 41) foods/d on gastrointestinal and immune health in adolescents (aged 12.7 ± 0.1 y). A variety of grain-based foods were delivered weekly to participants and their families. Participants were encouraged to eat 3 different kinds of study foods (e.g., bread, cereals, snacks)/d with goals of 0 g/d (RG) and 80 g/d (WG). Stool samples were obtained during the prebaseline and final weeks to measure bifidobacteria and lactic acid bacteria (LAB) using qPCR. Stool frequency was recorded daily. Blood was drawn at baseline and at final visits for immune markers. Across groups, total-grain intake increased by one serving. The intake of WG was similar at baseline (18 ± 3 g) between groups but increased to 60 ± 5 g in the WG group and decreased to 4 ± 1 g in the RG group. Fecal bifidobacteria increased from baseline with both interventions, but LAB increased (P < 0.05) from baseline [2.4 ± 0.2 log(10) genome equivalents (eq)] to wk 6 (3.0 ± 0.2 log(10) genome eq) in the WG group but not in the RG group (baseline: 2.9 ± 0.2 log(10) genome eq; wk 6: 3.0 ± 0.1 log(10) genome eq). There was no difference in stool frequency, serum antioxidant potential, or in vitro LPS-stimulated mononuclear cell production of inflammatory cytokines between groups. However, across both groups the number of daily stools tended to increase (P = 0.08) by 0.0034 stools/g WG or by 0.2 stools with 60 g WG, mean antioxidant potential increased by 58%, and mean production of TNF-α, IL-1β, and IL-6 decreased by 24, 22, and 42%, respectively, between baseline and wk 6. Overall, incorporating either WG or RG foods increased serum antioxidant concentrations and decreased inflammatory cytokine production; however, WG study foods had more of an effect on aspects of gastrointestinal health.

Lactobacillus gasseri KS-13, Bifidobacterium bifidum G9-1, and Bifidobacterium longum MM-2 Ingestion Induces a Less Inflammatory Cytokine Profile and a Potentially Beneficial Shift in Gut Microbiota in Older Adults: A Randomized, Double-Blind, Placebo-Controlled, Crossover Study

Objective: This study determined whether older adults who consumed a probiotic mixture would have a greater proportion of circulating CD4+ lymphocytes, altered cytokine production, and a shift in intestinal microbiota toward a healthier microbial community. Methods: Participants (70 ± 1 years [mean ± SEM]; n = 32) consumed a probiotic (Lactobacillus gasseri KS-13, Bifidobacterium bifidum G9-1, and Bifidobacterium longum MM2) or a placebo twice daily for 3 weeks with a 5-week washout period between intervention periods. Blood and stools were collected before and after each intervention. The percentage of circulating CD4+ lymphocytes and ex vivo mitogen-stimulated cell cytokine production were measured. In stools, specific bacterial targets were quantified via quantitative polymerase chain reaction (qPCR) and community composition was determined via pyrosequencing. Results: During the first period of the crossover the percentage of CD4+ cells decreased with the placebo (48% ± 3% to 31% ± 3%, p < 0.01) but did not change with the probiotic (44% ± 3% to 42% ± 3%) and log-transformed concentrations of interleukin-10 increased with the probiotic (1.7 ± 0.2 to 3.4 ± 0.2, p < 0.0001) but not the placebo (1.7 ± 0.2 to 2.1 ± 0.2). With the probiotic versus the placebo a higher percentage of participants had an increase in fecal bifidobacteria (48% versus 30%, p < 0.05) and lactic acid bacteria (55% versus 43%, p < 0.05) and a decrease in Escherichia coli (52% versus 27%, p < 0.05). Several bacterial groups matching Faeacalibactierium prausnitzii were more prevalent in stool samples with the probiotic versus placebo. Conclusions: The probiotic maintained CD4+ lymphocytes and produced a less inflammatory cytokine profile possibly due to the changes in the microbial communities, which more closely resembled those reported in healthy younger populations.

Gut microbiota correlates with energy gain from dietary fibre and appears to be associated with acute and chronic intestinal diseases

Improvements in high-throughput sequencing technologies have spurred a large number of studies aimed at obtaining a better understanding of the composition and the dynamics in gut microbiota and its associations with various human diseases, especially those in the intestinal tract. Here we briefly summarize results from three different such studies from our group, all of which used 454 based high-throughput 16S rRNA sequence analysis combined with other microbiota profiling methods to determine faecal microbiota composition. In the first study, a controlled feeding trial, we establish that energy gain from the consumption of up to 50 g/day of a resistant maltodextrin depends on the prevalent microbiota composition. Over time, resistant maltodextrin supplementation increased the proportion of total faecal bacteria as well as potentially beneficial bifidobacteria. Thus, energy gain from resistant maltodextrin in an individual appears to vary over time and depend on the adaptation of gut microbiota. We then illustrate the power of molecular tools for identifying (i) distortions in early microbiota development in pre-term infants and the presence of potentially novel pathogens contributing to necrotizing enterocolitis and (ii) a specific microbiota signature, based on discriminant analysis of the 16S rRNA sequences, that correlates with the prevalence of an early risk marker associated with colorectal carcinogenesis, intestinal adenoma, in elderly adults.

Bifidobacterium bifidum R0071 decreases stress-associated diarrhoea-related symptoms and self-reported stress: a secondary analysis of a randomised trial

Psychological stress is associated with gastrointestinal (GI) distress. This secondary analysis from a randomised, double-blind, placebo-controlled study examined whether three different probiotics could normalise self-reported stress-associated GI discomfort and reduce overall self-reported stress. Undergraduate students (n=581) received Lactobacillus helveticus R0052, Bifidobacterium longum ssp. infantis R0033, Bifidobacterium bifidum R0071, or placebo. Participants self-reported 2 outcomes for a 6-week period, which included final academic exams: daily level of stress (0=no stress to 10=extremely stressed) and weekly three diarrhoea-related symptoms (DS, 1=no discomfort to 7=severe discomfort) using the GI Symptom Rating Scale. Self-reported stress was positively related to DS (P=0.0068). Mean DS scores were lower with B. bifidum versus placebo at week 2 at the average level of stress and the average body mass index (BMI). DS scores were lower with B. bifidum at week 5 versus week 0 and 1 and with B. infantis R0033 at week 6 versus week 0. DS scores were higher when antibiotics were used in the prior week with placebo (P=0.0092). DS were not different with or without antibiotic use with the probiotics. Only B. bifidum had an effect on self-reported stress scores (P=0.0086). The self-reported stress score was also dependent on hours of sleep per day where it decreased by 0.13 for each additional hour of sleep. During a stressful period, B. bifidum R0071 decreases DS and self-reported stress scores. This trial was registered at clinicaltrials.gov as NCT01709825.

Probiotics (Lactobacillus gasseri KS-13, Bifidobacterium bifidum G9-1, and Bifidobacterium longum MM-2) improve rhinoconjunctivitis-specific quality of life in individuals with seasonal allergies: a double-blind, placebo-controlled, randomized trial

Background: Rhinoconjunctivitis-specific quality of life is often reduced during seasonal allergies. The Mini Rhinoconjunctivitis Quality of Life Questionnaire (MRQLQ) is a validated tool used to measure quality of life in people experiencing allergies (0 = not troubled to 6 = extremely troubled). Probiotics may improve quality of life during allergy season by increasing the percentage of regulatory T cells (Tregs) and inducing tolerance.Objective: The objective of this study was to determine whether consuming Lactobacillus gasseri KS-13, Bifidobacterium bifidum G9-1, and B. longum MM-2 compared with placebo would result in beneficial effects on MRQLQ scores throughout allergy season in individuals who typically experience seasonal allergies. Secondary outcomes included changes in immune markers as part of a potential mechanism for changes in MRQLQ scores.Design: In this double-blind, placebo-controlled, parallel, randomized clinical trial, 173 participants (mean ± SEM: age 27 ± 1 y) who self-identified as having seasonal allergies received either a probiotic (2 capsules/d, 1.5 billion colony-forming units/capsule) or placebo during spring allergy season for 8 wk. MRQLQ scores were collected weekly throughout the study. Fasting blood samples were taken from a subgroup (placebo, n = 37; probiotic, n = 35) at baseline and week 6 (predicted peak of pollen) to determine serum immunoglobulin (Ig) E concentrations and Treg percentages.Results: The probiotic group reported an improvement in the MRQLQ global score from baseline to pollen peak (-0.68 ± 0.13) when compared with the placebo group (-0.19 ± 0.14; P = 0.0092). Both serum total IgE and the percentage of Tregs increased from baseline to week 6, but changes were not different between groups.Conclusions: This combination probiotic improved rhinoconjunctivitis-specific quality of life during allergy season for healthy individuals with self-reported seasonal allergies; however, the associated mechanism is still unclear. This trial was registered at clinicaltrials.gov as NCT02349711.

Associations between diet, gut microbiota and markers of CRC risk

Background Recent advances in our understanding of the contributions of gut microbiota to host metabolism have renewed research interest in how distortions in microbiota activities correlate with health status. While extensive microbiota surveys have already revealed the complexity of microbiota dynamics there is a need to better understand mechanisms through which microbiota can affect host metabolism. We have recently shown that the amount of energy available to the host after intake of resistant maltodextrin varies among individuals and is associated with microbiota. Here we hypothesized that microbiota i) is associated with dietary habits, ii) correlates with colorectal cancer (CRC) risk and iii) can affect the methylation status in nearby gut epithelium, which might be a mechanism contributing to colorectal carcinogenesis.

Abstract A31: Correlations between diet, gut microbiota and epithelial methylation pattern with CRC risk

Purpose: To determine which dietary factors contribute to microbiota variation and identify correlations with gut epithelial methylation pattern associated with the presence of high-risk polyps. Dietary exposures have long been suggested as important risk factors for cancers, especially those of the gastrointestinal tract. Diet is known to affect the commensal gut microbiota, which contributes important activities to the human host that are required for maintaining normal health. However, distortions in microbiota composition and associated microbial activities might contribute to disease processes including colorectal carcinogenesis. High-throughput sequencing based microbiota studies have revealed a large degree of intra- and inter-individual variation, the sources of which are currently not well understood. As part of this project we explored various ‘Big Data’ approaches that can facilitate an efficient mining of the large dataset generated by combining dietary intake data with output from multiple high-throughput technologies. We hypothesized that specific nutrients can affect microbiota composition, and that microbiota correlates with an epithelial methylation pattern associated with CRC risk. Methods: The study included 126 human healthy volunteers undergoing a screening colonoscopy. Dietary habits were assessed using 4-day food records, FFQ (Block 98) and Meat Module Questionnaire. Microbiota composition was examined in a stool samples collected before colonoscopy and in multiple biopsy samples retrieved during the screening procedure. The colonoscopy results were used for ascertainment of polyp status. Microbiota composition was analyzed in DNA extracted from stool and biopsy samples using a 16S rRNA based approach. In addition, in a subset of 39 stool samples a shotgun metagenomic analysis was undertaken. Sequence reads were binned into operational taxonomic units (OTUs) using ESPRIT and further analyzed using our in house analytical pipeline as well as publicly available platforms such as QIIME. We then performed a discriminant analysis to identify a microbiota pattern associated with polyp prevalence. Methylation analysis was performed in DNA extracted from biopsies obtained from six high-risk cases and six matched controls using the Infinium HumanMethylation450 beadchip. We used regression analysis approaches as well as the popular apriori rule-mining algorithm to identify associations in the complex heterogeneous dataset. Results: We observed microbiota associations with dietary intake as well as differences in the presence of OTUs in subjects with and without polyps (p Conclusions: Our observations suggest complex interactions between diet and microbiota that correlate with epithelial methylation pattern and CRC risk. An efficient mining of this and other large datasets, generated by combining multiple layers of complex data, will require adaptation of ‘Big Data’ analytical approaches that can handle the unique characteristics of high dimensional but sparse data. Citation Format: Tyler Culpepper, Maria Ukhanova, Lusine Yaghjyan, Samantha Sites, Yijun Sun, Prosperi Mattia, Volker Mai. Correlations between diet, gut microbiota and epithelial methylation pattern with CRC risk [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr A31.