Introduction: Short- and long-term health outcomes of children stem from their first 1000 days of development (3 months prior to conception to 2 years postpartum). Research shows a correlation between poor maternal nutrition and adverse birth outcomes. Various factors such as human breast milk (HBM), gut microbiome (GM), and body mass index (BMI) correlate with nutrition. The purpose of this study was to determine if maternal factors such as BMI impact preterm infant microbiome and short chain fatty acid (SCFA) profiles. Methods: Sample Collection: In order to understand the effect of maternal health factors on neonatal GM, deidentified stool samples were collected from the NICU at the MUSC and were utilized for GM and SCFA analysis at ETSU. Microbiome Analysis: GM analysis was performed on stool samples using the Qiagen QIAmp PowerFecal Pro DNA Kit. DNA was sequenced using Amplicon sequence of the 16s rRNA region with a modified Klindworth et al method. GM was analyzed using CLC Genomics Workbench v. 23 where Alpha diversity indexes were calculated with the Abundance Analysis tool and the Beta diversity (inter-sample diversity) was calculated using the weighted Unifrac metric. Short Chain Fatty Acid Analysis: The stool samples were subjected to SCFA extraction and analysis via a modified Schwiertz et al. method. Results: Significance was observed between the groups in microbiome for; C-section (yes, no), gestation (<28, 28-32, 33-36 weeks), week of sample collection (1, 2, 3, 4, >4 weeks), and maternal BMI + antibiotics (no antibiotics + normal, overweight, or obese BMI and antibiotics + normal, overweight, or obese BMI). Significance was detected between the groups in fecal fermentation for; recreational drug use (use, no use), preeclampsia (preeclamptic, not), sepsis evaluation (yes, no), week of sample collection (1, 2, 3, 4, >4 weeks), and Fenton measurements for birth length, birth weight, and occipital frontal circumference (small, average, large for gestational age). Conclusions: These results provide valuable insights into the various maternal and neonatal factors on the GM and SCFA profiles of preterm infants, which can have implications for their overall health and development. It is possible for future adverse health outcomes of premature neonates to be attenuated through HBM ingested and GM.

Objectives: This study aimed to evaluate physiological and microbiome differences in a murine model consuming a Mediterranean (M) v. Western (W) diet. Methods Study design: To investigate the potential of diets in modifying the fecal microbiota, we used 16 ICR mice per diet split evenly between males and females. Mice were acclimated for 5 weeks, consuming regular chow, before switching to M or W diets. Four same sex mice were housed per cage and randomly assigned M or W diets with 2 cages of male and female mice/diet. All animals were weighed weekly, and stool samples were collected, freeze-dried, and ground. An aliquot was analyzed for short-chain fatty acid (SCFA) to determine the fecal fermentation profile (FFP). Diets: All animals were acclimated to their surroundings while consuming the standard mouse chow diet for 5 wks. Experimental diets were from Envigo for 10 wks. M v.. W diets were 24.2v. 17.3% protein, 42.3 v. 48.5% carbohydrate, 13 v. 21.2 % fat, 9 v. 5% fiber, and energy density of 3.6 v. 4.5 kcal/g, and were offered ad libitum. Fecal Fermentation Profile: Fecal samples had SCFA extracted and analyzed using a modified Schwiertz et al. method via gas chromatography. The resulting SCFA profiles were used to determine differences between diets. Statistical Model: General linear models examined main effects of diet, sex, and week while accounting for baseline value and a random effect for cage (SCFA) or mouse (weight). Results: Diet effects for M v. W diets for SCFA were Acetate 50.12 v. 38.45% (p< 0.01), Propionate 14.19 v. 7.98% (p< 0.01), Butyrate 8.37 v. 17.27% (p< 0.01), Caproate 16.19 v. 23.68% (p< 0.01), and Caprillic 0.44 v. 0.68% (p< 0.01). Sex comparisons showed higher percentages of Butyrate (p< 0.05) for males and higher percentages of Isobutyrate (p< 0.01), Isovalerate (p< 0.01), and Propionate (p=0.02) for female mice. On the Mediterranean diet, male mice gained more weight than female mice, 4.96g v. 2.86g compared to baseline (p<0.01). Animals on a Western diet approached significance by gaining more weight (p<0.0871).

Chronic conditions such as obesity, inflammatory bowel disease (IBD), and colitis are associated with gastrointestinal (GI) inflammation and compromised GI barrier integrity. Cocoa may be a potential dietary strategy to mitigate gut-related conditions and been shown to elicit anti-inflammatory, antioxidant, and prebiotic effects. Alkali treatment of cocoa was once thought to reduce its bioactivity, but new evidence suggests it may enhance cocoa's health properties, through the formation of new, potentially bioactive high molecular weight compounds. Inulin, a fructose-containing plant polymer, exerts prebiotic effects and has also been investigated in the mitigation of IBD. This study aims to 1) investigate effects of alkali processing on gut health related bioactivity and phytochemical composition of cocoa and 2) evaluate potential additive benefits of combining cocoa and inulin. Polyphenolic and flavanol compounds in natural cocoa, alkalized cocoa, and inulin powders were characterized using Folin-Ciocalteu (total polyphenols) and 4-dimethylaminocinnamaldehyde (total flavanols) assays, thiolysis , and HILIC UPLC-MS/MS. Treatments of cocoa and inulin were made in 1:2 cocoa:inulin and 1:4 cocoa:inulin mixtures for both natural and alkalized cocoas. Cocoa mixtures, in addition to both cocoa powders and inulin alone, were subjected to an in-vitro digestion to generate material for an in-vitro fecal fermentation. Samples collected from the fermentation at 0, 6, 12, and 24 hours were analyzed via HPLC-MS for microbial metabolites, applied to HT-29 colon cancer cells to assess anti-inflammatory activity, and applied to a florescence assay measuring PLA2 inhibitory activity. The alkalized cocoa powder was found to have a significantly lower concentration of total polyphenols and total flavanols, as well as a lower mDP, suggesting that alkalization may affect larger procyanidins more than smaller flavanol compounds. Inulin enhanced the inhibition of the PLA2 enzyme and enhanced the IL-8 anti-inflammatory properties of cocoa, although the trends were weak. Overall, we did not see any clear, significant effects of alkalization or the addition of inulin to cocoa's colonic metabolite formation or its gut bioactivity in vitro. However, we have demonstrated that colonic fermentation of cocoa may have a negative effect on its bioactivity in vitro. Future research should further explore flavanol DP and bioactivity, fiber's interaction with polyphenols, colonic metabolism of cocoa, and cocoa's gut health effects in vivo. / Master of Science in Life Sciences / Gut conditions like obesity-associated inflammation and inflammatory bowel disease are highly prevalent, debilitating, and currently have no cure. Cocoa has been investigated as a possible dietary strategy for the mitigation and prevention of chronic inflammatory gut conditions due to its anti-inflammatory and enzyme inhibiting properties. Most attribute these effects of cocoa to its abundance of compounds called polyphenols. It is widely thought that the ability of cocoa to promote health is lost when cocoa beans are processed, because of the loss of polyphenols. Alkalization, or "Dutching", is an optional step in cocoa processing that some manufacturers perform to enhance flavor and color formation. Dutching cocoa can promote the polymerization of many smaller, flavanol, protein, and other compounds into larger, indigestible compounds. These indigestible compounds will not be absorbed in the small intestine and may be broken down in the large intestine by colonic bacteria, forming new metabolites. We obtained cocoa powders, one natural (not alkalized) and one alkalized and compared them in terms of content of polyphenols, bioactivities, and anti-inflammatory abilities. Additionally, we added a known prebiotic, inulin, to our cocoa formulations to determine if there are additive benefits of cocoa and inulin together. Ultimately, we found that alkalized cocoa contained lower concentrations of all polyphenolic compounds, even the larger compounds. Inulin enhanced the inhibition of digestive enzymes and the anti-inflammatory properties of cocoa, though not significantly. Inulin also reduced the pH (i.e. increased the acidity) of a simulated gut environment, which may be beneficial. Alkalization did not significantly affect cocoa's enzyme inhibitory activity or anti-inflammatory activity. Overall, the addition of inulin to cocoa does not seem to be effective in increasing cocoa's ability to treat and prevent gut diseases, but more information is needed.

The gut microbiota and its metabolites have vast impacts on the human digestive system, immune system, and health outcomes. Short chain volatile fatty acids (SCVFAs) present in feces can be representative of the interactions of the microbiota present in the gut. Low microbiota diversity in the human gut is highly associated with obesity and adverse health outcomes. Furthermore, the maternal microbiome has a direct impact on neonatal microbiota through various pathways such as environment, skin flora, breast milk composition, and vaginal secretions. This study is aimed to further understand the associations between various factors (maternal adiposity, gestational time, length of life, delivery mode, and race/ethnicity ) and neonatal microbiome and its metabolites, SCFA. Data (pre-pregnancy BMI, gestational time, length of life at time of sample collection, delivery mode, race/ethnicity, SCVFA profiles, fecal fermentation profiles, and 16s rRNA sequences, n=75) was obtained from 75 mother-infant dyads. Qiagen CLC Genomics Workbench was used to process 16s RNA data, generate quantitative and qualitative measures of alpha and beta diversity, and generate an analysis of the composition of microbiomes for differential abundances. Multiple metrics were analyzed for alpha and beta diversity and no significant differences were found for acetic acid (A), propionic acid (P), butyric acid (B), or APB combined. Shannon diversity index, a measure of Alpha diversity, showed no significant difference between groups in each subset. BMI differences were significant for no c-section vs. c-section and Black vs. White race/ethnicity. There were no significant differences found in PERMANOVA, a measure of beta diversity, or found in differential abundances among the groups.

ABSTRACT Influence of fenugreek gum on bread and in vitro physiological effects Keisha T. Roberts Advisors: University of Guelph Dr. S.W. Cui Professor T.E. Graham This thesis examined the effect of fenugreek gum, from Canadian grown fenugreek on bread quality, when substituted for wheat flour at 5 % and 10 %, and the in vitro physiological effects of these breads, based on models of acute and long - term feeding. Study I determined bread could be produced with 10 % fenugreek gum, while maintaining quality parameters of volume and texture, comparable to a control. This was accomplished through the development of a novel bread production method, using the lamination procedure for puff pastry production. The behavior of fenugreek gum and starch (wheat flour) was determined by rapid visco analysis (RVA), farinograph and dynamic rheological measurement, while scanning electron microscopy of bread found fenugreek gum could be identified within the bread matrix. Study 2 in vitro starch digestion found fenugreek gum at 5 % and 10 % reduced glucose liberated from bread, with 10 % fenugreek gum causing a reduction of over 30 %. RVA of fenugreek breads highlighted differences in viscosity between breads and wheat flour substituted with the gum. This was substantial as viscosity measurements by RVA are conducted on raw ingredients and not the food as consumed, which reflects the possible reduction in viscosity with food processing. This study also determined extruding fenugreek gum may have caused morphological changes to the gum, which may possibly contribute to attenuation of glucose liberated in vitro. Study 3 evaluated the accumulation of short chain fatty acids (SCFA) from the fermentation of three substrates: Extruded fenugreek gum, bread with 10 % extruded gum and control bread, based on fecal microbiota from three donors. SCFA profiles varied with substrates and donors, with fenugreek gum having the highest accumulation of SCFA after 12 hours. Donors were a caucuasian Canadian, a black Jamaican and a black Trinidadian who was the only donor culturally exposed to fenugreek. This Trinidadian’s SCFA profiles were consistently higher for fenugreek gum than the other donors. These studies collectively showed fenugreek gum, though viscous could be successfully incorporated into bread and have potential as a functional food and nutraceutical.