Consumer awareness of the link between dietary fats and health outcomes has led to increased demand for food products enriched with bioactive fatty acids (FA). Ruminant-derived fats, such as dairy fats, contribute significantly to the American diet and contain many unique beneficial FA, such as short- and medium-chain FA, n-3 FA, conjugated linoleic acids (CLA), vaccenic acid (VA), as well as odd-and branched-chain FA (OBCFA). Increasing these FA in dairy products by altering farm management practices, such as breed, lactation stage, and nutrition, may improve human health without a change to the diet. The overarching goal of this dissertation was to evaluate on-farm strategies to increase the content of bioactive FA in bovine milk. The first objective was to enrich milk fat with bioactive FA via supplementation with echium oil, a terrestrial oil rich in n-3 FA. Treatments were 1.5% and 3.0% dry matter as lipid encapsulated echium oil (EEO) which were compared to a control (no EEO). Milk fat contents of n-3 FA increased with EEO supplementation but the transfer of n-3 FA from EEO into milk fat was rather low (< 5%). In a subsequent trial, ruminal protection of EEO and post-ruminal release of EEO-derived FA was examined. EEO-derived FA were preferentially incorporated into plasma lipid fractions unavailable to the mammary gland. Moreover, fecal excretion of EEO-derived FA ranged from 7-14% of intake, and VA and CLA, the biohydrogenation and metabolism products of n-3 FA, increased in milk and feces with EEO supplementation. Therefore, lipid-encapsulation provided inadequate digestibility and low transfer efficiency of n-3 FA into milk. The second objective was to compare the bacterial community structure and unique bioactive FA in bacterial membranes and milk fat between Holstein (HO), Jersey (JE), and HO x JE crossbreeds (CB) across a lactation. Lactation stage had a prominent effect on rumen bacterial taxa, with Firmicutes being most abundant during early lactation. The FA composition of bacterial cells was affected by both lactation stage and genetics, and OBCFA in bacterial cells were positively correlated with several bacteria of the Firmicutes phylum. HO and CB exhibited greater contents of various bioactive FA in milk than JE. The highest content of all bioactive FA occurred at early lactation, while OBCFA were highest at late lactation. The third objective was to determine the effects of grazing a monoculture vs. a diverse pasture on rumen bacterial and protozoal taxa, their membrane FA composition, and milk FA. Microbial communities shifted in response to grazing regime accompanied with changes in their membrane FA profiles. Rumen microbiota from cows grazing a diverse pasture had higher contents of n-3 FA and VA, but lower contents of OBCFA. Microbial membrane FA correlated with microbial taxa, the contents of ALA and n-3 FA were positively correlated with the bacterial genus Butyrivibrio and the protozoal genus Eudioplodinium. Milk contents of CLA and n-3 FA increased when cows grazed a diverse pasture, while grazing a monoculture led to greater milk contents of OBCFA. In conclusion, grazing cows on a diverse pasture, when compared to genetic effects and lipid supplementation, was the most efficacious strategy to increase the content of bioactive FA in milk.

Recent findings have shown an inverse association between the circulating levels of pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0) with the risk of pathological development in type 2 diabetes, cardio vascular disease and neurological disorders. From previously published research, it has been said that both these odd chain fatty acids are biomarkers of their dietary intake and are significantly correlated to dietary ruminant fat intake. However, there are profound studies that show the contrary where they do not display this biomarker correlation. Additionally, several astute studies have suggested or shown odd chain fatty acid endogenous biosynthesis, most often suggested via alpha oxidation; the cleavage of a single carbon unit from a fatty acid chain within the peroxisomes. To better understand the correlations and interactions between these two fatty acids with pathological development, the origin of these odd chain fatty acids needed to be determined, along with confirming their association with the disease aetiology. To minimise animal & human experimentation we made use of existing sample sets made available through institutional collaborations, which produced both animal and human interventional study samples suitable for odd chain fatty acid investigations. These sample collaborations allowed us to comprehensively investigate all plausible contributory sources of these odd chain fatty acids; including from the intestinal microbiota, from dietary contributions, and derived from novel endogenous biosynthesis. The investigations included two intestinal germ-free studies, two ruminant fat diet studies, two dietary fat studies and an ethanol intake study. Endogenous biosynthesis was assessed through: a stearic acid infusion, phytol supplementation, and an Hacl1 knockout mouse model. A human dietary intervention study was used to translate the results. Finally, a study comparing circulating baseline C15:0 and C17:0 levels with the development of glucose intolerance. We found that the circulating C15:0 and C17:0 levels were not significantly influenced by the presence or absence of intestinal microbiota. The circulating C15:0 levels were significantly and linearly increased when the C15:0 dietary composition increased; however, there was no significant correlation in the circulating C17:0 levels with intake. Circulating levels of C15:0 were affected by the dietary composition and factors affecting the dietary intake, e.g. total fat intake and ethanol, whereas circulating C17:0 levels were found to be independent of these variables. In our studies, the circulating C15:0 levels were not significantly affected by any expected variations in alpha oxidation caused by pathway substrate inhibition or gene knockout. However, C17:0 was significantly related, demonstrating it is substantially endogenously biosynthesised. Furthermore, we found that the circulating C15:0 levels, when independent of any dietary variations, did not correlate with the progression of glucose intolerance when induced, but the circulating C17:0 levels did significantly relate and linearly correlated with the development of glucose intolerance. To summarise, the circulating C15:0 and C17:0 levels were independently derived; the C15:0 levels substantially correlated with its dietary intake, whilst the C17:0 levels proved to be separately derived from its endogenous biosynthesis via alpha oxidation of stearic acid. C15:0 was found to be minimally endogenously biosynthesised via a single cycle of beta oxidation of C17:0 in the peroxisomes, however, this did not significantly contribute to the circulating levels of C15:0. Additionally, only the baseline levels of C17:0 significantly correlated with the development of glucose intolerance. These findings highlight the considerable differences between both of these odd chain fatty acids that were once thought to be homogeneous and similarly derived. On the contrary, they display profound dietary, metabolic, and pathological differences.

In the human gut, bacterial fermentation of dietary fibers and proteins produces metabolites, primarily as short-chain fatty acids (SCFA), that are highly beneficial for host health. However, unlike dietary fiber, bacterial fermentation of protein additionally generates potentially toxic substances such as ammonia, hydrogen sulfide, amines, and indoles. It is believed that most gut bacteria favor utilization of dietary fiber over that of protein for energy. Therefore, when fermentable dietary fiber is readily available to colonic bacteria, protein fermentation, and its subsequent potentially toxic metabolites, remains relatively low. Dietary intake primarily determines the quantity of dietary fiber and protein substrate available to the gut microbiota and the resulting profile of metabolites produced. Increased protein consumption is associated with deleterious health outcomes such as higher risk of colorectal cancer and type II diabetes. Conversely, diets following US dietary recommendations are high in fiber, which promote a healthy microbiome and are protective against disease. Diets following the recommendation are also moderate in protein intake so that, ultimately, far more fiber than protein is available for colonic bacterial fermentation. On the contrary, dietary fiber intake is chronically low in a standard Western diet, while protein consumption is above dietary recommendations, which results in nearly equal amounts of dietary fiber and protein available for gut microbial fermentation. Furthermore, the popularity of high-protein diets for athletes, as well as that of high-protein low-carbohydrate diets for weight loss, may flip fiber and protein substrate proportions upside down, resulting in more protein than fiber available in the gut for fermentation. The objective of this study was to elucidate how substrate ratios in protein-fiber mixtures affect protein fermentation and metabolites, as well as examine the degree to which fiber source may influence these outcomes. Each dietary fiber source [fructooligosaccharides (FOS), apple pectin (Pectin), a wheat bran and raw potato starch mixture (WB+PS), and an even mixture of the three aforementioned fibers (Even Mix)] and protein were combined in three ratios and provided as substrate for in vitro fecal fermentation to understand how low, medium, and high fiber inclusion levels influence fermentation outcomes. They were compared to 100% protein and fiber (each different fiber) controls. Branched-chain fatty acids (BCFAs), metabolites produced exclusively from protein fermentation, were used as a measure of protein fermentation; the data were normalized based on the initial quantity of protein within the substrate. In protein-fiber substrate mixtures, only FOS and Even Mix inhibited BCFAs (mM/g protein basis) and only when they made up at least half of the substrate. Unexpectedly, the rate of protein fermentation was increased when the protein-fiber substrate contained 25% WB+PS fiber, possibly due to the starch component of the fiber. There was evidence that when pH drops during fermentation, as was the case for protein-FOS mixtures, it played a significant role in suppressing protein fermentation. Ammonia production was not largely affected by increasing the proportion of dietary fiber. A significant reduction did not occur until FOS made up at least 50% of the protein-fiber substrate; for Pectin, WB+PS, and Even Mix fibers, 75% inclusion was required for a significant decrease in ammonia. Interestingly, protein was butyrogenic. Protein as the sole substrate produced more butyrate than either Pectin or Even Mix as the sole substrates, and in fact, addition of Pectin to protein significantly reduced butyrate concentrations. However, the possible benefits of butyrate produced via protein fermentation needs to be tempered by the production of potentially toxic compounds and the association between protein fermentation and colorectal cancer. Overall, the thesis findings showed protein fermentation to be relatively stable and not easily influenced by increasing the availability of dietary fiber, and no clear evidence of microbial preference for carbohydrates over protein was found.

No description available.

<p>The development of a novel charge inversion ion/ion reaction in conjunction with a mass spectrometry technique (collisional induced dissociation (CID)) to induce fragmentation of selected ions species in the gas-phase. The utility of this experiment allows identification of varying saturated and unsaturated classes of glycerophospholipids (GPLs) in a biological matrix. In this work, we are able to characterize GPLs species at the subclass, headgroup, fatty acyl sum compositional levels,  leaving the location(s) of carbon-carbon single bond (C-C), carbon-carbon double bond (C=C), cyclopropane moiety, branching site and differentiate isomeric species. </p> <p><br></p> <p>All data were collected on modified a Sciex QTRAP4000 hybrid triple quadrupole/linear ion trap mass spectrometer. Briefly, alternately, pulsed nano-electrospray ionization (nESI) was used for ion generation. Deprotonated lipid anions were generated via negative ion mode nESI, mass selected during transit through Q1, and transferred to q2 for storage. Next, the charge inversion (IIRXN) reagent doubly charged magnesium complex cations, were generated via positive ion mode nESI. To facilitate the ion/ion reaction, magnesium complex dictations and lipid anions were simultaneously stored in q2, resulting in the formation of charge-inverted lipid cations. Ion-trap CID of charge-inverted isomers resulted in distinctive fragmentation, facilitating differentiation of isomeric and localization of unsaturation sites in acyl chain constituents. </p>