An investigation into the properties of non-digestible carbohydrates that selectively promote colonic propionate production

Short chain fatty acids (SCFA), including propionate, are produced by the bacterial fermentation of carbohydrates in the colon. Propionate has many potential roles in health, including inhibiting cholesterol synthesis, de novo lipogenesis and increasing satiety. The profile of SCFA produced is determined by both the substrate available and the bacteria present and may be influenced by environmental conditions within the lumen of the colon. Whilst it may be beneficial to increase colonic propionate production, dietary strategies to achieve this are unproven. Adding propionate to food leads to poorer organoleptic properties, and oral propionate is absorbed in the small intestine. The optimum way to selectively increase colonic propionate would be to select fermentable carbohydrates that selectively promote propionate production. To date, few studies have undertaken a systematic assessment of the factors leading to increased colonic propionate production making the selection of propiogenic carbohydrates challenging. The aim of this thesis was to identify the best carbohydrates for selectively increasing propionate production, and to explore the factors which control propionate production. This work started with a systematic review of the literature for evidence of candidate carbohydrates, which led to a screen of ‘propiogenic’ substrates using in vitro batch fermentations and mechanistic analysis of the impact of pH, bond linkage and orientation using a range of sugars, polysaccharides and fibre sources. A new unit for SCFA production was developed to allow comparison of results from in vitro studies encompassing a range different methodologies found in the literature. The systematic review found that rhamnose yielded the highest rate and proportion of propionate production whereas, for polysaccharides, β-glucan ranked highest for rate and guar gum ranked highest for molar production, but this was not replicated across all studies. Thus, no single NDC was established as highly propiogenic. Some substrates appeared more propiogenic than others and when these were screened in vitro. Laminarin, and other β-glucans ranked highest for propionate production. Legume fibre and mycoprotein fibre were also propiogenic. A full complement of glucose disaccharides were tested to examine the role glycosidic bond orientation and position on propionate production. Of the glucose disaccharides tested, β(1-4) bonding was associated with increased proportion of propionate and α(1-1) and β(1-4) increased the rate and proportion of butyrate production. In conclusion, it appears that for fibre to affect satiety, high intakes of fibre are needed, and which a major mechanism is thought to occur via propionate. Within this thesis it was identified that rather than selecting specific fibres, increasing overall intakes of highly fermentable carbohydrates is as effective at increasing propionate production. Selecting carbohydrates with beta-bonding, particularly laminarin and other β(1-4) fermentable carbohydrates leads to marginal increases in propionate production. Compared with targeted delivery of propionate to the colon, fermentable carbohydrates examined in this thesis have lesser and variable effects on propionate production. A more complete understanding of the impact of bond configurations in polysaccharides, rather than disaccharides, may help selection or design of dietary carbohydrates which selectively promote colonic propionate production substrates for inclusion in functional foods. Overall this study has concluded that few substrates are selectively propiogenic and the evidence suggests that similar changes in propionate production may be achieved by modest changes in dietary fibre intake

Fiber, a component for health gastrointestinal endocrine system

Dietary fiber was classified according to its solubility in an attempt to relate physiological effects to chemical types of fiber. Soluble fibers (B-glucans, gums, wheat dextrin, psyllium, pectin, inulin) were considered to have benefits on serum lipids, while insoluble fibers (cellulose, lignin, pectins, hemicelluloses) were linked with laxation benefits. More important characteristics of fiber in terms of physiological benefits are viscosity and fermentability. Viscous fibers (pectins, B-glucans, gums, psyllium) are those that have gel-forming properties in the intestinal tract, and fermentable fibers (wheat dextrin, pectins, B-glucans, gum, inulin) are those that can be metabolized by colonic bacteria. Objective: To summarize the beneficial effects of dietary fiber, as nutraceuticals, in order to maintain a healthy gastrointestinal system. Methods: Our study is a systematic review. Electronic databases, including PubMed, Medline, with supplement of relevant websites, were searched. We included randomized and non-randomized clinical trials, epidemiological studies (cohort and case-control). We excluded case series, case reports, in vitro and animal studies. Results: The WHO, the U.S. Food and Drug Administration (FDA), the Heart Foundation and the Romanian Dietary Guidelines recommends that adults should aim to consume approximately 25–30 g fiber daily. Dietary fiber is found in the indigestible parts of cereals, fruits and vegetables. There are countries where people don’t eat enough food fibers, these people need to take some kind of fiber supplement. Evidence has been found that dietary fiber from whole foods or supplements may (1) reduce the risk of cardiovascular disease by improving serum lipids and reducing serum total and low-density lipoprotein (LDL) cholesterol concentrations, (2) decreases the glycaemic index of foods, which leads to an improvement in glycemic response, positive impact on diabetes, (3) protect against development of obesity by increasing satiety hormone leptin concentrations, (4) reduced risk of developing colorectal cancer by normalizes bowel movements, improve the integrity of the epithelial layer of the intestines, increase the resistance against pathogenic colonization, have favorable effects on the gut microbiome, wich is the second genomes of the microorganisms, (5) have a positive impact on the endocrine system by gastrointestinal polypeptide hormonal regulation of digestion, (6) have prebiotic effect by short-chain fatty acids (SCFA) production; butyrate acid is the preferred energy source for colonic epithelial cells, promotes normal cell differentiation and proliferation, and also help regulate sodium and water absorption, and can enhance absorption of calcium and other minerals. Although all prebiotics are fiber, not all fiber is prebiotic. This generally refers to the ability of a fiber to increase the growth of bifidobacteria and lactobacilli, which are beneficial to human health, and (7) play a role in improving immune function via production of SCFAs by increases T helper cells, macrophages, neutrophils, and increased cytotoxic activity of natural killer cells. Conclusion: Fiber consumption is associated with high nutritional value and antioxidant status of the diet, enhancing the effects on human health. Fibers with prebiotic properties can also be recommended as part of fiber intake. Due to the variability of fiber’s effects in the body, it is important to consume fiber from a variety of sources. Increasing fiber consumption for health promotion and disease prevention is a critical public health goal.

Production of bioactive metabolites by intestinal bacteria

The adult intestinal microbiota comprises a microbial ecosystem of approximately 100 trillion microorganisms, with specific bacterial communities holding distinct metabolic capabilities. Bacteria produce a range of bioactive compounds to survive unfavourable stimuli and to interact with other organisms, and generate several bioactive products during degradation of dietary constituents the host is not capable of digesting. This thesis addressed the impact of feeding potential probiotic bacteria and other dietary strategies such as pure fatty acids and prebiotics, on gut microbiota composition, short chain fatty acid (SCFA) production and modulation of metabolism in animal models. In the first experimental chapter (Chapter 2) a gas chromatography method for the quantification of SCFA was optimized and applied in the analysis of caecal samples obtained in animal studies described in other chapters of this thesis. In Chapter 3, t10, c12 CLA supplementation was shown to significantly alter murine gut microbiota composition and SCFA production rather than no supplementation. These changes were suggested to be extra factors affecting host lipid metabolism. Chapter 4 described the contrasting effects of CLA-producing strains, Bifidobacterium breve DPC 6330 and B. breve NCIMB 702258, on murine fat distribution/composition and gut microbiota composition, suggesting that these changes were most likely strain-dependent. In Chapter 5, dietary GABA-producing strain Lactobacillus brevis DPC 6108 was shown to significantly increase (p<0.05) serum insulin in healthy rats, leading to a second experiment using a type 1 diabetes rat model. Lb. brevis DPC 6108 administration did not change insulin levels in diabetic rats, but attenuated high levels of glucose when compared to diabetic control. However, an auto-immune-induced diabetes model was suggested as a better model to study GABA-related effects on diabetes. In Chapter 6 bovine milk oligosaccharides, 6’sialyllactose and Beneo Orafti P95 oligofructose supplementations were associated with depletion or reduction of less favourable bacteria, demonstrating that ingestion of these oligosaccharides might be a safe and effective approach to modulate populations of the intestinal microbiota. In Chapter 7 (General discussion) the major findings of all studies were reviewed and discussed.

New insights into vaginal environment during pregnancy: a multi-omics approach

The present thesis aims to provide a thorough comprehension of the vaginal ecosystem of pregnant women and enhance the knowledge of pregnancy pathophysiology. The first study emphasized the importance of limiting protein intake from animal sources, consuming carbohydrates, and avoiding starting pregnancy overweight to maintain a healthy vaginal environment characterized by lactobacilli and related metabolites. In the second paper, a reduction in bacterial diversity, an increase in Lactobacillus abundance, and a decrease in bacterial vaginosis-related genera were observed during pregnancy. Lactobacillus abundance correlated with higher levels of lactate, sarcosine, and amino acids, while bacterial vaginosis-related genera were associated with amines, formate, acetate, alcohols, and short-chain fatty acids. An association between intrapartum antibiotic prophylaxis for Group B Streptococcus and higher vaginal abundance of Prevotella was found. Moreover, women experiencing a first-trimester miscarriage displayed a higher abundance of Fusobacterium. The third study explored the presence of macrolides and tetracyclines resistance genes in the vaginal environment, highlighting that different vaginal microbiota types were associated with distinct resistance profiles. Lactobacilli-dominated ecosystems showed fewer or no resistance genes, while women with increased bacterial vaginosis-related genera were positive for resistance genes. The last two papers aimed to identify potential biomarkers of vaginal health or disease status. The fourth paper showed that positivity for Torquetenovirus decreased from the first to the third trimester, being more prevalent in women with higher vaginal leukocyte counts. Torquetenovirus-positive samples showed higher levels of cytokines, propionate, and cadaverine. Lactobacillus species decreased in Torquetenovirus-positive samples, while Sneathia and Shuttleworthia increased. The last work pointed out the association between clade 2 of Gardnerella vaginalis and bacterial vaginosis. Moreover, as the number of simultaneously detected G. vaginalis clades increased, bacterial vaginosis-associated bacteria also tended to increase. Additionally, sialidase gene levels negatively correlated with Lactobacillus and positively correlated with Gardnerella, Atopobium, Prevotella, Megasphaera, and Sneathia.

The effects of long (C20/22) and short (C18) chain omega-3 fatty acids on keel bone fractures, bone biomechanics, behavior, and egg production in free-range laying hens.

Keel fractures in the laying hen are the most critical animal welfare issue facing the egg production industry, particularly with the increased use of extensive systems in response to the 2012 EU directive banning conventional battery cages. The current study is aimed at assessing the effects of 2 omega-3 (n3) enhanced diets on bone health, production endpoints, and behavior in free-range laying hens. Data was collected from 2 experiments over 2 laying cycles, each of which compared a (n3) supplemented diet with a control diet. Experiment 1 employed a diet supplemented with a 60:40 fish oil-linseed mixture (n3:n6 to 1.35) compared with a control diet (n3:n6 to 0.11), whereas the n3 diet in Experiment 2 was supplemented with a 40:60 fish oil-linseed (n3:n6 to 0.77) compared to the control diet (n3:n6 to 0.11). The n3 enhanced diet of Experiment 1 had a higher n3:n6 ratio, and a greater proportion of n3 in the long chain (C20/22) form (0.41 LC:SC) than that of Experiment 2 (0.12 LC:SC). Although dietary treatment was successful in reducing the frequency of fractures by approximately 27% in Experiment 2, data from Experiment 1 indicated the diet actually induced a greater likelihood of fracture (odds ratio: 1.2) and had substantial production detriment. Reduced keel breakage during Experiment 2 could be related to changes in bone health as n3-supplemented birds demonstrated greater load at failure of the keel, and tibiae and humeri that were more flexible. These results support previous findings that n3-supplemented diets can reduce fracture likely by increasing bone strength, and that this can be achieved without detriment to production. However, our findings suggest diets with excessive quantities of n3, or very high levels of C20/22, may experience health and production detriments. Further research is needed to optimize the quantity and type of n3 in terms of bone health and production variables and investigate the potential associated mechanisms.

Characterization of hepatic fatty acids in mice with reduced liver fat by ultra-short echo time (1)H-MRS at 14.1 T in vivo.

Alterations in the hepatic lipid content (HLC) and fatty acid composition are associated with disruptions in whole body metabolism, both in humans and in rodent models, and can be non-invasively assessed by (1)H-MRS in vivo. We used (1)H-MRS to characterize the hepatic fatty-acyl chains of healthy mice and to follow changes caused by streptozotocin (STZ) injection. Using STEAM at 14.1 T with an ultra-short TE of 2.8 ms, confounding effects from T2 relaxation and J-coupling were avoided, allowing for accurate estimations of the contribution of unsaturated (UFA), saturated (SFA), mono-unsaturated (MUFA) and poly-unsaturated (PUFA) fatty-acyl chains, number of double bonds, PU bonds and mean chain length. Compared with in vivo (1) H-MRS, high resolution NMR performed in vitro in hepatic lipid extracts reported longer fatty-acyl chains (18 versus 15 carbons) with a lower contribution from UFA (61 ± 1% versus 80 ± 5%) but a higher number of PU bonds per UFA (1.39 ± 0.03 versus 0.58 ± 0.08), driven by the presence of membrane species in the extracts. STZ injection caused a decrease of HLC (from 1.7 ± 0.3% to 0.7 ± 0.1%), an increase in the contribution of SFA (from 21 ± 2% to 45 ± 6%) and a reduction of the mean length (from 15 to 13 carbons) of cytosolic fatty-acyl chains. In addition, SFAs were also likely to have increased in membrane lipids of STZ-induced diabetic mice, along with a decrease of the mean chain length. These studies show the applicability of (1)H-MRS in vivo to monitor changes in the composition of the hepatic fatty-acyl chains in mice even when they exhibit reduced HLC, pointing to the value of this methodology to evaluate lipid-lowering interventions in the scope of metabolic disorders.

Proton flux induced by free fatty acids across phospholipid bilayers: New evidences based on short-circuit measurements in planar lipid membranes

Free fatty acids (FFA) are important mediators of proton transport across membranes. However, information concerning the influence of the Structural features of both FFA and the membrane environment on the proton translocation mechanisms across phospholipid membranes is relatively scant. The effects of FFA chain length, unsaturation and membrane composition on proton transport have been addressed in this study by means of electrical measurements in planar lipid bilayers. Proton conductance (G(H)(+)) was calculated from open-circuit voltage and short-circuit current density measurements. We found that cis-unsaturated FFA caused a more pronounced effect on proton transport as compared to Saturated and trans-unsaturated FFA. Cholesterol and cardiolipin decreased membrane leak conductance. Cardiolipin also decreased proton conductance. These effects indicate a dual modulation of protein-independent proton transport by FFA: through a flip-flop mechanism and by modifying a proton diffusional pathway. Moreover the membrane phospholipid composition was shown to importantly affect both processes. (C) 2009 Elsevier Inc. All rights reserved.

The effect of long-chain polyunsaturated fatty acids intake during pregnancy on adiposity of healthy full-term offspring at birth

Objective - The adjusted effect of long-chain polyunsaturated fatty acid (LCPUFA) intake during pregnancy on adiposity at birth of healthy full-term appropriate-for-gestational age neonates was evaluated. Study Design - In a cross-sectional convenience sample of 100 mother and infant dyads, LCPUFA intake during pregnancy was assessed by food frequency questionnaire with nutrient intake calculated using Food Processor Plus. Linear regression models for neonatal body composition measurements, assessed by air displacement plethysmography and anthropometry, were adjusted for maternal LCPUFA intakes, energy and macronutrient intakes, prepregnancy body mass index and gestational weight gain. Result - Positive associations between maternal docosahexaenoic acid intake and ponderal index in male offspring (β=0.165; 95% confidence interval (CI): 0.031–0.299; P=0.017), and between n-6:n-3 LCPUFA ratio intake and fat mass (β=0.021; 95% CI: 0.002–0.041; P=0.034) and percentage of fat mass (β=0.636; 95% CI: 0.125–1.147; P=0.016) in female offspring were found. Conclusion - Using a reliable validated method to assess body composition, adjusted positive associations between maternal docosahexaenoic acid intake and birth size in male offspring and between n-6:n-3 LCPUFA ratio intake and adiposity in female offspring were found, suggesting that maternal LCPUFA intake strongly influences fetal body composition.

The Effect of Long-Chain Polyunsaturated Fatty Acids Intake During Pregnancy on Adiposity of Healthy Full-Term Offspring at Birth

OBJECTIVE: The adjusted effect of long-chain polyunsaturated fatty acid (LCPUFA) intake during pregnancy on adiposity at birth of healthy full-term appropriate-for-gestational age neonates was evaluated. STUDY DESIGN: In a cross-sectional convenience sample of 100 mother and infant dyads, LCPUFA intake during pregnancy was assessed by food frequency questionnaire with nutrient intake calculated using Food Processor Plus. Linear regression models for neonatal body composition measurements, assessed by air displacement plethysmography and anthropometry, were adjusted for maternal LCPUFA intakes, energy and macronutrient intakes, prepregnancy body mass index and gestational weight gain. RESULT: Positive associations between maternal docosahexaenoic acid intake and ponderal index in male offspring (β=0.165; 95% confidence interval (CI): 0.031-0.299; P=0.017), and between n-6:n-3 LCPUFA ratio intake and fat mass (β=0.021; 95% CI: 0.002-0.041; P=0.034) and percentage of fat mass (β=0.636; 95% CI: 0.125-1.147; P=0.016) in female offspring were found. CONCLUSION: Using a reliable validated method to assess body composition, adjusted positive associations between maternal docosahexaenoic acid intake and birth size in male offspring and between n-6:n-3 LCPUFA ratio intake and adiposity in female offspring were found, suggesting that maternal LCPUFA intake strongly influences fetal body composition.

Increased flow of fatty acids toward beta-oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransferase activity or synthesizing medium-chain-length fatty acids.

Synthesis of polyhydroxyalkanoates (PHAs) from intermediates of fatty acid beta-oxidation was used as a tool to study fatty acid degradation in developing seeds of Arabidopsis. Transgenic plants expressing a peroxisomal PHA synthase under the control of a napin promoter accumulated PHA in developing seeds to a final level of 0. 06 mg g(-1) dry weight. In plants co-expressing a plastidial acyl-acyl carrier protein thioesterase from Cuphea lanceolata and a peroxisomal PHA synthase, approximately 18-fold more PHA accumulated in developing seeds. The proportion of 3-hydroxydecanoic acid monomer in the PHA was strongly increased, indicating a large flow of capric acid toward beta-oxidation. Furthermore, expression of the peroxisomal PHA synthase in an Arabidopsis mutant deficient in the enzyme diacylglycerol acyltransferase resulted in a 10-fold increase in PHA accumulation in developing seeds. These data indicate that plants can respond to the inadequate incorporation of fatty acids into triacylglycerides by recycling the fatty acids via beta-oxidation and that a considerable flow toward beta-oxidation can occur even in a plant tissue primarily devoted to the accumulation of storage lipids.

Plasma phospholipid long-chain n-3 polyunsaturated fatty acids and body weight change.

OBJECTIVE We investigated the association between the proportion of long-chain n-3 polyunsaturated fatty acids (PUFA) in plasma phospholipids from blood samples drawn at enrollment and subsequent change in body weight. Sex, age, and BMI were considered as potential effect modifiers. METHOD A total of 1,998 women and men participating in the European Prospective Investigation into Cancer and Nutrition (EPIC) were followed for a median of 4.9 years. The associations between the proportion of plasma phospholipid long-chain n-3 PUFA and change in weight were investigated using mixed-effect linear regression. RESULTS The proportion of long-chain n-3 PUFA was not associated with change in weight. Among all participants, the 1-year weight change was -0.7 g per 1% point higher long-chain n-3 PUFA level (95% confidence interval: -20.7 to 19.3). The results when stratified by sex, age, or BMI groups were not systematically different. CONCLUSION The results of this study suggest that the proportion of long-chain n-3 PUFA in plasma phospholipids is not associated with subsequent change in body weight within the range of exposure in the general population.

Long-chain n-3 fatty acids and inflammation: potential application in surgical and trauma patients

Lipids used in nutritional support of surgical or critically ill patients have been based on soybean oil, which is rich in the n-6 fatty acid linoleic acid (18:2n-6). Linoleic acid is the precursor of arachidonic acid (20:4n-6). In turn, arachidonic acid in cell membrane phospholipids is the substrate for the synthesis of a range of biologically active compounds (eicosanoids) including prostaglandins, thromboxanes, and leukotrienes. These compounds can act as mediators in their own right and can also act as regulators of other processes, such as platelet aggregation, blood clotting, smooth muscle contraction, leukocyte chemotaxis, inflammatory cytokine production, and immune function. There is a view that an excess of n-6 fatty acids should be avoided since this could contribute to a state where physiological processes become dysregulated. One alternative is the use of fish oil. The rationale of this latter approach is that fish oil contains long chain n-3 fatty acids, such as eicosapentaenoic acid. When fish oil is provided, eicosapentaenoic acid is incorporated into cell membrane phospholipids, partly at the expense of arachidonic acid. Thus, there is less arachidonic acid available for eicosanoid synthesis. Hence, fish oil decreases production of prostaglandins like PGE2 and of leukotrienes like LTB4. Thus, n-3 fatty acids can potentially reduce platelet aggregation, blood clotting, smooth muscle contraction, and leukocyte chemotaxis, and can modulate inflammatory cytokine production and immune function. These effects have been demonstrated in cell culture, animal feeding and healthy volunteer studies. Fish oil decreases the host metabolic response and improves survival to endotoxin in laboratory animals. Recently clinical studies performed in various patient groups have indicated benefit from this approach.

Very long chain n-3 polyunsaturated fatty acids in the food chain in the UK and the potential of animal-derived foods to increase intake

The very long chain (VLC) n-3 polyunsaturated fatty acids (PUFA), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are widely recognised to have beneficial effects on human health. However, recommended intakes of VLC n-3 PUFA (450 mg/day) are not being met by the diet in the majority of the population mainly because of low consumption of oil-rich fish. Current mean intake of VLC n-3 PUFA by adults is estimated to be about 282 mg/day with EPA and DHA contributing about 244 mg/day. Furthermore, the fact that only about 27% of adults eat any oil-rich fish (excluding canned tuna) and knowledge of the poor conversion of α-linolenic acid to EPA and DHA in vivo, particularly in men, leads to the need to review current dietary sources of these fatty acids. Animal-derived foods are likely to have an important function in increasing intake and studies have shown that feeding fish oils to animals can increase the EPA and DHA content of the resulting food products. This paper highlights the importance of examining current and projected consumption trends of meat and other animal products when exploring the potential impact of enriched foods by means of altering animal diets. When related to current food consumption data, potential dietary intakes of EPA+DHA from foods derived from animals fed enriched diets are calculated to be about 231 mg/day. If widely consumed, such foods could have a significant impact on progression of conditions such as cardiovascular disease. Consideration is also given to the sources of VLC n-3 PUFA in animal diets, with the sustainability of fish oil being questioned and the need to investigate the use of alternative dietary sources such as those of algal origin.

Increased n-6 polyunsaturated fatty acids do not attenuate the effects of long-chain n-3 polyunsaturated fatty acids on insulin sensitivity or triacylglycerol reduction in Indian Asians

Background: Indian Asians in Western countries have a higher rate of coronary artery disease than do the indigenous white populations, and this higher rate may be influenced by a dietary imbalance of n-6 and n-3 polyunsaturated fatty acids (PUFAs). Objective: The objective of the study was to test the hypothesis that a high background dietary intake of n-6 PUFA attenuates the effects of fish-oil supplementation on insulin sensitivity and associated blood lipids of the metabolic syndrome. Design: Twenty-nine Indian Asian men were recruited to participate in a 12-wk dietary intervention trial. Volunteers were randomly assigned to receive either a moderate or a high n-6 PUFA diet featuring modified oils and spreads over a 6-wk period. After this 6-wk period, both groups were supplemented with 4.0 g fish oil/d (2.5 g eicosapentaenoic acid + docosahexaenoic acid) for an additional 6 wk in combination with the dietary treatment. Volunteers participated in a postprandial study and an insulin sensitivity test after the 6-wk dietary intervention and again after the fish-oil supplementation period. Results: There was no significant time X treatment interaction for blood lipids or insulin action after dietary intervention with the moderate or high n-6 PUFA diets in combination with fish oil. After the 6-wk period of fish oil supplementation, fasting and postprandial plasma triacylglycerol concentrations decreased significantly. Conclusion: The background dietary n-6 PUFA concentration did not modulate the effect of fish-oil supplementation on blood lipids or measures of insulin sensitivity in this ethnic group.