Buckwheat (Fagopyrum esculentum) Hulls Are a Rich Source of Fermentable Dietary Fibre and Bioactive Phytochemicals.

Pseudo-cereals such as buckwheat (Fagopyrum esculentum) are valid candidates to promote diet biodiversity and nutrition security in an era of global climate change. Buckwheat hulls (BHs) are currently an unexplored source of dietary fibre and bioactive phytochemicals. This study assessed the effects of several bioprocessing treatments (using enzymes, yeast, and combinations of both) on BHs' nutrient and phytochemical content, their digestion and metabolism in vitro (using a gastrointestinal digestion model and mixed microbiota from human faeces). The metabolites were measured using targeted LC-MS/MS and GC analysis and 16S rRNA gene sequencing was used to detect the impact on microbiota composition. BHs are rich in insoluble fibre (31.09 ± 0.22% as non-starch polysaccharides), protocatechuic acid (390.71 ± 31.72 mg/kg), and syringaresinol (125.60 ± 6.76 mg/kg). The bioprocessing treatments significantly increased the extractability of gallic acid, vanillic acid, p-hydroxybenzoic acid, syringic acid, vanillin, syringaldehyde, p-coumaric acid, ferulic acid, caffeic acid, and syringaresinol in the alkaline-labile bound form, suggesting the bioaccessibility of these phytochemicals to the colon. Furthermore, one of the treatments, EC_2 treatment, increased significantly the in vitro upper gastrointestinal release of bioactive phytochemicals, especially for protocatechuic acid (p < 0.01). The BH fibre was fermentable, promoting the formation mainly of propionate and, to a lesser extent, butyrate formation. The EM_1 and EC_2 treatments effectively increased the content of insoluble fibre but had no effect on dietary fibre fermentation (p > 0.05). These findings promote the use of buckwheat hulls as a source of dietary fibre and phytochemicals to help meet dietary recommendations and needs.

Hemp and buckwheat are valuable sources of dietary amino acids, beneficially modulating gastrointestinal hormones and promoting satiety in healthy volunteers.

PURPOSE: This study evaluated the postprandial effects following consumption of buckwheat, fava bean, pea, hemp and lupin compared to meat (beef); focussing on biomarkers of satiety, gut hormones, aminoacids and plant metabolites bioavailability and metabolism. METHODS: Ten subjects (n = 3 men; n = 7 women; 42 ± 11.8 years of age; BMI 26 ± 5.8 kg/m2) participated in six 1-day independent acute interventions, each meal containing 30 g of protein from buckwheat, fava bean, pea, hemp, lupin and meat (beef). Blood samples were collected during 24-h and VAS questionnaires over 5-h. RESULTS: Volunteers consumed significantly higher amounts of most amino acids from the meat meal, and with few exceptions, postprandial composition of plasma amino acids was not significantly different after consuming the plant-based meals. Buckwheat meal was the most satious (300 min hunger scores, p < 0.05).Significant increase in GLP-1 plasma (AUC, iAUC p = 0.01) found after hemp compared with the other plant-based meals. Decreased plasma ghrelin concentrations (iAUC p < 0.05) found on plant (hemp) vs. meat meal. Several plasma metabolites after hemp meal consumption were associated with hormone trends (partial least squares-discriminant analysis (PLS-DA): 4-hydroxyphenylpyruvic acid, indole 3-pyruvic acid, 5-hydoxytryptophan, genistein and biochanin A with GLP-1, PYY and insulin; 3-hydroxymandelic acid and luteolidin with GLP-1 and ghrelin and 4-hydroxymandelic acid, benzoic acid and secoisolariciresinol with insulin and ghrelin. Plasma branched-chain amino acids (BCAAs), (iAUC, p < 0.001); and phenylalanine and tyrosine (iAUC, p < 0.05) were lower after buckwheat comparison with meat meal. CONCLUSION: Plants are valuable sources of amino acids which are promoting satiety. The impact of hemp and buckwheat on GLP-1 and, respectively, BCAAs should be explored further as could be relevant for aid and prevention of chronic diseases such as type 2 diabetes. Study registered with clinicaltrial.gov on 12th July 2013, study ID number: NCT01898351.

Sapogenol is a Major Microbial Metabolite in Human Plasma Associated with High Protein Soy-Based Diets: The Relevance for Functional Food Formulations.

Legumes are a source of health-promoting macro- and micronutrients, but also contain numerous phytochemicals with useful biological activities, an example of which are saponins. Epidemiological studies suggest that saponins may play a role in protection from cancer and benefit human health by lowering cholesterol. Therefore, they could represent good candidates for specialised functional foods. Following the consumption of a soya-rich high-protein weight-loss diet (SOYA HP WL), the concentrations of Soyasaponin I (SSI) and soyasapogenol B (SSB) were determined in faecal samples from human volunteers (n = 10) and found to be between 1.4 and 17.5 mg per 100 g fresh faecal sample. SSB was the major metabolite identified in volunteers' plasma (n = 10) after consumption of the soya test meal (SOYA MEAL); the postprandial (3 h after meal) plasma concentration for SSB varied between 48.5 ng/mL to 103.2 ng/mL. The metabolism of SSI by the gut microbiota (in vitro) was also confirmed. This study shows that the main systemic metabolites of soyasaponin are absorbed from the gut and that they are bioavailable in plasma predominantly as conjugates of sapogenol. The metabolism and bioavailability of biologically active molecules represent key information necessary for the efficient development of functional foods.

Rapid method for quantification of anthocyanidins and anthocyanins in human biological samples.

Using simple solvent extraction and enzymatic hydrolysis, a rapid LC-MS/MS method for quantification of free and conjugated forms of anthocyanidins and anthocyanins in plasma and urine samples was developed and validated. A mixed enzymatic treatment containing ß-glucuronidase (100 U mL-1) and sulfatase (2.5 U mL-1) for 5 min (37 °C; pH 6) was optimal condition for deconjugation of anthocyanidins and anthocyanins in urine and plasma samples. The LC-MS/MS allowed quantifying thirteen different anthocyanidins and anthocyanins simultaneously. The developed LC-MS/MS method was precise and accurate over multiple days and nominal concentrations. The stability assessment study confirmed that the long-term storage and/or periodic use of plasma and urine samples might have a considerable impact on the stability of some anthocyanidins. The method was successfully applied to measure anthocyanidins and anthocyanins in plasma and urine samples following consumption of acute blueberry test meals.

Availability and dose response of phytophenols from a wheat bran rich cereal product in healthy human volunteers.

SCOPE: Phytophenols present in cereals are metabolised to compounds that could be partly responsible for the reduced risk of chronic diseases and all-cause mortality associated with fibre-rich diets. The bioavailability, form and in vivo concentrations of these metabolites require to be established. MATERIALS AND METHODS: Eight healthy volunteers consumed a test meal containing a recommended dose (40 g) and high dose (120 g) of ready-to-eat wheat bran cereal and the systemic and colonic metabolites determined quantitatively by LC-MS. CONCLUSION: Analysis of the systemic metabolomes demonstrated that a wide range of phytophenols were absorbed/excreted (43 metabolites) within 5 h of consumption. These included 16 of the 21 major parent compounds identified in the intervention product and several of these were also found to be significantly increased in the colon. Not all of the metabolites were increased with the higher dose, suggesting some limitation in absorption due to intrinsic factors and/or the food matrix. Many compounds identified (e.g. ferulic acid and major metabolites) exhibit anti-inflammatory activity and impact on redox pathways. The combination of postprandial absorption and delivery to the colon, as well as hepatic recycling of the metabolites at these concentrations, is likely to be beneficial to both systemic and gut health.

Nutritional B vitamin deficiency alters the expression of key proteins associated with vascular smooth muscle cell proliferation and migration in the aorta of atherosclerotic apolipoprotein E null mice.

Low B vitamin status is linked with human vascular disease. We employed a proteomic and biochemical approach to determine whether nutritional folate deficiency and/or hyperhomocysteinemia altered metabolic processes linked with atherosclerosis in ApoE null mice. Animals were fed either a control fat (C; 4 % w/w lard) or a high-fat [HF; 21 % w/w lard and cholesterol (0/15 % w/w)] diet with different B vitamin compositions for 16 weeks. Aorta tissue was prepared and global protein expression, B vitamin, homocysteine and lipoprotein status measured. Changes in the expression of aorta proteins were detected in response to multiple B vitamin deficiency combined with a high-fat diet (P < 0.05) and were strongly linked with lipoprotein concentrations measured directly in the aorta adventitia (P < 0.001). Pathway analysis revealed treatment effects in the aorta-related primarily to cytoskeletal organisation, smooth muscle cell adhesion and invasiveness (e.g., fibrinogen, moesin, transgelin, vimentin). Combined B vitamin deficiency induced striking quantitative changes in the expression of aorta proteins in atherosclerotic ApoE null mice. Deregulated expression of these proteins is associated with human atherosclerosis. Cellular pathways altered by B vitamin status included cytoskeletal organisation, cell differentiation and migration, oxidative stress and chronic inflammation. These findings provide new insight into the molecular mechanisms through which B vitamin deficiency may accelerate atherosclerosis.

Role of pathogen-derived cell wall carbohydrates and prostaglandin E2 in immune response and suppression of fish immunity by the oomycete Saprolegnia parasitica.

Saprolegnia parasitica is a freshwater oomycete that is capable of infecting several species of fin fish. Saprolegniosis, the disease caused by this microbe, has a substantial impact on Atlantic salmon aquaculture. No sustainable treatment against saprolegniosis is available, and little is known regarding the host response. In this study, we examined the immune response of Atlantic salmon to S. parasitica infection and to its cell wall carbohydrates. Saprolegnia triggers a strong inflammatory response in its host (i.e., induction of interleukin-1ß1 [IL-1ß1], IL-6, and tumor necrosis factor alpha), while severely suppressing the expression of genes associated with adaptive immunity in fish, through downregulation of T-helper cell cytokines, antigen presentation machinery, and immunoglobulins. Oomycete cell wall carbohydrates were recognized by fish leukocytes, triggering upregulation of genes involved in the inflammatory response, similar to what is observed during infection. Our data suggest that S. parasitica is capable of producing prostaglandin [corrected] E2 (PGE2) in vitro, a metabolite not previously shown to be produced by oomycetes, and two proteins with homology to vertebrate enzymes known to play a role in prostaglandin biosynthesis have been identified in the oomycete genome. Exogenous PGE2 was shown to increase the inflammatory response in fish leukocytes incubated with cell wall carbohydrates while suppressing genes involved in cellular immunity (gamma interferon [IFN-γ] and the IFN-γ-inducible protein [γ-IP]). Inhibition of S. parasitica zoospore germination and mycelial growth by two cyclooxygenase inhibitors (aspirin and indomethacin) also suggests that prostaglandins may be involved in oomycete development.

Anti-platelet effects of olive oil extract: in vitro functional and proteomic studies.

PURPOSE: Platelets play a key role in haemostasis and wound healing, contributing to formation of vascular plugs. They are also involved in formation of atherosclerosic plaques. Some traditional diets, like the Mediterranean diet, are associated with a lower risk of cardiovascular disease. Components in these diets may have anti-platelet functions contributing to their health benefits. METHODS: We studied the effects of alperujo extract, an olive oil production waste product containing the majority of polyphenols found in olive fruits, through measurement of effects on platelet aggregation and activation in isolated human platelets, and through identification of changes in the platelet proteome. RESULTS: Alperujo extract (40 mg/L) significantly decreased in vitro ADP- (p = 0.002) and TRAP- (p = 0.02) induced platelet activation as measured by the flow cytometry using the antibody for p-selectin (CD62p), but it did not affect the conformation of the fibrinogen receptor as measured by flow cytometry using the antibodies for anti-fibrinogen, CD42a and CD42b. Alperujo extract (100 mg/L) inhibited both collagen- and TRAP-induced platelet aggregation by 5% (p < 0.05), and a combination of hydroxytyrosol and 3,4-dihydroxyphenylglycol were, at least partly, responsible for this effect. Proteomic analysis identified nine proteins that were differentially regulated by the alperujo extract upon ADP-induced platelet aggregation. These proteins represent important mechanisms that may underlie the anti-platelet effects of this extract: regulation of platelet structure and aggregation, coagulation and apoptosis, and signalling by integrin αIIb/ß3. CONCLUSIONS: Alperujo extract may protect against platelet activation, platelet adhesion and possibly have anti-inflammatory properties.

Impact of obesity and leptin on protein expression profiles in mouse colon.

BACKGROUND: Elevated leptin levels in obesity are associated with increased risk of colon pathology, implicating leptin signaling in colon disease. However, leptin-regulated processes in the colon are currently uncharacterized. Previously, we demonstrated that leptin receptors are expressed on colon epithelium and that increased adiposity and elevated plasma leptin in rats are associated with perturbed metabolism in colon tissue. Thus, we hypothesize that obesity disrupts expression of proteins regulated by leptin in the colon. METHODS: A proteomic analysis was conducted to investigate firstly, differences in the colon of mice lacking leptin and leptin signaling (ob/ob and db/db, respectively) by comparing protein expression profiles with wild-type mice. Secondly, responses to leptin challenge in wild-type mice and ob/ob mice were compared to identify leptin-regulated proteins and associated cellular processes. RESULTS: Forty proteins were identified with significantly altered expression patterns associated with differences in leptin status in comparisons between all groups of mice. These proteins are associated with calcium binding, cell cycle, cell proliferation, electron transport chain, energy metabolism, protein folding and transport, redox regulation, structural proteins, and proteins involved in transport and regulation of mucus production. CONCLUSIONS: This study provides evidence that obesity and leptin significantly alter protein profiles of a number of proteins linked to cellular processes in colon tissues that may be linked to the increased risk of colon pathology associated with obesity.

Maternal diets deficient in folic acid and related methyl donors modify mechanisms associated with lipid metabolism in the fetal liver of the rat.

Previously we have examined the effects of diets deficient in folic acid ( - F) or folate deficient with low methionine and choline ( - F LM LC) on the relative abundance of soluble proteins in the liver of the pregnant rat. In the present study we report the corresponding changes in the fetal liver at day 21 of gestation. The abundance of eighteen proteins increased when dams were fed the - F diet. When dams were fed the - F LM LC diet, thirty-three proteins increased and eight decreased. Many of the differentially abundant proteins in the fetal liver could be classified into the same functional groups as those previously identified in the maternal liver, namely protein synthesis, metabolism, lipid metabolism and proteins associated with the cytoskeleton and endoplasmic reticulum. The pattern was consistent with reduced cell proliferation in the - F LM LC group but not in the - F group. Metabolic enzymes associated with lipid metabolism changed in both the - F and - F LM LC groups. The mRNA for carnitine palmitoyl transferase were up-regulated and CD36 (fatty acid translocase) down-regulated in the - F group, suggesting increased mitochondrial oxidation of fatty acids as an indirect response to altered maternal lipid metabolism. In the - F LM LC group the mRNA for acetyl CoA carboxylase was down-regulated, suggesting reduced fatty acid synthesis. The mRNA for transcriptional regulators including PPARalpha and sterol response element-binding protein-1c were unchanged. These results suggest that an adequate supply of folic acid and the related methyl donors may benefit fetal development directly by improving lipid metabolism in fetal as well as maternal tissues.

Gene and protein expression profiles in the foetal liver of the pregnant rat fed a low protein diet.

Foetal growth is particularly sensitive to the protein content of the mother's diet. Microarray data from the foetal liver of pregnant rats fed normal (HP) or reduced protein diets (LP) were compared by gene set enrichment analysis. Soluble proteins from a second portion of the liver were analysed by two-dimensional gel electrophoresis. Genes associated with progesterone, insulin-like growth factor-1 and vascular endothelial growth factor were upregulated in HP compared to LP, in addition to genes associated with cell differentiation and signalling from the extracellular matrix. In contrast, cytokine signalling was downregulated. Proteomics showed that proteins associated with amino acid metabolism, mitochondrial function and cell motility were differentially abundant in the HP compared to the LP groups. These growth factor and extracellular matrix signalling pathways linked to cell motility may be important mediators of the changes in liver structure that occur in utero and persist into adult life.

Aorta protein networks in marginal and acute zinc deficiency.

Human zinc deficiency is a global problem and may influence the development of cardiovascular disease. Our objective was to determine Zn deficiency affected pathways and protein interactions in rat aorta and their likely influence on stress-induced atherogenesis. In two separate studies, rats were given diets acutely (<1 mg Zn/kg) or marginally (6 mg Zn/kg) deficient in Zn. Both studies included Zn adequate controls (35 mg Zn/kg) and the acute deficiency study included a pair-fed group. After 6 wk, proteins from thoracic aorta were separated by 2-DE. Proteins affected by zinc deficiency were identified by principal component analysis. Multiple correlations of identified proteins indicated protein networks of related function. Proteins clusters decreased in zinc deficiency were related to fatty acid and carbohydrate metabolism. Structurally related proteins, including zyxin and over nine transgelin 1 proteins, were either increased or decreased by acute and marginal deficiencies. PKC alpha was significantly decreased in Zn deficiency suggesting that Zn may regulate the phosphorylation of target proteins. Zn deficiency-related changes in structural, carbohydrate and fatty acid-related proteins may be disadvantageous for maintaining vascular health and are consistent with a protective role for zinc in the development of atherosclerosis.

Profiling of mitochondrial associated proteins from rat colon.

Mitochondrial dysfunction, damage and mutations of mitochondrial proteins give rise to a range of ill understood patterns of disease. Although there is significant general knowledge of the proteins and the functional processes of the mitochondria, there is little knowledge of difference about how mitochondria respond and how they are regulated in different organs and tissues. Proteomic profiling of mitochondria and associated proteins involved in mitochondrial regulation and trafficking within cells and tissues has the potential to provide insights into mitochondrial dysfunction associated with many human diseases. The rat colon mitoproteome analysis presented here provides a useful tool to assist in identification and interpretation of mitochondrial dysfunction implicated in colon pathogenesis. 2DPAGE followed by LC/MS/MS was used to identify 430 proteins from mitochondrial enriched fractions prepared from rat colon, resulting in 195 different proteins or approximately 50% of the resolved proteins being identified as multiple protein expression forms. Proteins associated with the colon mitoproteome were involved in calcium binding, cell cycle, energy metabolism and electron transport chain, protein folding, protein synthesis and degradation, redox regulation, structural proteins, signalling and transporter and channel proteins. The mitochondrial associated proteins identified in this study of colon tissue complement and are compared with other recently published mitoproteome analyses from other organ tissues, and will assist in revealing potentially organ specific roles of the mitochondria and organ specific disease associated with mitochondrial dysfunction.

A novel class of CoA-transferase involved in short-chain fatty acid metabolism in butyrate-producing human colonic bacteria.

Bacterial butyryl-CoA CoA-transferase activity plays a key role in butyrate formation in the human colon, but the enzyme and corresponding gene responsible for this activity have not previously been identified. A novel CoA-transferase gene is described from the colonic bacterium Roseburia sp. A2-183, with similarity to acetyl-CoA hydrolase as well as 4-hydroxybutyrate CoA-transferase sequences. The gene product, overexpressed in an Escherichia coli lysate, showed activity with butyryl-CoA and to a lesser degree propionyl-CoA in the presence of acetate. Butyrate, propionate, isobutyrate and valerate competed with acetate as the co-substrate. Despite the sequence similarity to 4-hydroxybutyrate CoA-transferases, 4-hydroxybutyrate did not compete with acetate as the co-substrate. Thus the CoA-transferase preferentially uses butyryl-CoA as substrate. Similar genes were identified in other butyrate-producing human gut bacteria from clostridial clusters IV and XIVa, while other candidate CoA-transferases for butyrate formation could not be detected in Roseburia sp. A2-183. This suggests strongly that the newly identified group of CoA-transferases described here plays a key role in butyrate formation in the human colon.