Effect of oat and soybean rich in distinct non-starch polysaccharides on fermentation, appetite regulation and fat accumulation in rat.

Consumption of non-starch polysaccharides (NSP) is associated with reduced risk of obesity. This study aimed to compare the effects of cereals (oats) and legumes (soybean), rich in different classes of NSP, on appetite regulation and fat accumulation in rats. Soy pectin fermented more efficient than cereal arabinoxylan in rats. Soy pectin and oat ß-glucan were utilized mainly in the caecum of rats. Only small amount of maltodextrin, cello-oligosaccharides and xylo-oligosaccharides were detected in the digesta. Caecal fermentation of soy pectin produced significantly higher concentration of short chain fatty acids (SCFAs) compared to the control. Retroperitoneal (RP) fat-pad weight was significantly lower for rats fed with soybean meal enriched diet than for controls. An inverse correlation between rat RP fat-pad weight and concentration (and proportion) of butyrate was observed. Consumption of soy pectin and oat ß-glucan enriched foods to produce targeted SCFAs in vivo could be a potential strategy to lower fat mass accumulation and a potential tool to manage obesity.

Mass spectrometric characterisation of avenanthramides and enhancing their production by germination of oat (Avena sativa).

Avenanthramides are amides, with a phenylalkenoic acid (PA) and an anthranilic acid (AA) subunit, which are secondary metabolites of oat. Oat seeds were germinated, extracted, and the avenanthramides analysed by a combination of UHPLC with ion trap and high resolution ESI-MS. Typical fragmentation pathways with corresponding diagnostic fragments belonging to the PA and AA subunits were identified and summarised in a decision guideline. Based on these findings 28 unique avenanthramides were annotated in the oat seed(ling) extracts, including the new avenanthramide 6f (with a 4/5-methoxy AA subunit). Avenanthramide content increased by 25 times from seed to seedling. Avenanthramides 2p, 2c, and 2f, which are commonly described as the major avenanthramides, represented less than 20% of the total content in the seedlings. Future quantitative analyses should, therefore, include a wider range of avenanthramides to avoid underestimation of the total avenanthramide content.

Structure Dependent-Immunomodulation by Sugar Beet Arabinans via a SYK Tyrosine Kinase-Dependent Signaling Pathway.

There is much interest in the immunomodulatory properties of dietary fibers but their activity may be influenced by contamination with microbial-associated molecular patterns (MAMPs) such as lipopolysaccharide (LPS) and lipoteichoic acids, which are difficult to remove completely from biological samples. Bone marrow-derived dendritic cells (BMDCs) from TLR2x4 double-KO mice were shown to be a reliable approach to analyse the immunomodulatory properties of a diverse range of dietary fibers, by avoiding immune cell activation due to contaminating MAMPs. Several of the 44 tested dietary fiber preparations induced cytokine responses in BMDCs from TLR2x4 double-KO mice. The particulate fractions of linear arabinan (LA) and branched arabinan (BA) from sugar beet pectin were shown to be strongly immune stimulatory with LA being more immune stimulatory than BA. Enzymatic debranching of BA increased its immune stimulatory activity, possibly due to increased particle formation by the alignment of debranched linear arabinan. Mechanistic studies showed that the immunostimulatory activity of LA and BA was independent of the Dectin-1 recognition but Syk kinase-dependent.

Resolubilization of Protein from Water-Insoluble Phlorotannin-Protein Complexes upon Acidification.

Marine phlorotannins (PhT) from Laminaria digitata might protect feed proteins from ruminal digestion by formation of insoluble non-covalent tannin-protein complexes at rumen pH (6-7). Formation and disintegration of PhT-protein complexes was studied with ß-casein (random coil) and bovine serum albumin (BSA, globular) at various pH. PhT had similar binding affinity for ß-casein and BSA as pentagalloyl glucose, as studied by fluorescence quenching. The affinity of PhT for both proteins was independent of pH (3.0, 6.0, and 8.0). In the presence of PhT, the pH range for precipitation of tannin-protein complexes widened to 0.5-1.5 pH units around the isoelectric point (pI) of the protein. Complete protein resolubilization from insoluble PhT-protein complexes was achieved at pH 7 and 2 for ß-casein and BSA, respectively. It was demonstrated that PhT modulate the solubility of proteins at neutral pH and that resolubilization of PhT-protein complexes at pH deviating from pI is mainly governed by the charge state of the protein.

Enzymatic Browning in Sugar Beet Leaves (Beta vulgaris L.): Influence of Caffeic Acid Derivatives, Oxidative Coupling, and Coupled Oxidation.

Sugar beet (Beta vulgaris L.) leaves of 8 month (8m) plants showed more enzymatic browning than those of 3 month (3m). Total phenolic content increased from 4.6 to 9.4 mg/g FW in 3m and 8m, respectively, quantitated by reverse-phase-ultrahigh-performance liquid chromatography-ultraviolet-mass spectrometry (RP-UHPLC-UV-MS). The PPO activity was 6.7 times higher in extracts from 8m than from 3m leaves. Substrate content increased from 0.53 to 2.45 mg/g FW in 3m and 8m, respectively, of which caffeic acid glycosyl esters were most important, increasing 10-fold with age. Caffeic acid glycosides and vitexin derivatives were no substrates. In 3m and 8m, nonsubstrate-to-substrate ratios were 8:1 and 3:1, respectively. A model system showed browning at 3:1 ratio due to formation of products with extensive conjugated systems through oxidative coupling and coupled oxidation. The 8:1 ratio did not turn brown as oxidative coupling occurred without much coupled oxidation. We postulate that differences in nonsubstrate-to-substrate ratio and therewith extent of coupled oxidation explain browning.

The position of prenylation of isoflavonoids and stilbenoids from legumes (Fabaceae) modulates the antimicrobial activity against Gram positive pathogens.

The legume plant family (Fabaceae) is a potential source of antimicrobial phytochemicals. Molecular diversity in phytochemicals of legume extracts was enhanced by germination and fungal elicitation of seven legume species, as established by RP-UHPLC-UV-MS. The relationship between phytochemical composition, including different types of skeletons and substitutions, and antibacterial properties of extracts was investigated. Extracts rich in prenylated isoflavonoids and stilbenoids showed potent antibacterial activity against Listeria monocytogenes and methicillin-resistant Staphylococcus aureus at concentrations between 0.05 and 0.1% (w/v). Prenylated phenolic compounds were significantly (p<0.01) correlated with the antibacterial properties of the extracts. Furthermore, the position of the prenyl group within the phenolic skeleton also influenced the antibacterial activity. Overall, prenylated phenolics from legume seedlings can serve multiple purposes, e.g. as phytoestrogens they can provide health benefits and as natural antimicrobials they offer preservation of foods.

Effects of in vitro fermentation of barley ß-glucan and sugar beet pectin using human fecal inocula on cytokine expression by dendritic cells.

SCOPE: This study simulates the fermentation process of barley ß-glucan and sugar beet pectin in the human colon and monitors the degradation products formed. Additionally, immune effects of the degradation products were investigated. METHODS AND RESULTS: Immunostimulatory activity of fermentation digesta was investigated using bone marrow derived dendritic cells (BMDCs) from toll-like receptor 2/4 (TLR2/4) knockout mice, which were unresponsive to microbe-associated molecular patterns. Cytokine responses were elicited to dietary fibers and not to the SCFA and microbiota. The fermentation digesta were analyzed for their SCFA profiles and glycan metabolites over time. During fermentation the amount of insoluble precipitating fibers increased and induced as well as soluble molecules of lower molecular mass greater amounts of cytokines in BMDCs than the parental fiber. Additionally, high amounts of cytokines can be attributed to soluble galactose-rich beet pectin molecules. CONCLUSIONS: The fermentation of the two fibers led to fiber-specific amounts of SCFA, glycosidic metabolites, and different immunomodulatory properties. BMDC from TLR2/4 knockout mice did not respond to the digest microbiota and SCFA, making it a useful approach to study temporal effects of fermentation on the immunomodulatory effects of fibers.

Effects of pectin on fermentation characteristics, carbohydrate utilization, and microbial community composition in the gastrointestinal tract of weaning pigs.

SCOPE: We aimed to investigate the effects of three different soluble pectins on the digestion of other consumed carbohydrates, and the consequent alterations of microbiota composition and SCFA levels in the intestine of pigs. METHODS AND RESULTS: Piglets were fed a low-methyl esterified pectin enriched diet (LMP), a high-methyl esterified pectin enriched diet (HMP), a hydrothermal treated soybean meal enriched diet (aSBM) or a control diet (CONT). LMP significantly decreased the ileal digestibility of starch resulting in more starch fermentation in the proximal colon. In the ileum, low-methyl esterified pectin present was more efficiently fermented by the microbiota than high-methyl esterified pectin present which was mainly fermented by the microbiota in the proximal colon. Treated soybean meal was mainly fermented in the proximal colon and shifted the fermentation of cereal dietary fiber to more distal parts, resulting in high SCFA levels in the mid colon. LMP, HMP, and aSBM decreased the relative abundance of the genus Lactobacillus and increased that of Prevotella in the colon. CONCLUSION: The LMP, HMP, and aSBM, differently affected the digestion processes compared to the control diet and shaped the colonic microbiota from a Lactobacillus-dominating flora to a Prevotella-dominating community, with potential health-promoting effects.

Transgenic modification of potato pectic polysaccharides also affects type and level of cell wall xyloglucan.

BACKGROUND: Genes encoding pectic enzymes were introduced into wild-type potato Karnico. Cell wall materials were extracted from Karnico and transgenic lines expressing ß-galactosidase (ß-Gal-14) or rhamnogalacturonan lyase (RGL-18). Pectic polysaccharides from the ß-Gal-14 transgenic line exhibited rhamnogalacturonan-I structural elements with shorter galactan side chains, whereas the RGL-18 transgenic line had less rhamnogalacturonan-I structures than Karnico. Xyloglucan in primary cell walls interacts with pectin and other cell wall polysaccharides and controls cell growth. RESULTS: Xyloglucan extracts from transgenic lines had different levels of monosaccharides compared to wild-type. Most XXGG-type xyloglucans from Karnico and RGL-18 alkali-extractable extracts predominantly consisted of XXGG and XSGG building blocks. Karnico and RGL-18 4 mol L-1 extracts had small proportions of the XXXG-type xyloglucan, whereas ß-Gal-14 extracts also contained the XXXG-type xyloglucan. The peak ratios of XSGG/XXGG were 1.9, 2.4 and 1.1 for 4 mol L-1 extracts of Karnico, RGL-18 and ß-Gal-14 lines, respectively. CONCLUSION: After transgenic modification on pectin, the xyloglucan building blocks may have been changed. The ß-Gal-14 lines mostly present XXXG-type repeating units instead of the XXGG-type in 4 mol L-1 extracts. The ratio of XSGG/XXGG repeating units also changed, indicating that the transgenic modification of pectin altered xyloglucan structure during plant development. © 2016 Society of Chemical Industry.

Evaluation of both targeted and non-targeted cell wall polysaccharides in transgenic potatoes.

In this study, we analyze 31 transgenic lines and their respective untransformed background lines to determine the transgene effects on targeted structures including the pectin components rhamnogalacturonan I (RG-I) and homogalacturonan (HG), neutral side chains (galactan/arabinangalactan), acetylation of pectin, and cellulose level. Modification arising from the pectin backbone- or pectin side chain transgenic lines either increased or decreased the HG:RG-I ratio, side chain length, and methyl esterification of pectin in the tuber cell wall. The pectin esterification transgenic line exhibited only limited side effects. The cellulose level-targeting transgenic lines yielded an unexpectedly high HG:RG-I ratio and longer pectic side chains. These results clearly demonstrate that in effects of a transgene are not restricted to the direct activity of the targeted enzyme but have consequences for the structure of the cell wall matrix. Analysis of whole cell wall structure is therefore necessary to assess the complete effect, direct and indirect, of a transgene.

Effect of Plant Age on the Quantity and Quality of Proteins Extracted from Sugar Beet (Beta vulgaris L.) Leaves.

Effects of the developmental stage (e.g., young, mature, or senescent) of leaves on their chemical composition have been described in the literature. This study focuses on the variation in chemical composition and quantity and quality of proteins extracted from leaves due to variation in plant age (i.e., harvesting time), using leaves from sugar beets grown in a field (Rhino, Arrival) and in a greenhouse (Isabella). Within the same variety (Rhino, field; Arrival, field; Isabella, greenhouse) the protein content was similar for leaves from young and old plants (22 ± 1, 16 ± 1, and 10 ± 3% w/w db, respectively). Variation in final protein isolation yield was mostly due to variation in nitrogen extractability (28-56%), although no consistent correlation with plant age was found. A significant effect of plant age was observed on the quality (color) of the extracted protein, that is, brown (indicative of polyphenol oxidase activity) and yellow for extracts from old and young plants, respectively.

Annotation of Different Dehydrocatechin Oligomers by MS/MS and Their Occurrence in Black Tea.

Dehydrocatechins (DhC's), oligomeric oxidation products of (epi)catechins, were formed in model incubations of epicatechin with mushroom tyrosinase. DhC oligomers up to tetramers were detected by reversed-phase ultrahigh-performance liquid chromatography mass spectrometry (RP-UHPLC-MS) analysis. Measurements with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) showed formation of oligomers up to at least 15 catechin subunits. Isomeric DhC's were obtained, and a method based on MS(2) fragment ratios was set up to distinguish between the different interflavanic configurations of the isomers. In the model incubation, 8 dehydrodicatechins (DhC2's) and 22 dehydrotricatechins (DhC3's) were tentatively annotated by their MS(2) signature fragments. Three different interflavanic configuration types were determined for the DhC2's. DhC2's and DhC3's were shown to occur in a black tea extract for the first time. For the DhC2's, at least two isomeric types, i.e., DhC ß and DhC ε, could be annotated in black tea.

A tandem mass spectrometry method based on selected ions detects low-abundance phenolics in black tea - theatridimensins as products of the oxidative cascade.

RATIONALE: Mixtures of phenolics are widespread in plant-derived food products, for instance black tea. Detailed compositional analysis of phenolics present is important for quality control. Characterization of low-abundance compounds often requires extensive purification; hence, the need for rapid screening methods to annotate compounds in complex mixtures without extensive sample preparation. Opportunities of ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS) as tool in a rapid screening method are discussed for black tea analysis, with the two-step-oxidation product theatridimensin (T3D) as example. METHODS: Three MS screening methods were compared for their ability to tentatively annotate two-step-oxidation products in black teas without the need for prior fractionation: (i) full MS; (ii) tandem mass spectrometry (MS/MS) on selected ions; and (iii) selected reaction monitoring (SRM), in combination with post-analysis extracted ion chromatography. A model system of theaflavin (TF), epicatechin (EC) and tyrosinase was used to prepare the two-step-oxidation product T3D, consisting of three oligomerized catechin subunits. Commercial teas were screened for the occurrence of T3Ds. RESULTS: The MS(2) fragmentation pattern of T3D was compared with that of an isomeric catechin trimer from black tea, TFsEC. MS(2) signature fragments were found to distinguish the two isomers, i.e. m/z 617 for T3D and m/z 563 for TFsEC. The MS screening methods, MS/MS on selected ions and SRM, both enabled monitoring MS(2) data of compounds present in low abundance. The former provided the most complete MS(2) data set, which facilitated the discovery of another isomer, i.e. theaflavate A. T3Ds, TFsECs, and theaflavate A could be tentatively annotated in all tested tea samples. CONCLUSIONS: When exploring black tea for the occurrence of two-step-oxidation products, the use of MS/MS on selected ions combined with extracted ion chromatography proved to be the most suitable. The occurrence of T3Ds and T3Dgs in various black teas was shown for the first time and the 'oxidative cascade hypothesis' was extended with novel oxidation products. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of pectin supplementation on the fermentation patterns of different structural carbohydrates in rats.

SCOPE: We aimed to investigate and compare the effects of four types of pectins on dietary fiber (DF) fermentation, microbiota composition, and short chain fatty acid (SCFA) production throughout the large intestine in rats. METHODS AND RESULTS: Male Wistar rats were given diets supplemented with or without 3% structurally different pectins for 7 weeks. Different fermentation patterns of pectins and different location of fermentation of pectin and diet arabinoxylans (AXs) in the large intestine were observed. During cecal fermentation, sugar beet pectin significantly stimulated Lactobacillus (p < 0.01) and Lachnospiraceae (p < 0.05). The stimulating effects of sugar beet pectin on these two groups of microbes are stronger than both other pectins. In the cecum, low-methyl esterified citrus pectin and complex soy pectin increased (p < 0.05) the production of total SCFAs, propionate and butyrate, whereas high-methyl esterified pectin and sugar beet pectin did not. The fermentation patterns of cereal AXs in the cecum were significantly different upon supplementation of different pectins. These differences, however, became smaller in the colon due to an enhanced fermentation of the remaining DFs. CONCLUSION: Dietary supplementation of pectin is a potential strategy to modulate the location of fermentation of DFs, and consequently microbiota composition and SCFA production for health-promoting effects.

Modification of potato cell wall pectin by the introduction of rhamnogalacturonan lyase and ß-galactosidase transgenes and their side effects.

Genes encoding pectic enzymes were introduced to wild-type potato Karnico. Cell wall materials were extracted from Karnico and transgenic lines expressing ß-galactosidase (ß-Gal-14 mutant) or rhamnogalacturonan lyase (RGL-18 mutant). After sequential extraction, ß-Gal-14 hot buffer-soluble solids (HBSS) of pectin contained 54% less galactose than Karnico HBSS, representing shorter galactan side chains. The individual pectin populations of ß-Gal-14 HBSS showed different modifications extended to the two sub-populations as obtained by ion-exchange chromatography. Compared to wild-type, RGL-18 HBSS contained 27% more galacturonic acid and 55% less Gal on fresh potato weight basis, which was due to the removal of galactan-rich rhamnogalacturonan I (RG-I) segments. All pectin populations of RGL-18 showed consistently low levels of RG-I segments. Transgenic modification showed side effects on the methyl-esterification and acetyl substitution of RGL-18 HBSS (DM=53, DA=21), but not of the ß-Gal-14 HBSS in comparison to wild-type (DM=29, DA=54).

Alteration of cell wall polysaccharides through transgenic expression of UDP-Glc 4-epimerase-encoding genes in potato tubers.

Uridine diphosphate (UDP)-glucose 4-epimerase (UGE) catalyzes the conversion of UDP-glucose to UDP-galactose. Cell wall materials from the cv. Kardal (wild-type, background) and two UGE transgenic lines (UGE 45-1 and UGE 51-16) were isolated and fractionated. The galactose (Gal) content (mg/100g tuber) from UGE 45-1 transgenic line was 38% higher than that of wild-type, and resulted in longer pectin side chains. The Gal content present in UGE 51-16 was 17% lower than that of wild-type, although most pectin populations maintained the same level of Gal. Both UGE transgenic lines showed unexpectedly a decrease in acetylation and an increase in methyl-esterification of pectin. Both UGE transgenic lines showed similar proportions of homogalacturonan and rhamnogalacturonan I within pectin backbone as the wild-type, except for the calcium-bound pectin fraction exhibiting relatively less rhamnogalacturonan I. Next to pectin modification, xyloglucan populations from both transgenic lines were altered resulting in different XSGG and XXGG proportion in comparison to wild-type.

Compositional changes in (iso)flavonoids and estrogenic activity of three edible Lupinus species by germination and Rhizopus-elicitation.

The effects of germination and elicitation on (iso)flavonoid composition of extracts from three edible lupine species (Lupinus luteus, Lupinus albus, Lupinus angustifolius) were determined by RP-UHPLC-MS(n). The total (iso)flavonoid content of lupine increased over 10-fold upon germination, with the total content and composition of isoflavonoids more affected than those of flavonoids. Glycosylated isoflavones were the most predominant compounds found in lupine seedlings. Lesser amounts of isoflavone aglycones, including prenylated ones, were also accumulated. Elicitation with Rhizopus oryzae, in addition to germination, raised the content of isoflavonoids further: the total content of 2'-hydroxygenistein derivatives was increased considerably, without increasing that of genistein derivatives. Elicitation by fungus triggered prenylation of isoflavonoids, especially of the 2'-hydroxygenistein derivatives. The preferred positions of prenylation differed among the three lupine species. The change in isoflavone composition increased the agonistic activity of the extracts towards the human estrogen receptors, whereas no antagonistic activity was observed.

Variation in accumulation of isoflavonoids in Phaseoleae seedlings elicited by Rhizopus.

Seeds from seven species of tribe Phaseoleae, i.e. Phaseolus, Vigna, Lablab and Psophocarpus, were investigated for inducibility of isoflavonoids by germination with or without subsequent elicitation with Rhizopus oryzae. Germination alone poorly induced isoflavonoid production (in the range of 0.2-0.7 mg representative compound equivalents (RCE)/g DW), whereas application of Rhizopus onto the seedlings increased the isoflavonoid content considerably (in the range of 0.5-3.3 mg RCE/g DW). The inducibility of different isoflavonoid subclasses in seedlings with Rhizopus varied per species. Isoflavones and isoflavanones were mainly found in elicited seedlings of Phaseolus, Vigna and Lablab, whereas pterocarpans were mainly observed in those of Psophocarpus. Despite their phylogenetic relatedness, the seeds of various species within Phaseoleae appeared to respond differently towards elicitation by Rhizopus during germination. The kind of molecules induced followed the phylogenetic relationship of the various species, but their amounts induced during germination, alone or combined with elicitation, did not.

Altering the phenolics profile of a green tea leaves extract using exogenous oxidases.

Transformation from green tea leaves into black tea involves oxidation of catechins into theaflavins and other complex phenolics by endogenous enzymes in tea leaves. By employing tyrosinase and laccase, both from Agaricus bisporus, on green tea catechins, the oxidation process was directed towards a higher theaflavins content, which is considered an important quality parameter in tea. The main tea catechins were incubated with tyrosinase and laccase, and product formation was monitored by RP-UHPLC-PDA-ESI-MS. The kind of catechin, their substitution with a galloyl group, and the type of oxidase used were important factors determining theaflavin concentrations. In particular, incubation of epicatechin with epigallocatechin with tyrosinase gave a high, stable theaflavin content. In a green tea extract, tyrosinase increased the proportion of theaflavins by twofold compared to black tea. Laccase mainly formed insoluble complexes. Our results indicate that the phenolic profile of tea can be modulated by using commercially available exogenous oxidases.

Glyceollins and dehydroglyceollins isolated from soybean act as SERMs and ER subtype-selective phytoestrogens.

Seven prenylated 6a-hydroxy-pterocapans and five prenylated 6a,11a-pterocarpenes with different kinds of prenylation were purified from an ethanolic extract of fungus-treated soybean sprouts. The activity of these compounds toward both human estrogen receptors (hERα and hERß) was determined in a yeast bioassay and the activity toward hERα was additionally tested in an U2-OS based hERα CALUX bioassay. In the yeast bioassay, compounds with chain prenylation showed in general an agonistic mode of action toward hERα, whereas furan and pyran prenylation led to an antagonistic mode of action. Five of these antagonistic compounds had an agonistic mode of action in the U2-OS based hERα CALUX bioassay, implying that these compounds can act as SERMs. The yeast bioassay also identified 8 ER subtype-selective compounds, with either an antagonistic mode of action or no response toward hERα and an agonistic mode of action toward hERß. The ER subtype-selective compounds were characterized by 6a-hydroxy-pterocarpan or 6a,11a-pterocarpene backbone structure. It is suggested that either the extra D-ring or the increase in length to 12-13.5Å of these compounds is responsible for an agonistic mode of action toward hERß and, thereby, inducing ER subtype-selective behavior.