Enzymatic solubilization of arabinoxylans from isolated rye pentosans and rye flour by different endo-xylanases and other hydrolyzing enzymes. Effect of a fungal caccase on the flour extracts oxidative gelation

Water-extractable (WEP) and water-unextractable (WUP) pentosans were isolated from a rye flour. The effect of a commercial enzyme preparation, Grindamyl S 100 (GS100), containing pentosanase activities, was investigated on WEP, WUP, a mix of WEP and WUP, and the rye flour, with the aim to monitor the solubilization and depolymerization of high molecular weight arabinoxylans and the effect on the viscosity of the reaction medium. The effects of other hydrolyzing enzymes were also tested. Three xylanases were used: xylanase 1 (Xyl-1) from Aspergillus niger, the main activity present in GS100; xylanase 2 (Xyl-2) from Talaromyces emersonii; and xylanase 3 (Xyl-3) from Bacillus subtilis. Xyl-3 was used in combination with Xyl-1, (1,4)-beta-D-arabinoxylan arabinofuranohydrolase, endo-beta-D-glucanase, or ferulate esterase from A. niger, but no synergism was observed. GS100 and xylanases increased the arabinoxylan solubilization, Xyl-3 and Xyl-1 being those that presented the best yields of extraction without extensive depolymerization of water-extractable arabinoxylans. Both xylanases were affected by an inhibitor in rye flour. Flour treated with hot ethanol was used to study the oxidative gelation of flour extracts treated with xylanases, in the presence of laccase from Pycnoporus cinnabarinus. Two doses of xylanases were tested (0.5 and 2.5 units). Only the flour extracts treated with 0.5 unit of Xyl-1 thickened.     

Structural features of arabinoxylans extracted with water at different temperatures from two rye flours of diverse breadmaking quality

The water extractable (WE) arabinoxylans from two rye flours differing in baking quality were studied following sequential extraction with water at 4, 40, and 100 degrees C. Ammonium sulfate fractionation of the resulting WE fractions and subsequent analysis revealed substantial differences in the structure of the isolated materials. Furthermore, it allowed us to identify the factors contributing to arabinoxylan water extractability. Our results provide compelling evidence for the existence of separate polymers in rye WE arabinoxylans with different substitution degrees, ranging from quantitatively dominating, lowly substituted populations (arabinose to xylose ratio, Ara/Xyl approximately 0.5) to comparatively less abundant, highly substituted analogues (Ara/Xyl approximately 1.3). Generally, arabinoxylan water extractability was governed by the relative proportion of lowly and highly branched structures. A gradually increasing proportion of highly substituted populations was observed from cold to hot WE fractions. This was associated with the lower proportion of monosubstituted xylopyranosyl residues in the backbone, the higher proportion of disubstituted xylopyranosyl residues, and the higher level of substitution with feruloyl residues. Notable differences in the ratio of phenolic compounds to arabinose residues were observed between corresponding polymers isolated from rye flours of high and low baking quality, whereas the differences in their molecular weights were much less pronounced.     

Antioxidant activity of steryl ferulate extracts from rye and wheat bran

Antioxidant activity of steryl ferulates from other sources than rice have not yet been studied much, despite the fact that rice steryl ferulates (gamma-oryzanol) have been shown to possess good antioxidant activity. In this study, steryl ferulate extracts from wheat or rye bran were studied for their capability to inhibit hydroperoxide formation in bulk methyl linoleate and methyl linoleate emulsion. Further, their activity to scavenge DPPH radicals was analyzed. The activities were compared to synthetic steryl ferulates, rice steryl ferulates, ferulic acid, and alpha-tocopherol. Nonrice cereal extracts of steryl ferulates exhibited good antioxidant activity, especially in the bulk lipid system. The radical scavenging activity was similar to that of nonesterified ferulic acid, indicating that the ferulic acid moiety is responsible for the antioxidant properties. This study illustrates a new aspect to the health-promoting properties of rye and wheat.     

Production of cellulose II from native cellulose by near- and supercritical water solubilization

We explored conditions for dissolving microcrystalline cellulose in high-temperature and high-pressure water without catalyst and in order to produce cellulose II in a rapid and selective manner. For understanding reactions of microcrystalline cellulose in subcritical and supercritical water, its solubilization treatment was conducted using a continuous-flow-type microreactor. It was found that cellulose could dissolve in near- and supercritical water at short treatment times of 0.02-0.4 s, resulting in the formation of cellulose II in relatively high yield after the treatment. Next, characteristics of the cellulose II obtained were investigated. As a result, it was confirmed that the relative crystallinity index and the degree of polymerization of the cellulose II were high values ranging from 80 to 60% and from 50 to 30%, respectively. From these findings, it was suggested that this method had high potential as an alternative technique for the conventional cellulose II production method.     

Heterogeneity in the fine structure of alkali-extractable arabinoxylans isolated from two rye flours with high and low breadmaking quality and their coexistence with other cell wall components

The alkali extractable (AE) arabinoxylans from two rye flours differing in baking quality were studied following sequential extraction of water-unextractable and starch-free rye flour residue with saturated barium hydroxide solution, water and 1 M sodium hydroxide solution (Ba, BaH, and Na, respectively), and further fractionation of isolated fractions by ammonium sulfate precipitation. (1)H NMR and sugar analyses of AE subfractions provided evidence for the presence of lowly branched arabinoxylans (average arabinose-to-xylose ratio, Ara/Xyl approximately 0.5), containing mainly un- and monosubstituted xylopyranosyl residues (Xylp) in the chain. The proportion of this subfraction decreased from 50% in the Ba fraction to 35 and 17% in the Na and BaH fractions, respectively. Other subfractions, rich in both mono- and disubstituted Xylp, represented arabinoxylan populations with intermediate (Ara/Xyl approximately 0.8) and high substitution degree (Ara/Xyl approximately 1.1). The Ba and Na fractions contained phenolic compounds, whereas they were absent in the BaH fraction. The higher ratio of such phenolic compounds to arabinose (PhC/Ara) found in AE arabinoxylans from rye flour of inferior baking quality was one of the most pronounced differences between arabinoxylan populations from rye flours with high and low baking quality. The arabinoxylans from rye flour of high baking quality present in Ba and Na fractions had slightly higher apparent molecular weights (MWs) when compared to those from rye flour with low baking quality. The arabinoxylans present in the BaH fractions, characterized by the highest MWs, had similar MWs.     

Extraction of rye bran by supercritical carbon dioxide: influence of temperature, CO2, and cosolvent flow rates

Process parameter optimization for the supercritical CO(2) extraction of rye bran to obtain alkylresorcinols (AR) was studied by carrying out a two-level fractional design experiment. Four parameters, temperature, CO(2) flow rate, cosolvent percentage, and extraction time, presumed to influence the extraction process, were analyzed. A tentative fractionation of the crude extract was also carried out and is discussed. The best extracts were achieved when the CO(2) flow rate and extraction time or temperature and cosolvent addition were kept high. It was found that temperature increase was not statistically significant within the range of the study performed, and the extraction time was thus the most important factor. A preliminary fractionation process in two cyclone separators yielded two fractions, one rich in AR components with higher molecular weights and the other rich in AR components with low molecular weight.     

Effects of processing on availability of total plant sterols, steryl ferulates and steryl glycosides from wheat and rye bran

Rye and wheat bran are excellent natural sources of plant sterols in the diet. Their content, however, may vary according to processing. Thermal (roasting and heating in a microwave oven), mechanical (milling and cryogenic grinding), and enzymatic treatments (hydrolysis with xylanase or beta-glucanase or a mixture of these two enzymes) were performed, and their effect on sterol content, extractability of sterols and the characteristic steryl conjugates of cereals (steryl ferulates, steryl glycosides, and acylated steryl glycosides) were studied. Mechanical and enzymatic treatments increased the apparent sterol content, whereas aqueous processing without enzymes hindered the availability of total sterols, especially from rye bran. Changes were also seen in the amounts of steryl conjugates caused by the enzymatic treatments. On the basis of the results of this study, it can be speculated that a combination of fine particle size and enzymatic processing results in optimal sterol availability in cereal processing.     

Endoxylanases in durum wheat semolina processing: solubilization of arabinoxylans, action of endogenous inhibitors, and effects on rheological properties

Endoxylanases seriously affect the rheological properties of durum wheat (Triticum durum Desf.) semolina spaghetti doughs prepared with, and as evaluated, by the farinograph. Under the experimental conditions, control doughs (34.9% moisture content) made from two semolinas (semA and semB) yielded a maximal consistency of 525 and 517 farinograph units (FU), with, respectively, 19.4 and 16.4% of the total level of arabinoxylans (TOT-AX) being water-extractable (WE-AX). When 75.4 Somogyi units/50 g of semolina of the endoxylanases from Trichoderma viride (XTV), rumen microorganisms (XRM), Bacillus subtilis (XBS), and Aspergillus niger (XAN) were used, the maximal consistencies at 34.9% moisture decreased for semA to 467, 436, 448, and 417 FU, respectively. This was accompanied by increased WE-AX contents of 60.8, 71.2, 70.7, and 73.0%, respectively. Similar results were observed for semB. By reducing the total water content of doughs, it was possible to recover the maximal consistency of the original doughs. Both the decrease in maximal consistency and the amount of water to be omitted were significantly related to the decrease in molecular weight (MW) of the WE-AX and the percentage of WE-AX solubilized as a result of the enzymic action. At the same time, it was clear that endogenous endoxylanase inhibitors were present in the durum wheat semolinas and that they inhibited the endoxylanases used to different degrees. Part of the differences in effects between the different endoxylanases (decrease in maximal consistency, amount of AX solubilized, MWs of the WE-AX, and amount of water that could be omitted) could be ascribed to the differences in inhibition of the endoxylanases by endogenous inhibitors.     

Postprandial glycemia, insulinemia, and satiety responses in healthy subjects after whole grain rye bread made from different rye varieties. 2

Rye breads made from commercial rye blends lower the postprandial insulin demand and appear to facilitate glucose regulation. However, differences in metabolic responses may occur between rye varieties. In the present work, five rye varieties (Amilo, Evolo, Kaskelott, Picasso. and Vicello) and a commercial blend of rye grown in Sweden were investigated with regard to their postprandial insulin, glucose, and appetite regulation properties in a randomized crossover study in 20 healthy subjects. The rye flours were baked into whole grain breads, and a white wheat bread (WWB) was used as reference (50 g of available starch). Picasso and Vicello rye bread showed lower glycemic indices (GIs) compared with WWB (80 and 79, respectively) (P < .0.05). In addition to the GI, two measures of the glycemic profile (GP and GP(2)) were calculated by dividing the incremental duration of the plasma glucose curve with the incremental glucose peak and squared incremental glucose peak, respectively. Vicello and Picasso ryes were characterized by a higher GP(2) than that of the WWB, suggesting a better regulated course of glycemia. Rye bread made from not only Vicello and Picasso but also Amilo and Kaskelott displayed significantly lower insulin indices (IIs) than WWB (74-82). A high GP and GP(2) and a low GI were related to a lower II and insulin incremental peak. A high content of insoluble fibers and a high GP(2) were related to a higher subjective satiety in the early and late postprandial phase (tAUC 0-60 min and tAUC 120-180 min, respectively). The results suggest that there may be differences in the course of glycemia following different rye varieties, affecting postprandial insulin responses and subjective satiety.     

Postprandial glycemia, insulinemia, and satiety responses in healthy subjects after whole grain rye bread made from different rye varieties. 1

Rye products typically induce low insulin responses and appear to facilitate glucose regulation. The objective of this study was to investigate differences in postprandial glucose, insulin, and satiety responses between breads made from five rye varieties. Breads made from whole grain rye (Amilo, Rekrut, Dankowski Zlote, Nikita, and Haute Loire Pop) or a white wheat bread (WWB) were tested in a randomized cross-over design in 14 healthy subjects (50 g available starch). Metabolic responses were also related to the composition of dietary fiber and bioactive compounds in the breads and to the rate of in vitro starch hydrolysis. The Amilo and Rekrut rye breads induced significantly lower insulin indices (II) than WWB. Low early postprandial glucose and insulin responses (tAUC 0-60 min) were related to higher amounts of caffeic, ferulic, sinapic, and vanillic acids in the rye breads, indicating that the phenolic acids in rye may influence glycemic regulation. All rye breads induced significantly higher subjective feelings of fullness compared to WWB. A low II was related to a higher feeling of fullness and a lower desire to eat in the late postprandial phase (180 min). The data indicate that some rye varieties may be more insulin-saving than others, possibly due to differences in dietary fiber, rate of starch hydrolysis, and bioactive components such as phenolic acids.     

Antioxidant activity of alkylresorcinols from rye bran and their protective effects on cell viability of PC-12 AC cells

Alkylresorcinols (ARs) are phenolic lipids that are present in high amounts in the bran layer of different cereals. Rye samples, cultivar Hazlet, and a white rye genotype, RT202, were analyzed for their antioxidant properties and AR content and composition, based on six fractions of the bran, where 1 was the outermost fraction and 6 was the bran fraction closest to the endosperm. Gas chromatography-mass spectrometry (GC-MS) analysis demonstrated that the most commonly found AR homologue in Hazlet rye is C19:0 and that the total amount of ARs decreases from the outermost to innermost fractions. The antioxidant activity using oxygen radical absorbance capacity (ORAC) for both white rye genotype RT202 and Hazlet brans was determined to decrease from the outermost fraction (136.05 μmol TE/g for Hazlet fraction 1 and 186.57 μmol TE/g for white rye genotype RT202 fraction 1) to the innermost fraction (9.84 μmol TE/g for Hazlet fraction 6 and 78.75 μmol TE/g for white rye genotype RT202 fraction 2). A positive relationship was seen with GC-MS results. Treatment of PC-12 AC cells with Hazlet fraction 1 increased mitochondrial biogenesis as determined using mitochondrial fluorescent dyes. In the presence of a prooxidant (AAPH), PC-12 AC cells were better protected from free radical attack when treated with Hazlet fraction 1 than with all other bran fractions. The results suggest that higher AR content in bran fractions confers antioxidant protection against free radical damage.     

Differentiation of rennet from other milk-clotting enzymes by polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis was used to differentiate animal rennet and other milk-clotting enzymes. After electrophoresis, the separated components were visualized by staining with aniline blue-black. Two prominent proteins were found in calf and bovine rennet, while only 1 major protein was observed in pepsin and enzymes of microbial origin. These patterns provided a basis for distinguishing animal rennet and the other enzymes as well as a means of identifying each type of enzyme by the characteristic pattern shown.     

Inhibition by soil humic acids of native and acetylated proteolytic enzymes

The activities of acetyltrypsin and acetylcarboxypeptidase were unaffected by neutralized solutions of soil humic acids in concentrations which markedly inhibited the non-acetylated enzymes. Further, acetyltrypsin, unlike trypsin, did not coprecipitate with humic acids. The results support the conclusion that humic acids bind the proteases by a cationexchange mechanism whereby protease amino groups are linked to humic acid carboxyl groups.     

Formation of phenolic microbial metabolites and short-chain Fatty acids from rye, wheat, and oat bran and their fractions in the metabolical in vitro colon model

Rye bran and aleurone, wheat bran and aleurone, and oat bran and cell wall concentrate were compared in their in vitro gut fermentation patterns of individual phenolic acids and short-chain fatty acids, preceded by enzymatic in vitro digestion mimicking small intestinal events. The formation of phenolic metabolites was the most pronounced from the wheat aleurone fraction. Phenylpropionic acids, presumably derived from ferulic acid (FA), were the major phenyl metabolites formed from all bran preparations. The processed rye, wheat, and oat bran fractions contained more water-extractable dietary fiber (DF) and had smaller particle sizes and were thus more easily fermentable than the corresponding brans. Rye aleurone and bran had the highest fermentation rate and extent probably due to high fructan and water-extractable arabinoxylan content. Oat samples also had a high content of water-extractable DF, β-glucan, but their fermentation rate was lower. Enzymatic digestion prior to in vitro colon fermentation changed the structure of oat cell walls as visualized by microscopy and increased the particle size, which is suggested to have retarded the fermentability of oat samples. Wheat bran was the most slowly fermentable among the studied samples, presumably due to the high proportion of water-unextractable DF. The in vitro digestion reduced the fructan content of wheat samples, thus also decreasing their fermentability. Among the studied short-chain fatty acids, acetate dominated the profiles. The highest and lowest production of propionate was from the oat and wheat samples, respectively. Interestingly, wheat aleurone generated similar amounts of butyrate as the rye fractions even without rapid gas production.     

In vitro fermentation of arabinoxylan oligosaccharides and low molecular mass arabinoxylans with different structural properties from wheat (Triticum aestivum L.) bran and psyllium (Plantago ovata Forsk) seed husk

Ball milling was used for producing complex arabinoxylan oligosaccharides (AXOS) and low molecular mass arabinoxylans (AX) from wheat bran, pericarp-enriched wheat bran, and psyllium seed husk. The arabinose to xylose ratio of the samples produced varied between 0.14 and 0.92, and their average degree of polymerization (avDP) ranged between 42 and 300. Their fermentation for 48 h in an in vitro system using human colon suspensions was compared to enzymatically produced wheat bran AXOS with an arabinose to xylose ratio of 0.22 and 0.34 and an avDP of 4 and 40, respectively. Degrees of AXOS fermentation ranged from 28% to 50% and were lower for the higher arabinose to xylose ratio and/or higher avDP materials. Arabinose to xylose ratios of the unfermented fractions exceeded those of their fermented counterparts, indicating that molecules less substituted with arabinose were preferably fermented. Xylanase, arabinofuranosidase, and xylosidase activities increased with incubation time. Enzyme activities in the samples containing psyllium seed husk AX or psyllium seed husk AXOS were generally higher than those in the wheat bran AXOS preparations. Fermentation gave rise to unbranched short-chain fatty acids. Concentrations of acetic, propionic, and butyric acids increased to 1.9-2.6, 1.9-2.8, and 1.3-2.0 times their initial values, respectively, after 24 h incubation. Results show that the human intestinal microbiota can at least partially use complex AXOS and low molecular mass AX. The tested materials are thus interesting physiologically active carbohydrates.     

Crossability of tetraploid wheat landraces native to Sichuan, Shaanxi, Gansu and Xinjiang provinces, China with rye

The crossability percentages of 131 landraces of wheat (Triticum turgidum L.) native to Sichuan, Shaanxi, Gansu and Xinjiang provinces of China with rye (Secale cereale L.) were tested. 45 landraces showed crossability percentages higher than 5% with 5 landraces expressing crossability percentages over 60%. The geographical distribution of crossability of these tetraploid wheat landraces was reported. Some utilization of high crossability resources was also discussed.     

Sterol ferulates, sterols, and 5-alk(en)ylresorcinols from wheat, rye, and corn bran oils and their inhibitory effects on Epstein-Barr virus activation

Sterol ferulate, free sterol, and 5-alk(en)ylresorcinol constituents of wheat, rye, and corn bran oils were studied. Among the sterol ferulates, one novel compound, 24-methylenecholestanol ferulate (7), along with six known compounds, namely, 24-methylcholestanol ferulate (1), 24-methylcholesterol ferulate (2), 2-methyllathosterol ferulate (3), stigmastanol ferulate (4), sitosterol ferulate (5), and schottenol ferulate (6), were isolated and characterized. Five known free sterols, namely, 24-methylcholesterol (8), stigmastanol (9), sitosterol (10), schottenol (11), and stigmasterol (12), were isolated and identified. 5-Alk(en)ylresorcinols were found in wheat and rye bran oils but not in corn bran oil. Of these, one new compound, 5-n-(2'-oxo-14'-Z-heneicosenyl) resorcinol (19), and seven known compounds, namely, 5-n-heptadecyl- (13), 5-n-nonadecyl- (14), 5-n-heneicosyl- (15), 5-n-tricosyl- (16), 5-n-pentacosyl- (17), 5-n-(14'-Z-nonadecenyl)- (18), and 5-n-(2'-oxoheneicosyl)resorcinols (20), were isolated and characterized. These compounds were evaluated with respect to their inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells, which is known to be a primary screening test for antitumor promoters. Four compounds, 1, 2, 4, and 11, showed potent inhibitory effects on EBV-EA induction.     

Both wheat (Triticum aestivum) bran arabinoxylans and gut flora-mediated fermentation products protect human colon cells from genotoxic activities of 4-hydroxynonenal and hydrogen peroxide

Dietary fibers are fermented by the gut flora to yield short chain fatty acids (SCFAs), which inhibit the growth of tumor cells, induce glutathione S-transferases (GSTs), and protect cells from the genotoxic activity of 4-hydroxynonenal (HNE). Here, we investigated effects of wheat bran-derived arabinoxylans and fermentation products on these parameters of chemoprevention. Newly isolated water extractable (WeAx) and alkali extractable arabinoxylans (AeAx) were fermented under anaerobic conditions with human feces. Resulting fermentation supernatants (FSs) were analyzed for SCFAs and used to treat HT29 colon cancer cells. Cell growth, cytotoxicity, antigenotoxicity against hydrogen peroxide (H2O2) or HNE, and GST activity were determined. Nonfermented WeAx decreased H2O2-induced DNA damage by 64%, thus demonstrating chemoprotective properties by this nonfermented wheat bran fiber. The fermentation of WeAx and AeAx resulted in 3-fold increases of SCFA, but all FSs (including the control without arabinoxylans) inhibited the growth of the HT29 cells, reduced the genotoxicity of HNE, and enhanced the activity of GSTs (FS WeAx, 2-fold; FS AeAx, 1.7-fold; and control FS, 1.4-fold), which detoxify HNE. Thus, increases in SCFAs were not reflected by enhanced functional effects. The conclusion is that fermentation mixtures contain modulatory compounds that arise from the feces and might add to the effectiveness of SCFAs.     

亚临界丙烷萃取米糠油及其微量活性组分动力学模型

为了揭示亚临界流体萃取米糠油及其微量活性组分的动力学规律,该文以亚临界丙烷为萃取介质,研究了米糠油、γ-谷维素及α-生育酚等活性组分在不同萃取温度和时间的萃取率,并基于 Baümler 模型对目标物的萃取率进行模型拟合,建立相应的动力学模型并验证。结果表明,亚临界萃取过程中目标物的相对萃取率随时间增长递增,且在初始阶段（洗涤过程）的增长速率快于第二阶段（扩散过程）的增长速率;同时,升高萃取温度可同时提高目标产物的扩散系数和相对萃取率;基于Baümler模型可较好拟合亚临界丙烷萃取米糠油、γ-谷维素和α-生育酚的动力学过程（R2>0.95）,所得动力学模型具有较好的预测准确性。由Arrhenius方程可计算出米糠油、α-生育酚和γ-谷维素的亚临界萃取反应活化能,其中米糠油的活化能最低（5.23 kJ/mol）,α-生育酚次之（7.05 kJ/mol）,γ-谷维素最高（9.11 kJ/mol）,表明γ-谷维素最难萃取且对温度依赖程度最高;根据拟合所得动力学模型,对米糠油提取率进行预测,结果表明,该模型对米糠油萃取率的预测准确率达95.8%,而对γ-谷维素和α-生育酚萃取率的预测准确度稍低,分别达94.7%和94.4%,对三者均具有很好的预测准确性。总之,基于 Baümler 模型可以较好地建立米糠油及其有益伴随物的亚临界萃取动力学模型,从而较为准确的预测米糠油及其微量活性组分的萃取率,对米糠油的亚临界萃取工艺具有一定的理论指导意义。