Degradation of starchy endosperm cell walls in nongerminating sterilized barley by fungi

Strains of fungi from different origins, including isolates of the natural microflora of barley, were screened for their ability to modify barley starchy endosperm cell walls in situ. In an initial step, fungi were selected that degrade the major component of the cell walls, that is, (1-->3),(1-->4)-beta-D-glucan, in vitro on artificial media. Nongerminating, sterilized barley, obtained by gamma-irradiation, was inoculated with such fungi and subjected to solid state fermentation under conditions resembling those of a traditional malting process. For some strains of fungi, a clear correlation between the production of endo-beta-glucanase and the friability of the treated kernels was found. Image analysis of Calcofluor stained longitudinal sections of barley kernels fermented with the endo-beta-glucanase producing strains showed that starchy endosperm cell walls were modified. As malt quality is inversely related to its (1-->3),(1-->4)-beta-D-glucan content, the selected strains have high potential to be used as starter cultures during malt production, contributing to the processing quality of the final product.     

Deposition of cell wall polysaccharides in wheat endosperm during grain development: Fourier transform-infrared microspectroscopy study

The time course and pattern deposition of the cell wall polysaccharides in the starchy endosperm of wheat (Triticum aestivum cv. Recital) during grain development was studied using Fourier transform infrared microspectroscopy (micro-FTIR). Three stages of grain development identified as key stages for cell wall construction were retained as follows: the end cellularization, the beginning of cell differentiation, and the beginning of maturation. Micro-FTIR revealed that beta-(1-->3),(1-->4) glucans and arabinoglactan proteins are the main cell wall components of endosperm at the end of the cellularization stage, whereas arabinoxylans (AX) appeared only at the cell differentiation stage. Past the differentiation stage, FTIR spectra were dominated by AX features. Cell walls at the beginning of cell differentiation and at endosperm maturation could be distinguished by spectral features that were ascribed to AX substitution. AX appeared more substituted at the beginning of cell differentiation. Moreover, a difference in the degree of AX substitution was found between peripheral and central parts of the grain at the cell differentiation stage; AX in central cells was less substituted. Thus, dramatic changes in endosperm cell wall composition were detected during wheat grain development with respect to both the relative occurrence of individual constituents and the fine structure of the AX.     

A proton nuclear magnetic resonance method for the quantitative analysis on a dry weight basis of (1-->3)-beta-D-glucans in a complex, solvent-wet matrix

Health benefits of the polysaccharide (1-->3)-beta-D-glucan, reported to induce immunobiological, hypocholesterolemic, and hypoglycemic effects in humans and animals, have made the isolation, characterization, and assay of a viable glucan product critical. A new analytical method, based on internal standard proton NMR analysis, for the assay of solvent-wet samples containing (1-->3)-beta-D-glucan is presented. The method enables glucan identification, provides a solvent-free assay, and improves upon the previous multistep extraction and lyophilization procedure by reducing the 1-2 day analysis time to 1-2 h. NMR offers a rapid method for quantifying the glucan in commercial samples, such as nutraceuticals, as well as industrial samples enabling better evaluation of the efficacy of these carbohydrates in health-related applications.     

Mixed Linkage (1→3),(1→4)‐β‐d‐Glucans of Grasses

The mixed‐linkage (1→3),(1→4)‐β‐d ‐glucans are unique to the Poales, the taxonomic order that includes the cereal grasses. (1→3), (1→4)‐β‐Glucans are the principal molecules associated with cellulose microfibrils during cell growth, and they are enzymatically hydrolyzed to a large extent once growth has ceased. They appear again during the developmental of the endosperm cell wall and maternal tissues surrounding them. The roles of (1→3),(1→4)‐β‐glucans in cell wall architecture and in cell growth are beginning to be understood. From biochemical experiments with active synthases in isolated Golgi membranes, the biochemical features and topology of synthesis are found to more closely parallel those of cellulose than those of all other noncellulosic β‐linked polysaccharides. The genes that encode part of the (1→3),(1→4)‐β‐glucan synthases are likely to be among those of the CESA/CSL gene superfamily, but a distinct glycosyl transferase also appears to be integral in the synthetic machinery. Several genes involved in the hydrolysis of (1→3),(1→4)‐β‐glucan have been cloned and sequenced, and the pattern of expression is starting to unveil their function in mobilization of β‐glucan reserve material and in cell growth.     

Stability and antioxidant activity of black currant anthocyanins in solution and encapsulated in glucan gel

The effects of ferric and ferrous ions, pH, and temperature on the stability and antioxidant activity of black currant anthocyanins (BCA) were studied, and the recovery of BCA from glucan gel [mixed linked (1-->3,1-->4)-beta-D-glucan] after using different encapsulating procedures was determined. The degradation of individual anthocyanins follows first-order kinetics and shows Arrhenius temperature dependence. The activation energies of individual anthocyanins, evaluated over the temperature range 60-100 degrees C, decrease with an increase in pH. While the antioxidant activity of BCA, measured by the ferric reducing antioxidant power assay, decreased with the degradation of anthocyanins, the completely degraded products still exhibited approximately 30% of the initial antioxidant activity. Ferric ions have a detrimental effect on the stability of BCA, especially for delphinidins. Freeze drying of encapsulated BCA gives approximately 20% higher recovery of individual anthocyanins than infrared drying.     

Identification of phenolics for control of Aspergillus flavus using Saccharomyces cerevisiae in a model target-gene bioassay

The yeast Saccharomyces cerevisiae was used in a high-throughput bioassay to identify phenolic agents for control of the aflatoxigenic fungus Aspergillus flavus. Veratraldehyde, 1, cinnamic acid, 5, and the respective benzoic acid derivatives vanillin, 2, vanillic acid, 3, and vanillylacetone, 4, and cinnamic acid derivatives o-coumaric acid, 6, m-coumaric acid, 7, and p-coumaric acid, 8, showed significant antifungal activities (from highest to lowest, 2, 5 > 1 > 6, 7 > 4 > 3, 8) in the yeast system, with caffeic acid, 9, having little to no effect. Antifungal activity levels against A. flavus were similar. This similarity in antifungal activity demonstrated the usefulness of the S. cerevisiae bioassay for screening antifungal compounds. Assays using deletion mutants of yeast identified signal transduction and antioxidative stress response genes important to fungal tolerance. Targeting the antioxidative stress response system with certain compounds (e.g., 4) in combination with strobilurin fungicides had a synergistic effect against both fungi.     

Antioxidative activity of propolis extract in yeast cells

The antioxidative activities of propolis and its main phenolic compounds, caffeic acid, p-coumaric acid, ferulic acid, and caffeic acid phenethyl ester, were investigated in the yeast Saccharomyces cerevisiae. After 1 h of exposure of the yeast cells, their intracellular oxidation was measured using 2',7'-dichlorofluorescein. Yeast cells exposed to 96% ethanolic extracts of propolis in DMSO (EEP) showed decreased intracellular oxidation, with no significant differences seen for the individual phenolic compounds. However, cellular uptake was seen only for a moderately polar fraction of EEP (E2) and caffeic acid phenethyl ester. The EEP antioxidative activity thus resulted from this E2 fraction of EEP. The influence of EEP was also investigated at the mitochondrial proteome level, by analyzing its profile after 1 h of exposure of the yeast cells to EEP and E2. Changes in the levels of antioxidative proteins and proteins involved in ATP synthesis were seen.     

红酵母RS1的高耐铝能力与增厚的细胞壁有关而不来自体外铝离子的螯合作用

Aluminum (Al) is very toxic to many living organisms, including plants, animals and microorganisms. However, despite many studies on Al tolerance in plants, little has been reported concerning these mechanisms in microorganisms. In this study, a red yeast, which could tolerate Al 3+ concentrations as high as 200 mmol L-1 , was isolated from acidic soils, identified as Rhodotorula sp. and designated as RS1. As the medium compositions can greatly affect the responses of microorganisms to Al, two culture mediums, glucose medium (GM) and lysogeny broth medium containing soil extract (S-LBM), were used. During growth of RS1, the pH of medium decreased in GM but increased in S-LBM. These changes in the pH of the media were not induced by Al addition. No or little secretion of organic acids was observed in RS1 growth media. Importantly, the thickness of the cell walls and the ratio of cell wall to biomass of RS1 significantly increased in GM with high Al 3+ concentrations. In the presence of 100 mmol Al L-1 , 78.0% of the total Al of whole cells was present in the thickened cell walls. The Al in cell walls was mostly bound to OH, amide and CO groups of polysaccharides. These results suggest that thickening of the cell wall in response to the high Al 3+ concentrations may play an important role in the high tolerance of RS1 to Al and that pH increase of the medium and chelation of Al ions are not involved in Al tolerance of this organism.     

Enzymatic synthesis of a selective inhibitor for alpha-glucosidases: alpha-acarviosinyl-(1-->9)-3-alpha-D-glucopyranosylpropen

Here, we describe the enzymatic synthesis of novel inhibitors using acarviosine-glucose as a donor and 3-alpha-D-glucopyranosylpropen (alphaGP) as an acceptor. Maltogenic amylase from Thermus sp. (ThMA) catalyzed the transglycosylation of the acarviosine moiety to alphaGP. The two major reaction products were isolated using chromatographies. Structural analyses revealed that acarviosine was transferred to either C-7 or C-9 of the alphaGP, which correspond to C-4 and C-6 of glucose. Both inhibited rat intestine alpha-glucosidase competitively but displayed a mixed-type inhibition mode against human pancreatic alpha-amylase. The alpha-acarviosinyl-(1-->7)-3-alpha-D-glucopyranosylpropen showed weaker inhibition potency than acarbose against both alpha-glycosidases. In contrast, the alpha-acarviosinyl-(1-->9)-3-alpha-D-glucopyranosylpropen exhibited a 3.0-fold improved inhibition potency against rat intestine alpha-glucosidase with 0.3-fold inhibition potency against human pancreatic alpha-amylase relative to acarbose. In conclusion, alpha-acarviosinyl-(1-->9)-3-alpha-D-glucopyranosylpropen is a novel alpha-glucosidase-selective inhibitor with 10-fold enhanced selectivity toward alpha-glucosidase over alpha-amylase relative to acarbose, and it could be applied as a potent hypoglycemic agent.     

Effect of tocopherols in the antioxidative activity of oxidized lipid-amine reaction products

Phosphatidylethanolamine (PE), phosphatidylcholine (PC), lysine (Lys), and mixtures of them were tested for antioxidative activity in a tocopherol-stripped olive oil (TSO) and the same oil after addition of 250 microg of alpha-tocopherol g of oil/(tocopherol-added olive oil, TAO) to evaluate the role of tocopherol in the antioxidant activity of oxidized lipid-amine products. Neither PE nor PC nor Lys protected TSO when tested alone, but both PE and Lys increased the induction period (IP) of TAO. On the contrary, PE/Lys and PC/Lys mixtures, but not PC/PE mixtures, protected both TSO and TAO. These results were a consequence of both the formation of oxidized lipid-amine products, which were determined by gas chromatography-mass spectrometry after their conversion into volatile derivatives, and a synergism between alpha-tocopherol and the produced compounds. These results were confirmed by analyzing the antioxidative activity of two of the produced carbonyl-amine products: 6-amino-2-(1H-pyrrol-1-yl)hexanoic acid (1) and 2,3-dipalmitoylpropyl 2-(1H-pyrrol-1-yl)ethyl phosphate (2). The hydrophilic compound 1 was more antioxidant than the analogous lipophilic compound 2, and this antioxidative activity was observed in TAO and not in TSO. All these results suggested that antioxidative activity of carbonyl-amine products may be greatly increased with the addition of tocopherols, and those products derived from Lys are more antioxidant in bulk oils than those derived from PE.     

Behavior of a fenhexamid photoproduct during the alcoholic fermentation of Saccharomyces cerevisiae

The fungicide fenhexamid [N-(2,3-dichloro-4-hydroxyphenyl)-1-methylcyclohexanecarboxamide] degraded rapidly by UV or sunlight irradiation, yielding 7-chloro-6-hydroxy-2-(1-methylcyclohexyl)-1,3-benzoxazole (CHB) as a main photoproduct. CHB was isolated, and its effect on alcoholic fermentation of Saccharomyces cerevisiae was studied. The results indicate that the presence of CHB does not affect the extent of alcohol production. After 12 days, the amount of CHB in the fermentation medium decreased by ca. 65%. Only 25% of the missing CHB was recovered unchanged from yeasts, most likely because it was adsorbed on the yeast wall cell. The remaining part degraded during the fermentation process. Glucan and chitin, two potential adsorbents, which constitute yeast cell walls, exhibited affinity for CHB.     

In vitro antioxidant activities of edible artichoke (Cynara scolymus L.) and effect on biomarkers of antioxidants in rats

Artichoke (Cynara scolymus L.), an edible vegetable from the Mediterranean area, is a good source of natural antioxidants such as vitamin C, hydroxycinnamic acids, and flavones. The antioxidant activity of aqueous-organic extracts of artichoke were determined using three methods: (a) free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) scavenging, (b) ferric-reducing antioxidant power (FRAP), and (c) inhibition of copper(II)-catalyzed in vitro human low-density lipoprotein (LDL) oxidation. In addition, the present study was performed to investigate the ability of the edible portion of artichoke to alter in vivo antioxidative defense in male rats using selected biomarkers of antioxidant status. One gram (dry matter) had a DPPH(*) activity and a FRAP value in vitro equivalent to those of 29.2 and 62.6 mg of vitamin C and to those of 77.9 and 159 mg of vitamin E, respectively. Artichoke extracts showed good efficiency in the inhibition in vitro of LDL oxidation. Neither ferric-reducing ability nor 2,2'-azinobis(3-ethylbenzothiazolin-6-sulfonate) radical scavenging activity was modified in the plasma of the artichoke group with respect to the control group. Among different antioxidant enzymes measured (superoxide dismutase, gluthatione peroxidase, gluthatione reductase, and catalase) in erythrocytes, only gluthatione peroxidase activity was elevated in the artichoke group compared to the control group. 2-Aminoadipic semialdehyde, a protein oxidation biomarker, was decreased in plasma proteins and hemoglobin in the artichoke-fed group versus the control group. In conclusion, the in vitro protective activity of artichoke was confirmed in a rat model.     

Isolation and characterization of antioxidative peptides from gelatin hydrolysate of Alaska pollack skin

Gelatin extracted from Alaska pollack skin was hydrolyzed with serial digestions in the order of Alcalase, Pronase E, and collagenase using a three-step recycling membrane reactor. The fraction from the second step, which was hydrolyzed with Pronase E, was composed of peptides ranging from 1.5 to 4.5 kDa and showed high antioxidative activity. Two different peptides showing strong antioxidative activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration on a Sephadex G-25 column, ion-exchange chromatography on a SP-Sephadex C-25 column, and high-performance liquid chromatography on an ODS column. The isolated peptides, P1 and P2, were composed of 13 and 16 amino acid residues, respectively; and both peptides contained a Gly residue at the C-terminus and the repeating motif Gly-Pro-Hyp. The antioxidative activities of the purified peptides were measured using the thiobarbituric acid method, and the cell viability was measured with MTT assay. The results showed that P2 had potent antioxidative activity on peroxidation of linoleic acid. Moreover, the cell viability of cultured liver cells was significantly enhanced by addition of the peptide. These results indicate that the purified peptide, P2, from gelatin hydrolysate of Alaska pollack skin is a natural antioxidant which has potent antioxidative activity.     

Mechanisms of aluminum‐induced growth stimulation in tea (Camellia sinensis)

Beneficial effects of aluminum (Al) on plant growth have been reported for plant species adapted to acid soils. However, mechanisms underlying the stimulatory effect of Al have not been fully elucidated. The aim of this study was to determine the possible contribution of photosynthesis, antioxidative defense, and the metabolism of both nitrogen and phenolics to the Al‐induced growth stimulation in tea (Camellia sinensis [L.] Kuntze) plants. In hydroponics, shoot growth achieved its maximum at 50 μM Al suply (24 μM Al³⁺ activity). A more than threefold increase of root biomass was observed for plants supplied with 300 μM Al (125 μM Al³⁺ activity). Total root length was positively related to root Al concentrations (r = 0.98). Chlorophyll a and carotenoid concentrations and net assimilation rates were considerably enhanced by Al supply in the young but not in the old leaves. Activity of nitrate reductase was not influenced by Al. Higher concentrations of soluble nitrogen compounds (nitrate, nitrite, amino acids) and reduction of protein concentrations suggest Al‐induced protein degradation. This occurred concomitantly with enhanced net CO₂‐assimilation rates and carbohydrate concentrations. Aluminum treatments activated antioxidant defense enzymes and increased free proline content. Lowering of malondialdehyde concentrations by Al supply indicates that membrane integrity was not impaired by Al. Leaves and roots of Al‐treated plants had considerably lower phenolic and lignin concentrations in the cell walls, but a higher proportion of soluble phenolics. In conclusion, Al‐induced growth stimulation in tea plants was mediated by higher photosynthesis rate and increased antioxidant defense. Additionally, greater root surface area may improve water and nutrient uptake by the plants.     

Nasunin from eggplant consists of cis-trans isomers of delphinidin 3-[4-(p-coumaroyl)-L-rhamnosyl (1-->6)glucopyranoside]-5-glucopyranoside

Two major anthocyanins were isolated from the acidified methanolic extract of eggplant (Solanum melongena L.) by column chromatography and preparative high-performance liquid chromatography. These anthocyanins were interconvertible under room light illumination condition. By means of tandem time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy, their structures were identified and elucidated as delphinidin 3-[4-(cis-p-coumaroyl)-l-rhamnosyl(1-->6)glucopyranoside]-5-glucopyranoside (compound 1) and delphinidin 3-[4-(trans-p-coumaroyl)-l-rhamnosyl-(1-->6)glucopyranoside]-5-glucopyranoside (compound 2), respectively. The results indicated that nasunin comprised cis and trans isomers of the p-coumaric acid moiety in its structure.     

Antioxidative properties of press juice from herring (Clupea harengus) against hemoglobin (Hb) mediated oxidation of washed cod mince

The antioxidative effect of herring (Clupea harengus) light muscle press juice (PJ) against hemoglobin-(Hb-) mediated oxidation of washed cod mince during ice storage was tested. The PJ was fractionated into low-molecular-weight (LMW; <1 [corrected] kDa) and high-molecular-weight (HMW; >1, >3.5, and > 50 kDa) fractions; it was preheated (10 min, 100 degrees C) and tested with or without removing heat coagulated proteins. Its antioxidative effect was compared with that given by endogenous levels of two tentative antioxidant candidates: ascorbic acid and uric acid. Oxidation was followed by determining rancid odor, peroxide value, and redness. Whole herring PJ and the LMW-PJ fraction significantly (p < 0.001) extended the oxidation lag phase of controls, from 2 up to 8 and 7 days, respectively. The HWM-PJ fractions were significantly (p < 0.05) less efficient than the whole and LMW-PJ samples, giving only 3.5-4.5 days of lag phase. Heat-treated PJ, with and without the heat-coagulated proteins, gave 7 and 5 days of oxidation lag phase, respectively. Heating different batches of the LMW-PJ fraction grouped the results into two categories: one where heating almost fully destroyed the antioxidative activity (fractions prepared from spring-caught herring) and another where heating had no or a minor effect (fractions prepared from fall-caught herring). The spring LMW-PJ had low ascorbic acid levels (18-42.6 microM), and 50-100% were destroyed by the heating. In fall LMW-PJ, the levels were 76.2-137.6 microM, and only 43-51% were destroyed. Ascorbic acid fortification of heated spring LMW-PJ to reach the levels found in the corresponding unheated spring LMW-PJ sample and the heated fall LMW-PJ gave back most of the antioxidative activity, which proved an important role of ascorbic acid for the antioxidative activity of LMW-herring PJ. This conclusion is drawn despite the fact that pure solutions with endogenous levels of ascorbic acid (giving 8.4-19.6 microM in final model) only very slightly delayed Hb-mediated oxidation of the washed cod mince.     

Production and characterization of human extracellular superoxide dismutase in the methylotrophic yeast Pichia pastoris

Reactive oxygen species are associated with various diseases including cardiovascular diseases, neurological disorders, and pulmonary diseases. Extracellular superoxide dismutase (ECSOD) is an antioxidant enzyme secreted by cells to prevent overproduction of reactive oxygen species. We expressed an ECSOD gene isolated from a human aortic smooth muscle cDNA library in the methylotrophic yeast Pichia pastoris. A synthetic secretion cassette was constructed with the inducible promoter of the alcohol oxidase 1 gene (AOX1) and the yeast alpha-mating factor signal peptide. As much as 25% of the total protein was ECSOD in some transformants grown under inducing conditions. After 36 h of methanol induction, ECSOD was exported into the culture medium at a concentration of approximately 440 mg/L with an antioxidative activity of 760 +/- 20 U/mg ECSOD. Transformed yeast cells were more resistant to heat shock and H(2)O(2) oxidative stress, indicating that the human ECSOD expressed by P. pastoris had multiple biological functions. Our data suggest that the methylotrophic yeast inducible system is suitable for large-scale production of enzymatically active human ECSOD.     

Antioxidative and anti-glycation activity of garcinol from Garcinia indica fruit rind

Garcinol, a polyisoprenylated benzophenone derivative, was purified from Garcinia indica fruit rind, and its antioxidative activity, chelating activity, free radical scavenging activity, and anti-glycation activity were studied. Garcinol exhibited moderate antioxidative activity in the micellar linoleic acid peroxidation system and also exhibited chelating activity at almost the same level as citrate. It also showed nearly 3 times greater DPPH (1, 1-diphenyl-2-picrylhydrazyl) free radical scavenging activity than DL-alpha-tocopherol by weight in aqueous ethanol solution. In a phenazine methosulfate/NADH-nitroblue tetrazolium system, garcinol exhibited superoxide anion scavenging activity and suppressed protein glycation in a bovine serum albumin/fructose system. Thus, garcinol might be beneficial as a potent antioxidant and a glycation inhibitor under specified conditions.     

茶叶中多酚类物质抗氧化活性的量子化学计算研究

抗氧化剂活性的评价是医药、保健、食品和化妆品等领域研究的热点课题。本文以茶叶中的多酚类物质为研究对象,采用AM1法对多酚类物质的抗氧化活性进行了量子化学计算。结果表明,通过比较生成热(HOF)值可以直接表征多酚类物质的抗氧化活性,即HOF值越低,多酚类的抗氧化活性越强。同时,本文对多酚类物质的抗氧化机理提出了新的观点:在多酚类物质的抗氧化过程中,酚羟基、氢原子和醇羟基都发挥了抗氧化作用。     

Bioguided fractionation and isolation of free radical scavenging components from in vitro propagated chinese medicinal plants Dendrobium tosaense Makino and Dendrobium moniliforme SW

This study was performed to investigate the free radical scavenging active components from in vitro propagated medicinal herbs of the genus Dendrobium, namely, Dendrobium tosaense Makino and Dendrobium moniliforme SW, using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical antioxidative assay. Seeds of the capsules derived after 12 weeks of hand-pollination germinated asymbiotically (50-74%) on half-strength Murashige and Skoog's (MS) basal medium with 3% sucrose and solidified with 0.9% Difco agar. Active growth in the germinated seedlings was achieved by reculturing on full-strength MS basal medium supplemented with 8% banana homogenate, 8% potato homogenate, 8% coconut water, 1.5% sucrose, and 0.9% Difco agar. Healthy plantlets transferred to plastic trays containing moss or moss and tree fern successfully acclimatized (84-100%) in the greenhouse. Extracts were prepared from plants grown in the greenhouse for a period of 6 months. Methanolic extracts of D. tosaense and D. moniliforme scavenged DPPH at 95.9 and 83.4%, respectively, at a concentration of 0.4 mg/mL. Therefore, methanolic solubles of D. tosaense and D. moniliforme were subjected to bioguided fractionation and separation by column chromatographic methods individually. After chromatographic separation of these crude extracts, the obtained fractions (Dm 1, Dm 2, Dm 3, Dt 1, Dt 2, and Dt 3) were tested for their activity. Among them, fractions Dm 2 and Dt 1 showed significant antioxidant activity by DPPH radical antioxidative assay. Active fractions were purified further by column chromatography and resulted in identification of the antioxidant components alkyl ferulates from D. moniliforme and quercetin from D. tosaense.