Effects of dough mixing and oxidising improvers on free reduced and free oxidised glutathione and protein-glutathione mixed disulphides of wheat flour

Changes in free reduced glutathione (GSH), free oxidised glutathione (GSSG) and protein-glutathione mixed disulphides (PSSG) during dough mixing were monitored. After a rapid decrease in the GSH content and an increase in the GSSG content, the contents of both GSH and GSSG decreased progressively, whereas the PSSG content increased, as a simple flour-water dough was mixed. Total glutathione (GSH plus GSSG plus PSSG) levels in simple flour-water doughs and doughs treated with ascorbic acid or potassium bromate remained essentially constant during dough mixing, indicating that the reactions in which glutathione is involved are simple oxidations of sulphydryl (SH) groups to disulphide (SS) bonds and SH/SS interchange reactions. Yeast contributed high levels of GSH and GSSG to doughs, but analysis of dough aqueous phases (liquors) and the similarity of the PSSG contents of simple and yeasted flour-water doughs suggested that the yeast GSH and GSSG were largely unavailable to react with flour proteins. The GSH content of ascorbic-acid-treated and yeasted dough decreased rapidly on wetting the flour, the magnitude of the decrease indicating that the ascorbate oxidation system oxidised the yeast intracellular GSH as well as the flour GSH. With further mixing, the GSH content of the ascorbate dough remained constant at low levels similar to those of the simple flour-water dough. The GSSG content of the ascorbate dough increased rapidly on wetting the flour, but declined as dough mixing continued. The PSSG content of the doughs increased markedly as dough mixing proceeded to the optimum and then stabilised. The increase in the PSSG content lagged behind the rapid oxidation of GSH to GSSG. Potassium bromate caused a pattern of changes similar to those observed for ascorbate, but the changes in GSH and PSSG contents were smaller in magnitude. The results indicate that the changes in the different glutathione pools and the effects of oxidising bread improvers are rather more complex than envisaged previously, particularly the effects on PSSG.     

环境因子对发酵液中谷胱甘肽稳定性的影响

利用酵母生产还原型谷胱甘肽（GSH）时,GSH的稳定性直接影响细胞酶催化合成GSH的效率。在絮凝酵母摇瓶发酵培养24 h时,模拟胞外存在GSH的环境,向发酵液中添加200 mg/L的GSH,考察胞内外GSH稳定性情况;同时检测环境因子氧、温度、pH,以及金属离子（Zn2+、Mn2+、Cd2+）对胞内外GSH稳定性的影响。结果表明：正常环境条件下培养6 h,发酵液中GSH的回收率达到70%,导致GSH不稳定的主要因素是氧化,细胞内GSH浓度相对变化不明显;在温度≤25 ℃,pH3.5～4.5和厌氧发酵条件下,发酵液中GSH稳定性显著提高(P＜0.05);Zn2+、Mn2+和Cd2+对GSH稳定性影响不明显(P＞0.05)。     

Comparison of oxidized and reduced glutathione in the bread-making qualities of rice batter

The demand for gluten-free bread is growing as the recognition of celiac disease and wheat allergy has increased worldwide. In our previous study, reduced glutathione (GSH) was found to improve the gas-retaining properties of rice batter used for gluten-free bread. In this article, oxidized glutathione (GSSG) was shown to have the same effect. Moreover, sensory tests revealed that GSSG bread had a significantly reduced sulfurous odor. Analyses by a gas chromatography-flame photometric detector demonstrated the presence of hydrogen sulfide and methyl mercaptan in the headspace of GSH bread, and also their significant reduction in GSSG bread. The viscoelastic properties and microstructures of GSSG and GSH bread did not noticeably differ. These observations suggest the usefulness of GSSG in making gluten-free rice bread and extend our knowledge of the use of glutathione in food processing. Practical Application: Glutathione, a widely-distributed peptide in cells, improves the bread-making quality of gluten-free rice batter. While both the reduced (GSH) and oxidized (GSSG) glutathione are effective, GSSG-bread has significantly reduced sulfurous odor compared to GSH-bread.     

Identification and characterization of genes involved in glutathione production in yeast

Glutathione is a major peptide protecting cells against oxidative stress. To study the cellular processes affecting intracellular glutathione production, we screened Saccharomyces cerevisiae mutant collections and identified new eight yeast deletion mutants that produced more than 1.2-fold higher levels of intracellular glutathione: chc1, cst6, ddc1, def1, pep12, rts1, ubp6, and yih1. Furthermore, overexpression of the DEF1 and CYS4 genes led to a higher production of glutathione, similar to overexpression of GSH1. A multiplier effect on activation of glutathione synthesis was observed by a combination of overexpression of GSH1 and deletion of one of the eight genes. Metabolome analysis of the def1, pep12, and ubp6 deletion mutant, and DEF1-overexpressing strains showed that levels of intracellular methionine and oxidized glutathione were higher than in the control strains, suggesting that methionine biosynthesis was activated and the oxidative stress response was increased in these glutathione-overproductive strains. Moreover, overexpression of GSH1, CYS4, and DEF1 also increased glutathione production in Candida utilis. Taken together, these results will significantly contribute to more effective industrial production of glutathione using yeasts.     

Determination of free reduced and total glutathione in wheat flours by an isotope dilution assay

Summary An isotope dilution assay (IDA) for free reduced glutathione (GSH) and total glutathione (GSH, oxidised glutathione and protein-bound glutathione) was developed and its accuracy and sensitivity were established. The new method for GSH required extraction of flour samples with a buffer at pH 4.5 containingN-ethyl maleimide (NEMI) and¹⁴C-labelled S-(N-ethylsuccinimido) glutathione ([¹⁴C]GS-NEMI), purification of the labelled and unlabelled GS-NEMI by three Chromatographic steps and assay of the specific radioactivity of the GS-NEMI isolated. Total glutathione was assayed after reduction with dithioerythritpl. Applications of the IDA indicated that the levels of GSH (16–41 nmol/g) and total glutathione (170–185 nmol/g) were relatively low in flours with low ash contents, but increased with increasing extraction grade. The level of GSH was higher in a flour obtained from kernels that were ground in the absence of gaseous oxygen. Storage of flours reduced the GSH concentration. IDA of fractions obtained from flour showed that the extraction residue, mainly consisting of starch and glutelins, contained most of the bound glutathione.

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Bestimmung von freiem reduziertem und vom gesamten Glutathion in Weizenmehlen durch Isotopenverdünnungsanalyse

Zusammenfassung Eine Isotopenverdünnungsanalyse (IVA) wurde für das freie reduzierte (GSH) und das gesamte Glutathion (GSH, GSSG proteingebundenes Glutathion) entwickelt und auf seine Genauigkeit und Empfindlichkeit überprüft. Die neue Methode für GSH erfordert die Extraktion der Mehlprobe mit einem Puffer bei pH 4,5, der N-Ethylmaleinsäureimid (NEMI) und [¹⁴C]-GS-NEMI enthält, Reinigung des markierten und unmarkierten GS-NEMI durch drei chromatographische Schritte sowie die Bestimmung der spezifischen Radioaktivität von dem isolierten GS-NEMI. Das gesamte Glutathion wird nach Reduktion mit Dithioerythrit bestimmt. Anwendungen der IVA zeigten, daß die Konzentrationen von GSH (16 bis 41 nmol) und von gesamtem Glutathion (170 bis 185 nmol/g) relativ gering waren in Mehlen mit niedrigen Aschegehalten, aber daß sie mit steigendem Ausmahlungsgrad zunahmen. Die GSH-Konzentration war in einem Mehl höher, das aus Körnern stammte, die in Abwesenheit von Sauerstoff gemahlen worden waren. Die Lagerung von Mehlen erniedrigte den GSH-Gehalt. Die IVA von Mehlfraktionen zeigte, daß der Extraktionsrückstand, der hauptsächlich aus Stärke und Glutelinen bestand, das meiste gebundene Glutathion enthielt.

     

还原型谷胱甘肽在果酒酿造中的应用

谷胱甘肽是一种广泛存在于生物细胞中的活性三肽,分为还原型谷胱甘肽（GSH）和氧化型谷胱甘肽（GSSG）两种,但绝大部 分是以GSH的形式存在的。 由于GSH具有重要的生理功能,所以它被广泛用于食品加工领域,可以起到增加食品营养价值和强化食品 风味的作用。 文章综述了外源GSH的添加对果酒品质特性的影响,讨论了GSH在果酒酿造中扮演的抗氧化、抑制褐变、改善果酒风味 以及促进苹果酸-乳酸发酵的作用,同时阐述了富集谷胱甘肽非活性干酵母制剂（g-IDY）在果酒中的应用现状,为GSH作为一种添加 剂应用于果酒酿造提供了理论依据。     

Relationship between free glutathione content and quality assessment parameters of wheat cultivars (Triticum aestivum L.)

Freshly milled flour samples from ten cultivars of bread wheat were analysed for free reduced glutathione (GSH) and oxidised glutathione (GSSG) by an enzymic method. Dough rheological properties for each flour were assessed by standard Brabender techniques. Flour quality differences were also measured by Pelshenke fermentation and Zeleny sedimentation techniques. Reduced glutathione levels were negatively correlated with oxidised glutathione levels, Farinogram mixing tolerance, Extensogram resistance, Pelshenke time and Zeleny volume. The ratios of oxidised to reduced glutathione (GSSG: GSH) were positively correlated with all dough and flour quality parameters, except Extensogram extensibility. It is concluded that there are differences in the endogenous contents of reduced and oxidised glutathione between flour samples from ten cultivars of wheat. These differences may contribute significantly to variations in the quality assessment parameters established between cultivars in this study.     

Patulin reduces glutathione level and enzyme activities in rat liver slices

In the present study, an attempt was made to identify glutathione (GSH) adducts of patulin in precision-cut rat liver slices, which were used as a model system to study the metabolism and biological effects of this mycotoxin. Patulin disappeared in the slices but none of the GSH adducts, previously demonstrated in the chemical reaction of patulin with GSH, could be detected by HPLC. After incubation with various concentrations of patulin, a concentration-dependent decline of the GSH level was observed in the slices. For example, only 25% of the GSH of controls was found with 200 microM patulin. The activities of glutathione-S-transferase (GST) and of drug metabolizing phase I and phase II enzymes, assayed by the hydroxylation and conjugation of testosterone, were also reduced. On the other hand, incubation with patulin markedly increased lipid peroxidation in the slices. The effects of patulin on enzyme activities and lipid peroxidation may be a consequence of the GSH decline, which cannot be accounted for by a direct reaction of patulin with GSH due to the high concentration of GSH in hepatocytes. The decrease of GSH level and GST activity may be related to the putative mutagenic and carcinogenic potential of patulin.     

Changes in glutathione content in human hair follicle keratinocytes as a function of age of donor: relation with glutathione dependent enzymes

Glutathione (GSH) plays an important role in cellular protection during ageing. The hair plucking technique is a non-invasive method for the direct biochemical study of keratinocytes. Hair was taken from the suboccipital area of 63 volunteers (men and women whose ages ranged from 13 to 103 years). The results show a diminution in the glutathione content as a function of age. We compare two groups of population: group A (less than 80) and group B (more than 80). A remarkable fact is observed: group B displays a weak dispersion of the values as compared to A. The glutathione content (nmol 10⁻³ mg DNA) is 5.42 ± 0.60 for A and 1.86 ± 0.35 for B. A reduction of 88% was observed in glutathione reductase activity and of 78% in the activity of glutathione-S-transferase from group A to group B. The glutathione peroxidase activity remains relatively constant. The decrease in the GSH concentration and the constancy of the glutathione peroxidase suggest that the capacity of the cell to protect itself from peroxides remains unchanged but that the GSH concentration may become the limiting factor.     

酿酒酵母谷胱甘肽产量与镉盐抗性关系的研究与应用

酿酒酵母谷胱甘肽（GSH）生成量与其镉盐抗性关系验证实验和利用镉盐抗性筛选高产GSH酿酒酵母突变株实验证明,酿酒酵母GSH生成量越高,其镉盐抗性越强;反之,酿酒酵母镉盐抗性越强,其GSH生成量不一定越高。在紫外诱变实验中利用镉盐抗性筛选高产GSH酿酒酵母突变株,正突变率达到23.5%,最终获得突变株BV54和MV16,其胞内GSH含量较出发菌株B-3和M8分别提高了32.53%和36.55%。结果表明,这种利用镉盐抗性筛选高产GSH酿酒酵母突变株的育种方法可行且高效。     

Biosynthesis of phytochelatins in the fission yeast. Phytochelatin synthesis: a second role for the glutathione synthetase gene of Schizosaccharomyces pombe

By complementation screening of a cadmium-sensitive Schizosaccharomyces pombe mutant deficient in phytochelatin synthesis, but with 44% of the wild-type glutathione content, we cloned a DNA fragment involved in phytochelatin synthesis. Sequence analysis revealed that it encodes the second enzyme involved in glutathione (GSH) biosynthesis, glutathione synthetase (GSH2) (E.C.6.3.2.3, Wang and Oliver, 1997). The mutant allele shows a single base-pair exchange at the 3' end of the reading frame leading to a single amino acid change from glycine to aspartate. This mutation leads to a significant reduction of phytochelatin synthesis, whereas glutathione synthesis is impaired to a far lesser extent. Complementation with the Arabidopsis thaliana GSH2 cDNA led to a partial restoration of phytochelatin synthesis. These data strongly suggest that the GSH2 gene encodes a bifunctional enzyme that is able to catalyse both the synthesis of GSH by adding glycine to the dipeptide (gammaGlu-Cys) and the synthesis of phytochelatins. The sequence has been submitted to EMBL, Accession No. Y08414.     

采后ASM处理对苹果果实抗坏血酸-谷胱甘肽循环系统的影响

采后100mg·L-1的苯丙噻重氮（ASM）溶液浸泡苹果果实10min,24h后损伤接种P.expansum,测定常温贮藏期间果实病斑直径及抗坏血酸-谷胱甘肽循环系统的变化。结果表明,ASM处理显著降低了果实病斑直径,并提高了果实过氧化氢（H2O2）含量,抑制了过氧化氢酶（CAT）活性,提高了果实抗坏血酸过氧化物酶（APX）、谷胱甘肽还原酶（GR）、单脱氢抗坏血酸还原酶（MDAR）、脱氢抗坏血酸还原酶（DHAR）活性及抗坏血酸（As A）和还原型谷胱甘肽（GSH）含量。由此表明,ASM处理通过抑制CAT活性促进果实前期H2O2的积累,从而启动果实体内其他防卫反应提高抗病性,抗坏血酸-谷胱甘肽循环系统中抗氧化酶活性和抗氧化剂含量的提高有利于消除过量的H2O2对果实的伤害。      

Browning susceptibility of white wine and antioxidant effect of glutathione

Glutathione (GSH) content and other physicochemical parameters of thirteen white wines originated from Lebanon were studied in relation to their browning capacity; the impact of pH, total sulphur dioxide and total phenol content into the browning susceptibility of the wine was shown. The effect of GSH, added to the wine at different concentrations, on the total phenol content was studied at 55 °C during 8 days of accelerated oxidation. GSH addition resulted in significantly higher total phenol content only on day 0 of the test. GSH was shown to be readily oxidised in these conditions: even on day 0, there was a considerable reduction (up to 73%) in GSH level. Moreover, after 8 days of oxidation, GSH concentration in all studied samples ranged from 4.79 to 5.11 mg L^?1; these values were close to GSH value in control wine (without added GSH) on day 0. On the contrary, GSH appeared to contribute significantly to the wine stability, which might have been via the increase of reduced phenolic pool. GSH addition appeared to have an improving effect on the organoleptic qualities of the wine.     

还原型谷胱甘肽的特性、工业生产及在大健康行业的应用研究进展

文章概述了还原型谷胱甘肽(glutathione, GSH)的结构特征和生理活性,及其在生物体内的代谢途径,总结了GSH的制备、提取纯化、应用及工业生产等方面的研究进展,并展望了其功能活性、工业生产和产品应用等方面今后的研究方向。     

前体氨基酸对热带假丝酵母产谷胱甘肽的影响

以能利用木糖发酵产谷胱甘肽(GSH)的热带假丝酵母突变株CV26为实验菌株,分别研究了添加L-半胱氨酸、谷氨酸、甘氨酸3种前体氨基酸对CV26产GSH的影响。在单因素实验基础上,对3种前体氨基酸的添加进行了正交实验,结果表明:在GSH发酵的12h添加6mmol/L的甘氨酸和2mmol/L的谷氨酸,24h添加3mmol/L的L-半胱氨酸,GSH的产量和胞内含量都有较大的提高,分别达到149.28mg/L和20.43mg/g,比对照组分别提高了51.5%和57.8%。     

丙酮酸钠促进S腺苷蛋氨酸和谷胱甘肽联合高产及其生理机制

为了考察丙酮酸钠对S-腺苷蛋氨酸(SAM)和谷胱甘肽(GSH)联产发酵的影响,本文通过在不同培养时间向摇瓶中添加不同浓度丙酮酸钠,发现0 h添加2 g/L丙酮酸钠最有利于SAM和GSH生物合成。在该条件下进行分批发酵培养,结果表明:SAM和GSH联产量在27 h时达到最大值402.3 mg/L,比不添加丙酮酸钠的对照提高了35.1%。进一步地,对酵母胞内SAM合成酶、γ-谷氨酰半胱氨酸合成酶、己糖激酶、异柠檬酸脱氢酶的活性以及NADH和ATP含量进行测定,结果发现:丙酮酸钠添加不仅提高了SAM和GSH代谢途径中的关键酶活性,还提高了胞内能量代谢物质的水平,最终提升了SAM和GSH的合成能力,实现了SAM和GSH的联合高产。该研究结果为类似耗能合成化合物的高效生产提供了一种可行的思路。     

Stability of metal-glutathione complexes during oxidation by hydrogen peroxide and Cu(II)-catalysis

Thiol-containing ligands such as glutathione (GSH) are expected to degrade in the presence of oxygen; however, complexation by Hg, Ag, and other trace metals may protect free thiol functional groups (R-S-) from oxidation, leading to persistence in surface water environments. In this study, the stability of GSH complexes with Hg2+, Ag+, and other metals including Cd2+, Zn2+, and Pb2+ was assessed during exposure to two potential environmental oxidants: H202 and Cu2+. The results indicated that Hg-(GSH)2 and Ag(GSH) complexes were completely stable for at least 2 days in the presence of either H202 or Cu2+. In contrast, free GSH oxidized within minutes to hours. Complexation by Cd, Zn, and Pb slightly decreased or did not significantly affect the oxidation rate of GSH, depending upon the pH (tested between pH 6 and 9). Thermodynamic modeling of GSH speciation demonstrated that the observed oxidation rates were not consistent with predicted free GSH3- concentration. These results indicated that Cd-, Zn-, and Pb-GSH complexes were susceptible to oxidation by a mechanism that differs from GSH3- oxidation. In contrast, Hg- and Ag-GSH complexes were inert for days, suggesting that they are stable for relatively long periods in the oxic water column. These results demonstrate that coordination of Hg(II) and Ag(I) to thiol-containing ligands can potentially increase persistence and transport in surface waters.     

能量策略对酶法合成谷胱甘肽效率影响

通过比较和调控细胞酶催化前体氨基酸合成谷胱甘肽(GSH)反应过程中的能量类型和添加策略,跟踪反应过程中合成GSH的浓度变化,考察能量种类和添加模式对酶法合成GSH转化效率的影响。结果表明,葡萄糖作为能量碳源对酵母细胞酶催化合成GSH转化率的影响明显,初始反应液添加100 mmol/L葡萄糖酶法合成GSH转化率为27.88%,分批定点流加葡萄糖(第2、3、4小时各添加27.78 mmol/L)酶法合成GSH转化率可达到35.81%;在酶反应过程能量供应策略中,初始反应液中添加0.5 g/L的腺苷时,酶法合成GSH的转化率提高到36.37%;初始反应液添加0.5 g/L腺苷结合分批定点流加葡萄糖(第2、3、4小时各添加27.78 mmol/L),酶法合成GSH转化率达到41.57%。