酸胁迫对S-腺苷甲硫氨酸和谷胱甘肽生物合成的影响及其生理机制

在高密度培养条件下,考察不同p H值环境对产朊假丝酵母生物合成S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)和谷胱甘肽(glutathione,GSH)的影响。结果发现弱酸胁迫(pH 3.5)有利于提升酵母细胞的SAM和GSH合成能力,实现SAM和GSH联合高产。通过对关键酶活性、能量物质供给和胞内氧化还原环境进行测定,发现弱酸胁迫提高了SAM合成酶、γ-谷氨酰半胱氨酸合成酶、己糖激酶、ATP合成酶以及过氧化氢酶的活性,提高了胞内NADH和ATP的水平和再生能力,为SAM和GSH的过量合成提供了充足的物质和能量保障。研究结果揭示了弱酸胁迫促进SAM和GSH生物合成的生理机制,也为类似小分子活性化合物的过量合成提供可行的思路。     

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。     

蛋清源活性肽对过氧化氢诱导的HEK293细胞谷胱甘肽抗氧化系统的影响

研究了蛋清源活性肽WNWAD、WNW、WAD、WN、AD的体外氧自由基吸收能力(ORAC活性),以及其对过氧化氢诱导的HEK293细胞还原型谷胱甘肽(GSH)含量、氧化型谷胱甘肽(GSSG)含量、GSH/GSSG比值以及γ-谷氨酰半胱氨酸合成酶(γ-GCS)表达的影响。结果表明,浓度为5.0μmol/L的蛋清源活性肽WNWAD、WNW、WAD、WN的ORAC活性均强于Trolox,AD没有ORAC活性。过氧化氢诱导的HEK293细胞氧化损伤模型组细胞中GSH含量由(173.52±2.87)μmol/L降至(115.85±2.03)μmol/L(P0.01),GSSG含量由(0.90±0.08)μmol/L升至(33.23±0.68)μmol/L(P0.01);细胞中加入不同浓度的蛋清源活性肽,GSH含量与损伤组相比,显著升高(P0.05),GSSG含量与损伤组相比明显降低(P0.01),在0.5μmol/L WNWAD作用下GSSG含量由(33.23±0.68)μmol/L降至(0.96±0.02)μmol/L (P0.01),接近对照组细胞中GSSG含量;GSH/GSSG比值由损伤组的3.49±0.68提至174.22±0.13(P0.01)。蛋清源活性肽处理过的细胞中γ-GCS mRNA表达量相对于损伤组有不同程度增加,其中5.0μmol/L WN处理过的细胞中γ-GCS m RNA表达量最大,为8.53±0.06。以上研究结果表明,蛋清源活性肽能有效清除活性氧,减少GSH消耗,从而使细胞内γ-GCS含量减少。蛋清源活性肽能促进γ-GCS mRNA的表达,使GSH的合成增加,起到氧化应激抑制作用。     

两阶段pH控制方式提高富硒产朊假丝酵母的性能

为了提高富硒产朊假丝酵母的性能,本文在摇瓶培养和分批培养的基础上,考察了不同pH控制方式对流加培养条件下富硒产朊假丝酵母细胞生长、谷胱甘肽（GSH）合成和有机硒转化的影响,结果发现：在12 h将pH从3.5切换至5.5（pH 3.5→5.5）的两阶段pH控制方式最有利于富硒产朊假丝酵母胞内GSH和有机硒含量的提高。在pH 3.5→5.5条件下,富硒产朊假丝酵母胞内GSH含量、有机硒含量和有机硒转化率分别达到最大值13.09 mg/g、1.88 mg/g和94.69%。通过对酵母胞内GSH合成关键酶活性、氧化还原酶活性和能量代谢物质ATP水平进行测定,发现pH 3.5→5.5两阶段pH控制方式提高了富硒酵母胞内过氧化氢酶活性,降低了丙二醛含量,为酵母进行有机硒富集与转化以及GSH合成与积累提供了合适的氧化还原环境,并最终提高了富硒酵母的性能。     

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。     

外源性氧化胁迫对酿酒酵母突变株Y518合成谷胱甘肽的影响

利用外源过氧化氢对酿酒酵母突变株Y518进行氧化胁迫来研究外源性氧化胁迫对Y518合成谷胱甘肽的影响。结果表明:添加过氧化氢会抑制Y518细胞生长,但会促进其合成谷胱甘肽;当过氧化氢的添加时间为24h,添加量为0.6mmol/L时,Y518胞内谷胱甘肽含量达到(13.69±0.28)mg/g,比未添加过氧化氢时提高了约14%;在24h时添加0.6 mmol/L过氧化氢会对Y518产生外源性氧化胁迫,激活转录调节子YAP1和SKN7调控γ-谷氨酰半胱氨酸合成酶和谷胱甘肽合成酶编码基因GSH I和GSH II的表达,提高γ-谷氨酰半胱氨酸合成酶和谷胱甘肽合成酶的活力,以促进Y518合成谷胱甘肽。因此,添加外源过氧化氢能够使酿酒酵母突变株Y518产生外源性氧化胁迫,促进Y518进一步合成谷胱甘肽。     

基于两阶段氨基酸添加的谷胱甘肽发酵高产方法

为提高产朊假丝酵母(Candida utilis SZU 07-01)发酵生产谷胱甘肽(glutathione,GSH)的产量,分别考察L-蛋氨酸和L-半胱氨酸在GSH分批发酵和流加发酵中的作用,结果发现L-蛋氨酸提升了酵母细胞在生长期的GSH合成能力,而L-半胱氨酸显著提高了细胞生长结束后的GSH产量。在此基础上,提出了两阶段氨基酸添加策略,即在分批发酵初始时(0 h)添加60 mmol/L L-蛋氨酸,在流加发酵过程中细胞干质量达到最大值时(第27小时)一次性添加30 mmol/L L-半胱氨酸。最终,GSH产量和胞内GSH含量进一步提高,分别达到1 247.1 mg/L和24.1 mg/g。该两阶段氨基酸添加策略在GSH发酵高产中的成功应用,对其他类似化合物的高效生产具有一定的借鉴意义。     

基于S-亚硝基化解析一氧化氮对哈密瓜采后抗环血酸-谷胱甘肽循环的影响

为了探究一氧化氮(NO)对哈密瓜采后贮藏期的抗氧化作用，该研究以“西州蜜17”为试材，采用外源NO精准熏蒸方法，分析测定哈密瓜生理指标、S-亚硝基化水平和抗氧化指标的变化，从S-亚硝基化水平的角度探讨NO对哈密瓜采后抗坏血酸-谷胱甘肽循环(glutathione-ascorbate cycle, AsA-GSH cycle)的影响。结果表明，NO熏蒸能较好地维持哈密瓜贮藏品质，降低果实H₂O₂、丙二醛(malondialdehyde, MDA)含量，显著提高果实内源NO和S-亚硝基硫醇(S-nitrosothiols, SNO)含量，抑制S-亚硝基谷胱甘肽还原酶(S-nitrosoglutathione reductase, GSNOR)活性升高。NO熏蒸可以维持较高的抗坏血酸(ascorbic acid, AsA)和脱氢抗坏血酸(dehydroascorbate, DHA)的比值(AsA/DHA)、还原型谷胱甘肽(reduced glutathione, GSH)和氧化型谷胱甘肽(oxidized glutathione, GSSG)的比值...     

奎宁酸的抗氧化活性和神经保护作用研究

奎宁酸是一种小米来源的类黄酮类物质,为探究奎宁酸的神经保护作用,该研究建立了H₂O₂诱导的SH-SY5Y细胞氧化应激模型,通过MTT法测定奎宁酸对SH-SY5Y细胞活性影响,同时测定了抗氧化酶超氧化物歧化酶(superoxide dismutase, SOD)的活性和氧化应激标志分子谷胱甘肽(glutathione, GSH)、丙二醛(malondialdehyde, MDA)的含量,利用实时荧光定量PCR检测了抗氧化酶和炎症因子表达情况,并用Western blot测定了炎症因子的蛋白表达量。研究结果表明,100μg/mL的奎宁酸处理显著抑制H₂O₂引起的SH-SY5Y细胞存活率下降;对细胞抗氧化指标的检测显示,奎宁酸处理可以增强细胞SOD的活性,增加GSH的水平并降低细胞的MDA水平,提高SOD、过氧化氢酶(catalase, CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase, GPx)等抗氧化酶基因的表达水平,降低氧化损伤;另外,奎宁酸还可以降低H₂     

卷烟烟气总粒相物诱导A549细胞的氧化应激研究

为研究卷烟烟气总粒相物(TPM)对人肺腺癌细胞(A549)的氧化损伤,捕集3R4F参比卷烟主流烟气的TPM,对A549细胞进行染毒,采用中性红细胞毒性法测试TPM与A549细胞存活率的剂量-效应关系,检测了细胞内谷胱甘肽(GSH)和细胞外超氧化物歧化酶(EC-SOD)两种氧化应激指标。结果表明:①TPM与A549细胞存活率之间存在良好的剂量-效应关系。②细胞发生氧化应激时,细胞内还原态谷胱甘肽和氧化态谷胱甘肽比值(GSH/GSSG)相对于阴性对照组有显著降低,且不随染毒时间的增加而变化;EC-SOD在染毒4 h后没有显著增加,但24 h后有显著增加。      

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.     

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.     

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.     

The effect of selenium on antioxidant system in erythrocytes and liver of the carp (Cyprinus carpio L.)

The effect of selenium‐supplemented diet (sodium selenate and selenium yeast) on antioxidant in erthrocytes and liver of the carp (Cyprinus carpio L.) fingerlings was studied. With this goal, the activities of glutathione peroxidase (GSH‐Px), catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione‐S‐transferase (GST), as well as glutathione (GSH + GSSG) level, were determined. In the group supplemented with sodium selenate, no significant changes in the activity of the above enzymes were recorded in both the erythrocytes and in the liver, with the exception of GST activity that was significantly reduced in the plasma compared with the controls. Glutathione content was at the control level. In the group supplemented with selenium‐yeast, the activities of GSH‐Px, CAT, and SOD were significantly increased in erythrocytes, whereas GST activity and plasma content of GSH + GSSG were reduced compared with the controls. At the same time, the activities of hepatic SOD and GST were increased compared with the controls. These results demonstrate that organically bound selenium (selenium‐yeast) acts more efficiently on antioxidant system of the carp fingerlings than inorganic selenium salts.     

Influence of dietary supplementation with dextrin or oligofructose on the hepatic redox balance in rats

We assessed the impact of oligofructose (OFS) and dextrin (DEX) as diet supplements on hepatic redox state. Rats were fed either a 10% OFS or a 10% DEX supplemented diet for 9 wk. In the DEX diet group, the levels of hepatic protein carbonylation were decreased by 63%. Total glutathione and reduced glutathione (GSH) contents were reduced in the OFS and DEX diet groups by around 20%. DEX supplementation significantly reduced oxidized glutathione (GSSG) levels resulting in a 33% increase in the GSH/GSSG ratio. The activity of the hepatic antioxidant enzymes was not changed by either OFS or DEX supplementation. OFS supplementation caused a decrease in serum levels of triglycerides (36%), cholesterol (24%), HDL (16%) and LDL (17%). DEX supplementation only reduced triglycerides (32%) and urea (22%). Both diets increased serum levels of acetate by fivefold and propionate by twofold, but DEX diet decreased butyrate levels by 75%. Due to their different composition/structure these two dietary fibers affected metabolism in different ways. Diet supplementation with 10% DEX can potentially improve host health, by protecting the liver from protein carbonylation and by improving GSH/GSSG ratio and diet supplementation with 10% OFS can improve the lipid profile.     

Bioactive compounds in rye flours with different extraction rates

Rye flours with extraction rate of 100% (wholemeal flour), 95% (brown flour), 90% (brown flour) and 70% (light flour) were prepared in order to study the relation between flour extraction rates and content of bioactive compounds. The following compounds were analysed: total phenolic compounds (TPC), total flavonoids (TF), inositol hexaphosphate (IP6), reduced (GSH) and oxidized (GSSG) glutathione, tocopherols (T) and tocotrienols (T3). The reduced/oxidized glutathione status (GSH/GSSG) of the flours was examined as a potential index of flour resistance against oxidative stress. The following observations were made in relation to the flour extraction rates and bioactive compounds contents: (a) milling process caused decrease in content in TPC, TF, IP6, GSH and GSSG, T and T3, (b) the most resistant against oxidation processes were suggested a brown flours, then light and finally wholemeal flour, (c) the ratio of tocotrienols to tocopherols (T3/T) was the highest in rye flours with extraction rate of 100–90% whereas light flour was the poorest source of tocopherols and tocotrienols. The provided data support current trend to increase number of rye products from wholemeal or brown flours.     

Bestimmung von reduziertem und oxydiertem Glutathion in Weizenmehlen und -teigen

Zusammenfassung Für die quantitative Analyse von reduziertem (GSH) und oxydiertem (GSSG) Glutathion in Cerealien wurde das folgende Verfahren entwickelt: Extraktion eines Mehles oder Teigs und Carboxymethylierung des GSH, Gelfiltration an Sephadex G-50, Chromatographic an Dowex 50 WX8 und nach alkalischer Spaltung Bestimmung des abgetrennten GSSG mit 5,5-Dithio-bis(2-nitrobenzoesaure), Abtrennung des carboxymethylierten GSH an Dowex 1X8 und Bestimmung mit 2,4,6-Trinitrobenzoesulfonsäure. In drei Sorten von Weizenmehlen wurden 0,27–0,46 mol/g GSH and 0,26–0,38 mol/g GSSG gefunden. Beim Kneten (5 min) verschwand das GSH und das GSSG nahm entsprechend zu.

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Determination of reduced and oxidised glutathione in wheat flours and doughs

Summary A quantitative method for the analysis of reduced (GSH) and oxidised (GSSG) glutathione in cereals has been developed. The steps are: Extraction of the flour or dough and carboxymethylation of GSH, gel filtration on Sephadex G-50, chromatography on Dowex 50 WX8 and after basic cleavage determination of the separated GSSG with 5,5-dithio-bis(2-nitrobenzoic acid), separation of carboxymethylated GSH on Dowex 1X8 and determination with 2,4,6-trinitrobenzenesulfonic acid. When analysing for the amount of GSH and GSSG in flours of three varieties of wheat the former ranged from 0.27 to 0.46 mol/g and the latter from 0.26 to 0.38 mol/g. After kneading for 5 min to a dough the GSH disappeared with a corresponding increase in GSSG.

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Herrn Prof. Dr. L. Acker zu seinem 65. Geburtstag gewidmet

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Wir danken der AIF und dem Forschungskreis der Ernährungsindustrie für die finanzielle Unterstützung derArbeit. Frl. Ch. Hopfer danken wir für die geschickte experimentelle Mitarbeit     

Evaluation of Equol Function on Anti- or Prooxidant Status in vivo

ABSTRACT:  The present study was performed to investigate the effects of equol on oxidative stress and the antioxidant defense system in the livers of mice. Mice were orally administered equol at either 5 or 25 mg/kg body weight/day for 1, 3, or 7 wk. Equol administration significantly inhibited biomarkers of oxidative stress (thiobarbituric acid-reactive substances value, carbonyl content, and serum 8-OH-dG) at all doses and for all durations of administration, and this phenomenon was most pronounced at 3 wk. Moreover, catalase and total superoxide dismutase (SOD) activities and their mRNA expression were significantly increased by equol. Although equol increased the glutathione peroxidase (GSH-px) activity in mice treated with equol for 1 wk, long-term administration of equol (7 wk) caused a decrease in the ratio of reduced/oxidized glutathione (GSH/GSSG) and the activities of GSH-px and glutathione reductase (GR). Taken together, these results suggest that equol may act as an antioxidant through an inhibition of oxidative stress and stimulation of catalase and SOD, but can also cause prooxidant effects such as reduction of the GSH/GSSG ratio, depending on the treatment period.