In vitro potential antioxidant activity of (1-->3),(1-->6)-beta-D-glucan and protein fractions from Saccharomyces cerevisiae cell walls

(1-->3),(1-->6)-Beta-D-Glucan, a cell wall polysaccharide in many microorganisms, fungi and algae, is a well-known biological response modifier. Recently, it was found that (1-->3)-beta-D-glucan from Saccharomyces cerevisiae also exhibits antioxidative capabilities. In this study the antioxidative activity of the cell wall fractions of brewer's yeast was investigated. Particular emphasis was put on the question to which extent glucan is responsible for the antioxidative activity of the cell walls and how the other cell wall components might contribute. For the experiments yeast cell walls from brewery fermentations were used. Glucan was isolated by a three-step extraction procedure including a combination of hot water and enzymatic treatment. The level of (1-->3),(1-->6)-beta-D-glucan in the cell walls was analyzed enzymatically. The antioxidant activity was determined by electron paramagnetic resonance spectrometry and Trolox equivalent antioxidant capacity assay. The results show that the antioxidative activity of yeast cell wall proteins exceeds that of beta-glucan greatly. Especially aromatic side chains and free thiols from denatured proteins seem to work as antioxidants.     

Identification of cysteinylated aroma precursors of certain volatile thiols in passion fruit juice

Together with 3-mercaptohexan-1-ol and 3-mercaptohexyl acetate, already known to contribute to the aroma of passion fruit (Passiflora edulis), 3-mercapto-3-methylbutan-1-ol and 3-mercapto-3-methylbutyl acetate have been identified for the first time in this fruit. 3-Mercaptohexan-1-ol and 3-mercapto-3-methylbutan-1-ol may be produced in vitro from nonvolatile extracts of this fruit by the enzymatic action of a cell-free extract of Eubacterium limosum, which has a beta-lyase activity on S-cysteine conjugates (EC 4.4.1.13). This release strongly suggests that these volatile thiols are present in combined form, as S-cysteine conjugates. It was possible to identify the precursor of 3-mercaptohexan-1-ol as S-(3-hexan-1-ol)-L-cysteine, in the form of trimethylsilylated derivatives from the juice of this fruit, using GC/MS analysis. The presence of free and combined forms of these volatile thiols in this fruit has now been demonstrated.     

钙处理对葡萄柚果实细胞壁物质代谢及其相关基因表达的影响

【目的】研究采前、 采后钙处理对葡萄柚果实细胞壁组分、 细胞壁降解酶活性变化及其相关基因表达的影响,可为了解钙与果实细胞壁物质代谢之间的关系,揭示钙对果实软化的作用机理,为调控葡萄柚果实膳食纤维含量,提高果实质地品质提供理论依据。【方法】试验于2011年2月至11月在云南省玉溪市葡萄柚果园进行,供试品种为‘里约红’葡萄柚,该品种于2005年嫁接于当地砧木,株行距为3 m×3 m。试验由采前和采后钙处理两部分组成。采前钙处理在幼果初期、 幼果末期、 膨大初期、 膨大末期、 转色期,叶面喷施2% CaCl2; 采后钙处理在果实成熟采后浸于2% CaCl2溶液5 min, 室温贮藏。之后每15天取样一次,每次取10个果实,测定葡萄柚果肉细胞壁组分、 细胞壁降解酶活性及其基因表达量。【结果】随葡萄柚果实后熟软化,紧密结合型果胶(共价结合型果胶)解聚为松散结合型果胶(水溶性果胶、 离子结合型果胶),紧密结合型半纤维素(24% KOH可溶性半纤维素)解聚使其含量下降,而松散结合型半纤维素含量增加(4%KOH可溶性半纤维素)。果实PG、 PME、 Cx、 α-L-Af和β-Gal酶活性及其基因表达量均随果实软化呈不同程度增加。PME活性在果实采收后表现出较高含量,而PG活性在果实贮藏前期急剧增加,其酶基因的表达量与酶活性变化趋势基本一致。Cx、 α-L-Af和β-Gal活性在贮藏中、 后期上升较快,相关酶基因的表达量亦明显增加。钙处理显著地降低果实细胞壁降解酶活性和基因表达水平,其中采后钙处理对α-L-Af和β-Gal活性和基因表达在贮藏中、 后期的调控作用较显著,酶活性和基因表达均维持在较低水平。【结论】外源钙处理降低细胞壁降解酶活性及其基因表达,抑制了细胞壁物质的解聚,采后钙处理对细胞壁物质代谢的调控效果优于采前钙处理。外源钙处理抑制了细胞壁降解酶基因表达水平,降低了细胞壁降解酶活性,减缓了果胶、 半纤维素的解聚,从而达到调控果实膳食纤维含量、 维持果实质地品质、 延长果实货架期寿命的目的。     

Impact of UV-C irradiation on the cell wall-degrading enzymes during ripening of tomato (Lycopersicon esculentum L.) fruit

The effect of a hormic dose of UV-C (254 nm) on changes in fruit firmness and cell wall-degrading enzyme (CWDE) activity was determined using tomato fruit. Throughout the storage period, a decrease in firmness was jointly observed with an increase of the CWDE (polygalacturonase, pectin methyl esterase, cellulase, xylanase, beta-D-galactosidase, and protease) activity for all treatments, suggesting the involvement of these enzymes in the ripening process. However, the enhancement in the activity of the CWDE was significantly less in fruit subjected to the hormic dose of UV-C. This reduction may explain why irradiated fruit were firmer than control and consequently may explain how UV-C could delay the ripening and senescence process. We suggest that the CWDE are one of the targets of the UV-C, and by this action, irradiation contributed to a delay of the cell wall degradation and consequently retarded softening of the tomato fruit tissues.     

Pectin methylesterase from kiwi and kaki fruits: purification, characterization, and role of pH in the enzyme regulation and interaction with the kiwi proteinaceous inhibitor

Pectin methylesterase was purified from kiwi (Actinidia chinensis) and kaki fruit (Diospyros kaki). The pH values of the fruit homogenates were 3.5 and 6.2, respectively. The kiwi enzyme is localized in the cell wall and has a neutral-alkaline pI, whereas the kaki enzyme is localized in the soluble fraction and has a neutral-acidic pI. The molecular weights of the kiwi and kaki enzymes were 50 and 37 kDa, respectively. The two enzymes showed a similar salt and pH dependence of activity, and a different pH dependence of the inhibition by the kiwi proteinaceous inhibitor.     

猕猴桃果实贮藏期间细胞壁多糖物质降解特性及组织结构差异分析

为了深入了解猕猴桃果实采后细胞壁多糖物质降解及组织结构变化与果实贮藏性的关系,本研究以红阳和华特猕猴桃果实为试材,对25和4℃贮藏期间的细胞壁多糖物质含量及果胶降解酶活性进行测定,并比较两品种果实在25℃贮藏期间的细胞显微结构和钙组分含量差异。结果表明,在25和4℃贮藏条件下,随着贮藏时间的延长,红阳和华特猕猴桃果实中半纤维素（HCL）、纤维素（CL）和共价结合型果胶（CSP）含量不断降低,水溶性果胶（WSP）含量不断上升,离子结合型果胶（ISP）含量相对稳定。红阳猕猴桃各细胞壁多糖组分含量变化速度较快。两品种猕猴桃果实硬度均与WSP含量呈显著负相关,与CSP含量呈显著正相关。果胶降解酶活性检测结果显示,25℃贮藏前期,红阳猕猴桃中果胶酸裂解酶（PL）和β-半乳糖苷酶（β-Gal）活性显著高于华特;贮藏中后期,红阳中果胶甲酯酶（PME）活性显著高于华特。4℃贮藏期间,红阳中PME活性仍显著高于华特;4℃贮藏前期,红阳中β-Gal活性与华特无显著差异,而PL活性低于华特。相关性分析表明,25℃贮藏期间,与红阳和华特果实软化显著相关的果胶降解酶分别是PME和PL;4℃贮藏期间,与华特果实软...     

Guava fruit (Psidium guajava L.) as a new source of antioxidant dietary fiber.

Guava (Psidium guajava L.) is a tropical fruit, widely consumed fresh and also processed (beverages, syrup, ice cream, and jams). Pulp and peel fractions were tested, and both showed high content of dietary fiber (48.55-49.42%) and extractable polyphenols (2.62-7.79%). The antioxidant activity of polyphenol compounds was studied, using three complementary methods: (i) free radical DPPH* scavenging, (ii) ferric reducing antioxidant power assay (FRAP), and (iii) inhibition of copper-catalyzed in vitro human low-density lipoprotein (LDL) oxidation. All fractions tested showed a remarkable antioxidant capacity, and this activity was correlated with the corresponding total phenolic content. A 1-g (dry matter) portion of peel contained DPPH* activity, FRAP activity, and inhibition of copper-induced in vitro LDL oxidation, equivalent to 43 mg, 116 mg, and 176 mg of Trolox, respectively. These results indicate that guava could be a suitable source of natural antioxidants. Peel and pulp could also be used to obtain antioxidant dietary fiber (AODF), a new item which combines in a single natural product the properties of dietary fiber and antioxidant compounds.     

Great differences in antioxidant properties exist between 56 apple cultivars and vegetation seasons

The contents of ascorbate, thiols, and phenolic compounds and antioxidative enzyme activity were measured in the apple peel of 56 genotypes after harvest in two vegetation seasons, 2003 and 2004. The main reason of great interest in these bioactive compounds is their well-established physiological role in all living systems. The biggest differences between tested genotypes were noted for ascorbate peroxidase and glutathione reductase (GR) activity, followed by total ascorbate, phenolics, and glutathione concentration; the least difference was observed in the case of catalase. A large cultivar variation was noted in the anthocyanins and flavonols contents. Distinguishing the cultivars with the lowest, highest, relatively stable or those in which antioxidant content greatly differed depending on growing seasons was attempted. The GR activity is proposed as an environmental stress marker of apple fruit.     

混施微生物菌剂和有机肥对‘新红星’苹果解袋后果实品质的影响

以5年生‘新红星’苹果果树为试材,探究不同施肥处理(T1,有机肥;T2,微生物菌剂+有机肥;T3,微生物菌剂;CK,对照)对‘新红星’苹果解袋后果实内在品质、呼吸速率、乙烯释放率、果实硬脆性及相关酶和基因表达的影响。结果表明,与传统单一施肥相比,施加含微生物菌剂的有机肥显著提高了果实可溶性固形物含量、可溶性糖含量及糖酸比。解袋后0~12 d果实呼吸速率及乙烯释放速率显著上升,12~15 d趋于平稳。果实解袋后第9、12、15 d,T2组果实可溶性固形物含量相对于CK组高出2.63%、5.89%、8.78%;可溶性糖高出6.89%、6.71%、12.86%;糖酸比高出15.85%、19.25%、17.08%,且差异显著。施加含微生物菌剂的有机肥处理(T2)显著提高了果实的总香气含量,其含量相对于CK、T1、T3组分别高出16.69%、9.06%、6.25%。混施微生物菌剂和有机肥有利于解袋后苹果果实着色,使果皮细胞排列紧密整齐,对提升果实脆度,减缓果实硬度下降具有重要作用。细胞壁关键酶果胶甲酯酶(PME)、多聚半乳糖醛酸酶(PG)活性在解袋后一直处于上升状态,其中T2组PME、PG活性及MdPME、MdPG表达量均低于CK组,因此果实在解袋后保持较高的硬脆度。综上所述,混施微生物菌剂和有机肥(T2)对提高‘新红星’苹果果实品质及延长货架期具有重要作用。     

酸性硫酸钙处理对水蜜桃采后匍枝根霉致病力的影响

为研究酸性硫酸钙(ACS)溶液对水蜜桃果实采后致腐匍枝根霉(Rhizopus stolonifer)的抑菌效果,在离体和体内条件下,探究其对匍枝根霉细胞膜渗透率、细胞壁降解酶、呼吸相关酶活性和桃果发病率的影响。结果表明,不同稀释倍数的ACS溶液对匍枝根霉均有不同程度的抑菌作用,其中ACS-10^-3处理组的抑菌效果最强。在作用120 min时,ACS-10^-3组菌丝细胞膜渗透率达到52.76%,较CK高出39.72%;匍枝根霉在含有50%ACS稀释液的带药培养基中培养9 d时,ACS-10^-3组的PG、PMG、C_X和GLU等细胞壁降解酶活性得到了抑制,较CK分别降低38.41%、41.96%、59.31%和35.52%;且在很大程度上抑制了呼吸能量代谢系统中酶活性的上升,培养第9天时匍枝根霉的SDH、MDH和IDH活性分别为0.02±0.01、0.68±0.12、106.13±3.54 nmol·min^-1·g^-1,均显著低于CK(P<0.05)。同时,ACS稀释液可显著降低损伤接种桃果实的匍枝根霉病斑面积和接种发病率,与CK相比,ACS-10^-3组桃果实的匍枝根霉病斑面积和接种发病率分别降低了99.61%和73.33%。因此,适宜稀释倍数的ACS溶液能够有效抑制水蜜桃采后因匍枝根霉侵染引起的软腐病。     

Biochemical and genomic analysis of neoculin compared to monocot mannose-binding lectins

Neoculin occurring in an edible tropical fruit is a heterodimeric protein which has both sweetness and a taste-modifying activity that converts sourness to sweetness. Both the primary and the overall tertiary structures of neoculin resemble those of monocot mannose-binding lectins. This study investigated differences in biochemical properties between neoculin and the lectins. Structural comparison between the mannose-binding sites of lectins and the corresponding regions of neoculin showed that there is at least one amino acid substitution at each site in neoculin, suggesting a reason for the lack of its mannose-binding ability. This was consistent with hemagglutination assay data demonstrating that neoculin had no detectable agglutinin activity. DNA microarray analysis indicated that neoculin had no significant influence on gene expression in Caco-2 cell, whereas kidney bean lectin (Phaseolus vulgaris agglutinin) greatly influenced various gene expressions. These data strongly suggest that neoculin has no lectin-like properties, encouraging its practical use in the food industry.     

Fluctuations of formamide hydrolase activity in a barley field soil after drying and rewetting

Formamide hydrolase activity in a barley field soil was described by a two-component, rather than a one-component model of Michaelis–Menten kinetics. The two-component model had both a high-affinity (K_m values of 0.5–1.0 mM) and a low-affinity (K_m values of 30–60 mM) activity. Rapid and transient increases in both the overall heterotrophic activity (CO₂ production) and the high-affinity component of formamide hydrolase activity were observed in rewetted soil samples, suggesting that the high-affinity component represented enzyme activity of active microorganisms. Short-term field variations in the high-affinity component during a barley growth season were consistent with rapid, transient responses of microbial activity to precipitation and soil wetting. An assay of the high-affinity component of formamide hydrolase activity as approximated by rate determination at a single low (1 mM) formamide concentration can provide an easy and rapid indication of enzyme activity related directly to active microorganisms in the soil.     

Temporal sequence of cell wall disassembly events in developing fruits. 2. Analysis of blueberry (Vaccinium species)

Softening and pathogen susceptibility are the major factors limiting the marketing of blueberries as fresh fruits, and these traits are associated with fruit cell wall structure. However, few studies that characterize wall modifications occurring during development and ripening have been reported for this fruit. In this study the ripening-associated modifications of blueberry fruit cell walls (composition, pectin and hemicellulose solubilization, and depolymerization) at five stages of ripeness have been analyzed. Xylose was found to be the most abundant noncellulosic neutral sugar associated with fruit walls, and the observed high Xyl/Glc ratio suggested that xylans, which are usually a minor hemicellulosic fruit wall component, are abundant in blueberry. The pectic matrix showed increased solubilization at early and intermediate stages of ripening, but no changes were detected in late ripening. Furthermore, little reduction in pectin polymer size occurred during blueberry ripening. In contrast, hemicellulose levels decreased as ripening progressed, and a clear depolymerization of these components was observed. A model for cell wall degradation in this fruit is discussed.     

Screening and identification of antioxidants in biological samples using high-performance liquid chromatography-mass spectrometry and its application on Salacca edulis Reinw

In this study, a new approach was developed for screening and identifying antioxidants in biological samples. The approach was based on significant decreases of the intensities of ion peaks obtained from high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) upon reaction with 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals. HPLC-MS/MS was further applied to elucidate structures of antioxidant peaks characterized in a spiking test. The new approach could also be used to monitor the reactivity of antioxidants in biological sample with free radicals. The approach was successfully applied to the identification of antioxidants in salak (Salacca edulis Reinw), a tropical fruit that is reported to be a very good source of natural antioxidants, but it was still not clear which compounds were responsible for its antioxidant property. The antioxidants in salak were identified to be chlorogenic acid, (-)-epicatechin, and singly linked proanthocyanidins that mainly existed as dimers through hexamers of catechin or epicatechin. In salak, chlorogenic acid was identified to be an antioxidant of the slow reaction type as it reacted with free radicals much more slowly than either (-)-epicatechin or proanthocyanidins. The new approach was proved to be useful for the characterization and identification of antioxidants in biological samples as a mass detector combined with an HPLC separation system not only serves as an ideal tool to monitor free radical active components but also provides their possible chemical structures in a biological sample.     

Enzyme-assisted processing increases antimicrobial and antioxidant activity of bilberry

The effects of nine cell wall-degrading enzymes on the antimicrobial and antioxidant activities of bilberry were studied. Antimicrobial activity was measured using the human pathogens Salmonella enterica sv. Typhimurium and Staphylococcus aureus as test strains. Enzyme treatments liberated phenolics from the cell wall matrix, which clearly increased the antimicrobial activity of berry juices, press cakes, and berry mashes on the basis of plate counts. Antibacterial effects were stronger against Salmonella than against Staphylococcus bacteria. In general, the increase in activity measured as colony-forming units per milliliter was 3-5 logarithmic units against Salmonella and 1-2 units against Staphylococcus bacteria. Increase in antimicrobial activity was observed only in acidic conditions, which is also the natural environment in various berry products, such as juices. The activity profile of the pectinase preparation affected the chemistry of the phenolics due to the presence of deglycosylating activities in some preparations. The difference in phenolic profiles was reflected in the antimicrobial effects. Bilberry mashes treated with Pectinex Ultra SP-L, Pectinex 3 XL, and Pectinex BE XXL were most efficient against Salmonella bacteria, whereas mashes treated with Pectinex Smash, Pectinex BE 3-L, and Biopectinase CCM showed the strongest antimicrobial activity against Staphylococcus bacteria. Due to the liberation of phenolics from the cell wall matrix the antioxidant activity measured as radical scavenging activity was also increased on average about 30% by the enzymatic treatments. The highest increase in phenolic compounds was about 40%. Highest increases in anthocyanins and in antioxidant activity were observed in berry mash treated with Pectinex Smash XXL enzyme, and the lowest increase was observed after treatment with Pectinex BE 3-L. Enzyme-assisted processing is traditionally used to improve berry and fruit juice yields. However, enzymatic treatments also have an impact on the functional properties of the products. The increased liberation of phenolics from the cell wall matrix can prolong the shelf life of berry products by limiting the growth of contaminants during processing or storage. The increased amount of phenolic compounds may also have a positive effect on gut well-being.     

Molecular cloning and biochemical characterization of an endo-β-mannanase gene from soybean for soybean meal improvement

Soybean meal is the most commonly used protein source in animal feeds. Among the undesirable attributes of soybean meal is the high level of β-mannan, which was determined to be detrimental to the growth performance of animals. β-Mannan is a type of hemicellulose in the plant cell wall and can be hydrolyzed by endo-β-mannanase. The goal of this study is to isolate and characterize an endo-β-mannanase gene from soybean that can be used for genetic improvement of soybean meal. From the sequenced soybean genome, 21 putative endo-β-mannanase genes were identified. On the basis of their relatedness to known functional plant endo-β-mannanases, four soybean endo-β-mannanase genes (GmMAN1 to GmMAN4) were chosen for experimental analysis. GmMAN1 and GmMAN4 showed expression in the soybean tissue examined, and their cDNAs without the sequences for signal peptide were cloned and expressed in Escherichia coli to produce recombinant enzymes. Only GmMAN1 showed endo-β-mannanase hydrolase activity. Further gene expression analysis showed that GmMAN1 is specifically expressed in cotyledons of seedlings, suggesting a role of GmMAN1 in degrading mannan-rich food reserves during soybean seedling establishment. Purified recombinant GmMAN1 exhibited an apparent K(m) value of 34.9 mg/mL. The catalytic efficiency (k(cat)/K(m)) of GmMAN1 was determined to be 0.7 mL/(mg·s). GmMAN1 was also shown to be active in hydrolyzing the β-mannan-rich cell wall of soybean seeds.     

海藻酸钠寡糖提高水稻幼苗对镉胁迫的抗性

通过水培试验,研究了海藻酸钠寡糖对水稻镉(Cd)毒害的缓解效果,并从Cd的吸收分布、非蛋白巯基(NPT)含量和抗氧化酶活性等方面,初步探讨了其可能的生理机制。结果表明,海藻酸钠寡糖预处理一定程度上可缓解水稻Cd毒害,其中20 mg/L处理的效果较明显,表现为促进了幼苗生长,提高了光合色素含量。进一步分析显示,海藻酸钠寡糖处理的水稻地上部和地下部Cd含量及转移效率均显著降低,Cd在根部细胞壁中的含量及所占比例则显著增加,叶片和根中NPT含量也分别提高了23.5%和14.9%,植物螯合肽(PCs)含量分别增加28.0%和15.0%,抗氧化酶活性和脯氨酸含量也有不同程度提高,丙二醛含量显著下降27.1%。这些结果初步说明细胞壁固持、巯基分子螯合和抗氧化系统均参与了海藻酸钠寡糖对水稻Cd毒害的缓解。     

The effect of peatland drainage and rewetting (ditch blocking) on extracellular enzyme activities and water chemistry

Extensive areas of European peatlands have been drained by digging ditches in an attempt to improve the land, resulting in increased carbon dioxide fluxes to the atmosphere and enhanced fluvial dissolved organic carbon (DOC) concentrations. Numerous peatland restoration projects have been initiated which aim to raise water tables by ditch blocking, thus reversing drainage‐induced carbon losses. It has been suggested that extracellular hydrolase and phenol oxidase enzymes are partly responsible for controlling peatland carbon dynamics and that these enzymes are affected by environmental change. The aim of this study was to investigate how drainage and ditch blocking affect enzyme activities and water chemistry in a Welsh blanket bog, and to study the relationship between enzyme activity and water chemistry. A comparison of a drained and undrained site showed that the drained site had higher phenol oxidase and hydrolase activities, and lower concentrations of phenolic compounds which inhibit hydrolase enzymes. Ditch blocking had little impact upon enzyme activities; although hydrolase activities were lowered 4–9 months after restoration, the only significant difference was for arylsulphatase. Finally, we noted a negative correlation between β‐glucosidase activity and DOC concentrations, and a positive correlation between arylsulphatase activity and sulphate concentration. Phenol oxidase activity was negatively correlated with DOC concentrations in pore water, but for ditch water phenol oxidase correlated negatively with the ratio of phenolics to DOC. Our results imply that drainage could exacerbate gaseous and fluvial carbon losses and that peatland restoration may not reverse the effects, at least in the short term.     

Temporal sequence of cell wall disassembly events in developing fruits. 1. Analysis of raspberry (Rubus idaeus)

Raspberry fruits were harvested at five developmental stages, from green to red ripe, and the changes in cell wall composition, pectin and hemicellulose solubilization, and depolymerization were analyzed. Fruit softening at intermediate stages of ripening was associated with increased pectin solubilization, which occurred without depolymerization. Arabinose was found to be the most abundant noncellulosic neutral sugar in the cell wall and showed dramatic solubilization late in ripening. No changes in pectin molecular size were observed even at the 100% red stage. Subsequently, as fruit became fully ripe a dramatic depolymerization occurred. In contrast, the hemicellulosic fractions showed no significant changes in content or polymer size during ripening. The paper discusses the sequence of events leading to cell wall disassembly in raspberry fruit.