Purification and characterization of hydrolase with chitinase and chitosanase activity from commercial stem bromelain

A hydrolase with chitinase and chitosanase activity was purified from commercial stem bromelain through sequential steps of SP-Sepharose ion-exchange adsorption, HiLoad Superdex 75 gel filtration, HiLoad Q Sepharose ion-exchange chromatography, and Superdex 75 HR gel filtration. The purified hydrolase was homogeneous, as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited chitinase activity for hydrolysis of glycol chitin and 4-methylumbelliferyl beta-D-N,N',N' '-triacetylchitotrioside [4-MU-beta-(GlcNAc)(3)] and chitosanase activity for chitosan hydrolysis. For glycol chitin hydrolysis, the enzyme had an optimal pH of 4, an optimal temperature of 60 degrees C, and a K(m) of 0.2 mg/mL. For the 4-MU-beta-(GlcNAc)(3) hydrolysis, the enzyme had an optimal pH of 4 and an optimal temperature of 50 degrees C. For the chitosan hydrolysis, the enzyme had an optimal pH of 3, an optimal temperature of 50 degrees C, and a K(m) of 0.88 mg/mL. For hydrolysis of chitosans with various N-acetyl contents, the enzyme degraded 30-80% deacetylated chitosan most effectively. The enzyme split chitin or chitosan in an endo-manner. The molecular mass of the enzyme estimated by gel filtration was 31.4 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 5.9. Heavy metal ions of Hg(2+) and Ag(+), p-hydroxymercuribenzoic acid, and N-bromosuccinimide significantly inhibited the enzyme activity.     

Ascorbic acid-induced browning of (+)-catechin in a model wine system

The ability of ascorbic acid to induce browning of (+)-catechin in a model wine system has been studied. A significant increase in absorbance at 440 nm was observed over 14 days when ascorbic acid was incubated at 45 degrees C with (+)-catechin in a model wine base. The onset of browning was delayed for about 2 days, although the length of the lag period was dependent on the amount of molecular oxygen in the headspace of the reaction system. The lag period was not observed when a preoxidized solution of ascorbic acid was used, suggesting that a product of ascorbic acid oxidation is responsible for the onset of browning. Hydrogen peroxide, when added directly to (+)-catechin in the model system, was not capable of producing the same degree of browning as that generated by ascorbic acid. Liquid chromotography evidence is presented to show that different reaction products are produced by ascorbic acid and hydrogen peroxide.     

Release of lipids during yeast autolysis in a model wine system

The release of lipids during the aging of sparkling wines in contact with yeast can influence wine sensory attributes and, especially, foam characteristics. Model systems allow study of the autolysis process in a reasonable period of time compared to natural conditions, at which it can last several months. In this paper, the release of the different classes of lipids during the autolysis of three commercial yeast strains in a model wine medium has been monitored. Due to the absence of accurate quantitative methods, an HPLC method for separating and quantifying the different neutral and polar yeast lipid classes was developed. Lipids were eluted through a YMC PVA-Sil column with a complex solvent mixture. Detection was carried out with a light-scattering detector. The yeasts were suspended in the model wine buffer and incubated at 30 degrees C for up to 12 days. A release of triacylglycerols, 1,3-diacylglycerols, 2-monoacylglycerols, free fatty acids, sterol esters, and sterols was observed over the first 2 days, a period that corresponded to the maximum loss of yeast viability. A decrease in most of these lipids was observed from day 2, possibly indicative of the release of yeast hydrolytic enzymes due to the breakdown of the cell wall. Phospholipids were not detected in any of the autolysates. The mean lipid content in the autolysates as a percentage of the total lipid content in the yeasts was 8.6% for sterol esters, 3.8% for sterols, 2% for triacylglycerols, and <2% for 1,3-diacylglycerols and free fatty acids.     

Influence of wine structurally different polysaccharides on the volatility of aroma substances in a model system

The influence of native polysaccharides on wine organoleptic quality has not been yet clarified. Hence, the effect of purified fractions of arabinogalactan-proteins (AGPs), monomeric and dimeric rhamnogalacturonans II (mRG-II and dRG-II), and mannoproteins (MPs) on the volatility of various aroma substances was examined using the exponential dilution technique. The volatility of isoamyl acetate and ethyl hexanoate in a model wine system was not affected upon addition of wine polysaccharides in the range 5-20 g/L. At higher levels, the two esters were significantly retained in solution in the presence of the protein-rich polysaccharides MP0 and AGP0 and weakly salted out in the presence of the uronic acid-rich fractions AGP4, mRG-II, and dRG-II. In addition, the volatility increase caused by dRG-II on the esters was reversed when water was used as the solvent. 1-Hexanol exhibited retention in water with four fractions in the strength order AGP0 > dRG-II > mRG-II > AGP4 but was strongly salted out in the presence of fraction MP0. Furthermore, the diacetyl activity in water was overall not modified increasing only at high concentrations of AGP4. Reference polysaccharides dextrin and dextran were used for binding strength comparison.     

Characterization of the nitrogen compounds released during yeast autolysis in a model wine system

The nitrogen composition of wines aged with yeast for a long period of time, as in the case of sparkling wines, depends on the composition of the base wine and on the compounds released by the yeast. In this paper, the release of the different classes of nitrogen compounds during autolysis of one of the strains of yeast used in the manufacture of sparkling wines has been studied. The yeast, Saccharomyces bayanus, was suspended in a model wine buffer, pH 3.0 and 10% ethanol, and incubated at 30 degrees C. Samples of the autolysate were taken after 4, 24, 48, 72, 168, and 360 h of autolysis. An electrophoretic and chromatographic study was conducted of the proteins, peptides with molecular weights higher and lower than 700 Da, and amino acids released during the autolysis. Using SDS-PAGE, it was observed that it was predominantly polypeptides with molecular weights lower than 10 000 that were released. Through HPLC of the fraction lower than 10 000 Da, it was observed that it is polypeptides with molecular weights of between 10 000 and 700 Da that are released first and that these later break up to give rise to peptides with molecular weights lower than 700 Da, which in turn break down into amino acids. This indicates that the nature of the nitrogen compounds present in wines aged with yeast depends on the aging time, being less polymerized as the aging time increases.     

Iron(III) tartrate as a potential precursor of light-induced oxidative degradation of white wine: studies in a model wine system

The potential for iron(III) tartrate to act as a photoactivator in light-induced oxidative degradation of white wine is described. Using a tartaric-acid-based model wine system containing 5 mg/L iron, exposure to light from a xenon arc lamp led to the oxidative degradation of tartaric acid and the production of glyoxylic acid. The critical wavelength of light for the degradation process was found to be below 520 nm. No glyoxylic acid was formed in the absence of iron and/or light. Flint glass offered little protection from the light-induced photodegradation of tartaric acid. Antique Green glass offered more protection but did not stop the photodegradation process.     

Impact of glutathione on the formation of methylmethine- and carboxymethine-bridged (+)-catechin dimers in a model wine system

This study was performed to assess the impact of glutathione on the reaction between (+)-catechin and carbonyl compounds in wine-related conditions. (+)-Catechin (0.50 mM) and either glyoxylic acid (0.25 mM) or acetaldehyde (0.25 mM) were added to a model wine system with 0.0, 0.25, and 2.5 mM of glutathione added. UPLC-DAD and LC-MS analysis showed that the formation of carbonyl-bridged (+)-catechin dimers was inhibited in the samples with a glutathione to carbonyl ratio of 10:1 compared to the samples without glutathione. At a ratio of 1:1, glutathione inhibited the acetaldehyde-bridged dimers but only had a minor impact on the glyoxylic acid-bridged dimers. Further investigations showed that this trend of inhibition by glutathione on the glyoxylic acid-derived dimer was independent of temperatures, 20 °C vs 45 °C, or the presence of metal ions, 0.2 mg/L copper(II) and 5 mg/L iron(II). (1)H NMR analysis and LC-MS analysis provided evidence that glutathione inhibited dimer formation via different mechanisms depending on the carbonyl compound. For acetaldehyde-derived dimers, the main mode of inhibition was the ability of glutathione to form a (methyl-glutathionyl-methine)-(+)-catechin complex. Alternatively, the formation of a glutathione-glyoxylic acid addition product impeded the reaction between glyoxylic acid with (+)-catechin. These results demonstrate that glutathione, at sufficient concentration, can have a substantial impact on carbonyl-derived polymerization reactions in wine-like conditions.     

Effect of aluminum on variations in the proteins in pineapple roots

Abstract

Aluminum (Al) is biotoxic, often active in acid soil and retards the growth of crop roots. Cayenne is a type of pineapple cultivar that can be well cultivated in a strongly acid environment containing AlCl₃ concentrations up to 300?μmol?L^?1. In addition to organic acids, variations in the proteins in root apices are regarded as the mechanism involved in Al resistance. The objective of the present study was to explore the responsive proteins of Al stress in a known Al-resistant pineapple cultivar (i.e. Cayenne). After root emergence, pineapple seedlings were exposed to hydroponic solutions each containing 0 and 300?μmol?L^?1 AlCl₃ for 4?weeks. The total proteins in the root apices were separated using 2-D electrophoresis and a total of 17 apparently differential spots were identified by mass spectrometry, with 10 upregulated and seven downregulated proteins. The root apices of Cayenne under Al stress could be characterized by cellular activities involved in, for example, carbohydrate metabolism, organic acid production, energy metabolism, alleviating redox damage and root phenotypical change, which are critical for plant survival under Al toxicity. In contrast, there are one hypothetical and three unknown proteins that play unknown roles in Al resistance and warrant further investigation. The present study may provide an important clue to future proteomic research on Al-resistant mechanisms in pineapple.     

Sorption behavior of volatile phenols at the oak wood/wine interface in a model system

The sorption in a model system of aroma compounds of enological interest (mixture of the eight derivatives from guaiacol, 4-ethylphenol, and whiskylactone) onto wood was investigated to assess the influence of wood on the concentration of these volatiles during the aging of wine. To evaluate the influence of the solubility of aroma compounds in sorption phenomena, this parameter was determined for each volatile compound in model wine at 10 and 25 degrees C. The solubility is significantly higher in the model wine than in water and remains constant in the range of temperatures studied, except for guaiacol and vanillin. Kinetic and equilibrium sorptions were investigated. Sorption kinetics showed that the sorption equilibrium for all aroma compounds was reached after 6-7 days. From the study of the individual sorption isotherms, two distinct kinds of sorption behavior were observed depending on the presence or not of an ethylenic para substituent conjugated to the phenyl ring. K(ww) partition coefficients between the wood and the model wine were determined, which exhibited an unusual positive variation with temperature.     

Molecular basis of the interaction between proteins of plant origin and proanthocyanidins in a model wine system

Plant proteins are being used as a replacement for animal proteins in wine fining. The surface hydrophobicity of plant proteins in four commercial preparations differing for their origin and processing was assessed by using a fluorescent hydrophobic probe in wine-like media. Displacement of the probe by addition of wine phenolics was measured as a way to compare and predict to some extent the efficiency of these proteins in wine fining. It was found that the binding of polyphenols was much more specific than that of the hydrophobic probe. Further analysis of the polyphenol pattern in protein-treated wine-like solutions pointed out two relevant facts: (1) proteins may interfere with the chemistry of the interactions between polyphenols and other wine components; (2) individual protein preparation having different surface hydrophobicities also have different specificities in binding different polymeric forms of the polyphenols and in their substitution products. These findings are related to the possible carry-over of transition metals and may be worth exploring for custom tailoring the fining process. Whether the practical application of the latter finding will call for production and/or screening of plant-derived proteins with features appropriate to this task remains to be investigated. However, the approaches presented in this study may be used for large-scale screening of protein suitability for fining application under laboratory conditions, providing guidelines for their use in actual winemaking applications.     

Measurement of free zinc concentration in wine with AGNES

AGNES (absence of gradients and Nernstian equilibrium stripping), a voltammetric technique recently introduced to measure free metal concentration in solution and checked with different natural and synthetic aqueous media, has been applied here to determine free Zn concentration in wine. The content of ethanol in a solution increases its viscosity, and, so, the diffusion coefficient decreases. Another added effect in ethanolic solutions is the increase of the activity of the metal ions, due to the decrease of the permittivity in the alcoholic medium with respect to the aqueous one. With this taken into account, a specific methodology has been developed to apply AGNES in ethanolic media. A relevant point in this methodology has been the introduction of a new kind of blank, the EDTA blank, able to be applied in the same natural sample and with the same potential program. The free Zn concentrations of the two wines analyzed, a red and a white Raimat wine, were 4.5(2) x 10 (-7) and 7.2(4) x 10 (-7) M, respectively. These represent around 5% of the total Zn content. In the wine samples analyzed, it was checked that intermetallic formation of Zn-Cu does not affect the measurement of free Zn in a significant way.     

Degradation of free tryptophan in a cookie model system and its application in commercial samples

The stability of free tryptophan (Trp) was examined in five cookie-resembling models at varying baking temperatures and durations. Trp was measured by HPLC coupled with a fluorescent detector. Trp degradation was significantly greater in cookies formulated with glucose compared with sucrose, regardless of the temperatures and durations of baking. A lag period was clearly observed in cookies formulated with sucrose. The type of sugar used in the dough formulation affected not only the thermal destruction kinetics but also the degree of degradation of free Trp. However, the type of leavening agent (ammonium bicarbonate versus sodium bicarbonate) did not affect the rate of Trp destruction as happens in Maillard-driven reactions. In addition, the free Trp content was analyzed in nine different flours and sixty-two commercial cookies, and it was found that free Trp varied from 0.4 to 1287.9 mg/kg for rice and wheat bran, respectively. It was found that free Trp was significantly higher in dietetic commercial samples formulated with wheat bran compared with other flours.     

Aroma compound sorption by oak wood in a model wine

Oak wood used for wine barrels was immersed into a model wine containing eight aroma compounds (e.g., aromatic and terpene alcohols, ethyl esters, and aldehyde), for which activity coefficients in water and model wine were determined using the mutual solubility measurement. A mass balance of these volatiles considering their reactivity in model wine was established. For most of the studied aroma compounds, and mainly for linalool and ethyl octanoate, a sorption behavior into wood was reported for the first time. This phenomenon was selective and could not be related to the solubilities in model wine and hydrophobicities of the studied aroma compounds, suggesting that acid-base and polar characteristics of wood were more involved in this sorption mechanism. This study has also shown that the level of sorption is a function of the ratio of wood surface area/solution volume.     

Effect of a polyphenols-enriched chardonnay white wine in diabetic rats

A Chardonnay white wine enriched in polyphenols was obtained by modification of winemaking and characterized by its enrichment in total polyphenolic content (1346 mg/L as compared to 316 mg/L for traditional Chardonnay) and in various individual polyphenols (catechin, epicatechin, procyanidins dimers B1-B4, gallic acid, cafeic acid, and caftaric acid), as determined from HPLC coupled to a diode array detector. The polyphenols-enriched white wine (W) or its ethanol-free derivative (EFW) was then administered by gavage (10 mL/kg, twice a day) for 6 weeks to rats that have been rendered diabetic by a single iv injection of streptozotocin (55 mg/kg). Treatments had no effect on the symptoms associated with hyperglycemia. However, while a reduction in plasma antioxidant capacity was associated with the diabetic state, administration of W or EFW restored plasma antioxidant capacities to a level not significantly different from that of nondiabetic control animals. In addition, the effect of both treatments was manifested by the enlargement of mesenteric arteries, as determined by quantitative histomorphometry. In summary, our study indicates that white wine, when enriched in polyphenols, is able to induce ethanol-independent in vivo effects in a model of insulin-deficient diabetes characterized by a major oxidative stress.     

叶面追施硝态氮肥抑制菠萝营养生长效应

采用叶面淋施的盆栽试验方法,以我国菠萝主栽品种-巴厘为试材,研究不同形态氮素对盆栽菠萝营养生长和菠萝叶片黄化的影响,为菠萝氮肥合理施用提供参考。试验结果表明,叶面淋施硝态氮处理的菠萝根、茎叶生物量显著低于叶面淋施铵态氮、酰胺态氮,黄叶数显著高于叶面淋施铵态氮、酰胺态氮。与铵态氮相比,硝态氮处理的菠萝总叶数、根数目、根重、茎叶重分别减少18.7%、26.5%、49.7%、43.5%,黄叶数增加192.7%。叶面淋施硝态氮抑制菠萝营养生长主要机理是硝态氮提高了土壤p H值,减少了铁吸收,降低菠萝叶片中全铁、活性铁、叶绿素含量(与铵态氮相比,分别减少25.9%、66.9%、23.2%)。     

Effect of copper on the volatility of aroma compounds in a model mouth system

Copper is thought to influence aroma perception by affecting volatility of aroma compounds in the mouth through interaction with salivary components, especially proteins. Our objective was to identify the effect of copper on the volatility of aroma compounds and the role of copper-protein interaction in volatile chemistry in the mouth. Copper (2.5 mg/L) and four aroma compounds (hexanal, butyl acetate, 2-heptanone, and ethyl hexanoate, 0.5 microL/L each) were added to model systems containing water, electrolytes, and artificial saliva at different pH levels. Headspace concentration of each volatile was measured using SPME-GC analysis. Copper in the model systems increased headspace concentration of volatiles at pH 6.5, but no change in volatility was observed at pH 7.0. At pH 7.5, the presence of copper in the artificial saliva system containing mucin and alpha-amylase decreased headspace volatile concentration, whereas histatin did not cause any changes in volatility. Effect of copper on volatiles at pH 6.5 may be due to increased solubility of copper at lower pH. Salivary proteins seem to interact with copper at pH 7.5. The interaction may change configuration of binding sites for aroma compounds in mucin.     

Effect of hexanal and iron on color development in a glucose/phenylalanine model system

Aqueous glucose/phenylalanine (0.1 M with respect to each reactant) systems were heated in an autoclave for 30 min at 140 degrees C, in the presence of hexanal (0.04, 0.1, and 0.2 M) or FeCl(2) (0.01 M). Results show that hexanal significantly inhibited color development at pH 5 and 6 and led to an increase of 5-(hydroxymethyl)furfural. Iron addition had similar effects at pH 5, but only small effects at pH 6. The reaction routes proposed give key roles to the alpha-dicarbonyl compounds, to the Strecker aldehyde, and to the Schiff base formed by the reaction between hexanal and phenylalanine.     

Effect of beta-glycosidase activity of Oenococcus oeni on the glycosylated flavor precursors of Tannat wine during malolactic fermentation

Under traditional wine-making conditions, this work examines the beta-glycosidic activity of Oenococcus oeni on glycosylated aroma compounds of Tannat wines during malolactic fermentation (MLF) by comparing the changes on selected aglycones liberated. MLF diminished the content of all the glycosylated compounds. The level of the free aroma components was slightly modified by the action of the malolactic fermentation so that the cleavage of the glycosidic linkage by the beta-glycosidic activity of O. oeni did not appear to increase significatively the aglycone contents. The consequences of further chemical rearrangements of the algycones under wine conditions were explored using synthesized glycoconjugates on synthetic medium. Bacteria could also be responsible for the cleavage of aroma glycosylated compounds, being the aglycone adsorbed on polysaccharides or peptidoglycans and was released into the external medium. This hypothesis was studied through the evaluation of a stable arrangement of aroma compounds with polysaccharides produced by lactic acid bacteria. A possible retaining of free-made aroma compounds into the whole cells of O. oeni was also investigated through cell culture analysis. Through the results obtained, we assume stable linkage of aroma compounds with bacterial polysaccharides.     

开放式空气CO2浓度升高对水稻/小麦轮作土壤速效钾的影响

采用FACE（Free air carbon dioxide enrichment）技术,研究了不同施N水平下,大气CO2浓度升高对水稻/小麦轮作土壤速效钾的影响。结果表明,相对于对照处理,在不同氮水平下CO2浓度升高使作物生物量增加,导致作物生长季对土壤钾的吸收增加,但并没有降低作物主要生长期土壤（0―5、5―15 cm土层）速效钾的含量;CO2浓度升高使土壤速效钾增加的幅度在作物根际达6.3%~22.3%,在行间达3.7%~11.2%,且土壤速效钾增加的幅度在小麦季大于水稻季。表明根系对土壤速效钾的影响很大,因此,短期内土壤钾含量不会成为限制因素而影响作物对高CO2浓度的响应,反而会增加土壤钾的有效性。但在土壤肥力较低的土壤上可能会产生消极影响。     

Effect of citric acid and glycine addition on acrylamide and flavor in a potato model system

Acrylamide levels in cooked/processed food can be reduced by treatment with citric acid or glycine. In a potato model system cooked at 180 degrees C for 10-60 min, these treatments affected the volatile profiles. Strecker aldehydes and alkylpyrazines, key flavor compounds of cooked potato, were monitored. Citric acid limited the generation of volatiles, particularly the alkylpyrazines. Glycine increased the total volatile yield by promoting the formation of certain alkylpyrazines, namely, 2,3-dimethylpyrazine, trimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, tetramethylpyrazine, and 2,5-diethyl-3-methylpyrazine. However, the formation of other pyrazines and Strecker aldehydes was suppressed. It was proposed that the opposing effects of these treatments on total volatile yield may be used to best advantage by employing a combined treatment at lower concentrations, especially as both treatments were found to have an additive effect in reducing acrylamide. This would minimize the impact on flavor but still achieve the desired reduction in acrylamide levels.