Partial purification of polyphenol oxidase from Chinese cabbage Brassica rapa L

Polyphenol oxidase (PPO) was purified and characterized from Chinese cabbage by ammonium sulfate precipitation and DEAE-Toyopearl 650M column chromatography. Substrate staining of the crude protein extract showed the presence of three isozymic forms of this enzyme. The molecular weight of the purified enzyme was estimated to be approximately 65 kDa by gel filtration on Toyopearl HW-55F. On SDS-PAGE analysis, this enzyme was composed of a subunit molecular weight of 65 kDa. The optimum pH was 5.0, and this enzyme was stable at pH 6.0 but was unstable below pH 4.0 or above pH 7.0. The optimum temperature was 40 degrees C. Heat inactivation studies showed temperatures >40 degrees C resulted in loss of enzyme activity. PPO showed activity to catechol, pyrogallol, and dopamine (K(m) and V(max) values were 682.5 mM and 67.6 OD/min for catechol, 15.4 mM and 14.1 OD/min for pyrogallol, and 62.0 mM and 14.9 OD/min for dopamine, respectively). The most effective inhibitor was 2-mercaptoethanol, followed in decreasing order by ascorbic acid, glutathione, and L-cysteine. The enzyme activity of the preparation was maintained for 2 days at 4 degrees C but showed a sudden decreased after 3 days.     

Determination of total N-nitroso compounds and their precursors in frankfurters, fresh meat, dried salted fish, sauces, tobacco, and tobacco smoke particulates.

Total N-nitroso compounds (NOC) and NOC precursors (NOCP) were determined in extracts of food and tobacco products. Following Walters' method, NOC were decomposed to NO with refluxing HBr/HCl/HOAc/EtOAc and NO was measured by chemiluminescence. NOC were determined after sulfamic acid treatment to destroy nitrite, and NOCP were determined after treatment with 110 mM nitrite and then sulfamic acid. Analysis without HBr gave results < or =20% of those with HBr. This NOC method was efficient for nitrosamines but not nitrosoureas. The standard nitrosation for determining NOCP gave high yields for readily nitrosated amines, including 1-deoxy-1-fructosylvaline, but not for simple amines, dipeptides, and alkylureas. Mean NOC and NOCP results were (respectively, in micromol/kg of product) 5.5 and 2700 for frankfurters, 0.5 and 660 for fresh meat, 5.8 and 5800 for salted, dried fish, and 660 and 2900 for chewing tobacco (all for aqueous extracts) and 220 and 20000 nmol/cigarette for MeCN extracts of cigarette smoke filter pads.     

Partial purification and characterization of pectin methylesterase from acerola (Malpighia glabra L.)

The enzyme pectin methylesterase (PME) is present in acerola fruit and was partially purified by gel filtration on Sephadex G-100. The results of gel filtration showed different PME isoforms. The total PME (precipitated by 70% salt saturation) and one of these isoforms (fraction from Sephadex G-100 elution) that showed a molecular mass of 15.5 +/- 1.0 kDa were studied. The optimum pH values of both forms were 9.0. The total and the partially purified PME showed that PME specific activity increases with temperature. The total acerola PME retained 13.5% of its specific activity after 90 min of incubation at 98 degrees C. The partially purified acerola (PME isoform) showed 125.5% of its specific activity after 90 min of incubation at 98 degrees C. The K(m) values of the total PME and the partially purified PME isoform were 0.081 and 0.12 mg/mL, respectively. The V(max) values of the total PME and the partially purified PME were 2.92 and 6.21 micromol/min/mL/mg of protein, respectively.     

Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.     

Extraction and purification of isoflavones from soybeans and characterization of their estrogenic activities

Soybean isoflavones have multiple beneficial health effects especially on estrogen-deficient diseases such as menopausal symptoms. In this study, isoflavones were produced from soybean flour, and the extraction and purification parameters were optimized to give a high yield of total isoflavones, about 0.62 mg of aglycones/g of soybean flour, which is >2 times the initial yield. HPLC analysis and MTT cell proliferation assay using MCF-7 cells revealed that the product thus obtained not only contained a high content of isoflavone aglycones but also had estrogenic activity. MTT data also revealed that both genistein and daidzein exhibited estrogenic effects at lower concentrations and antiproliferative effects at higher concentrations, and 1 microM genistein and 10 microM daidzein exerted significant estrogenic activities, which were not more than that of the endogenous level of 17beta-estradiol (E2). The production method developed can be used as a guideline for manufacturing soy isoflavones, and the MTT assay was demonstrated to be suitable for quality control on isoflavone products. The results on the estrogenic properties of isoflavones can be used as reference data for their effective and safe usages in estrogenic therapy.     

Partial purification and immobilization/stabilization on highly activated glyoxyl-agarose supports of different proteases from flavourzyme

The fractioning of some components and their immobilization of Flavourzyme, a commercial protease/aminopeptidase preparation, has been investigated to improve its specificity and stability. Adsorption of Flavourzyme on two ionic exchangers yielded two fractions with endoprotease activity and one fraction containing aminopeptidase activity. The use of an amine agarose gel has made it possible to purify a 43 kDa protein with only endoprotease activity. Immobilization of this endoprotease and the original Flavourzyme preparation onto glyoxyl-agarose provided derivatives that were more thermostable than their soluble counterparts. Tests using immobilized Flavourzyme and immobilized purified endoprotease for the hydrolysis of chickpea proteins showed that both preparations can be used for the production of protein hydrolysates and compare very favorably with the original crude Flavourzyme in terms of reducing the production of free amino acids. This was especially so in the case of immobilized endoprotease, which produced only 0.2% free amino acids. Keeping free amino acids content low is very important in protein hydrolysates for nutritional use to avoid excessive osmotic pressure.     

Partial purification and kinetic characterization of a carotenoid cleavage enzyme from quince fruit (Cydonia oblonga)

For the first time, a cytosolic carotenoid cleavage enzyme isolated from quince (Cydonia oblonga) fruit is described. The enzyme was partially purified by using centrifugation, acetone precipitation, ultrafiltration (300 kD, 50 kD), isoelectric focusing (pH 3-10), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (7.5%). In this way, an enzymatically active protein fraction was obtained that contained three similar proteins, all exhibiting molecular weights in the range of 20 kD. Using beta-carotene as substrate, the enzyme activity was detected spectrophotometrically at a wavelength of 505 nm. The time constant of the reaction was 8.2 min, the Michaelis constant (K(m)) was 11.0 micromol x L(-1), and the maximum velocity (v(max)) was 0.083 micromol x L(-1) x min(-1) x mg(protein)(-1). The optimum temperature was above 50 degrees C.     

银杏花粉黄酮组分分离纯化及其清除DPPH自由基能力

为了分离纯化银杏花粉中具有潜在抗氧化活性的黄酮组分及研究其潜在抗氧化活性,该文对银杏花粉的总黄酮进行提取和分步萃取,采用1,1-diphenyl-2-picrylhydrazyl(DPPH)自由基清除率检测和组分分析锁定待纯化萃取相。通过DPPH-HPLC-PAD技术筛选目标清除自由基组分,并采用制备型液相色谱技术分离纯化这些组分,然后采用紫外可见吸收光谱、质谱、核磁共振氢谱、碳谱等技术进行结构鉴定,最后通过测定DPPH自由基清除率初步研究其潜在抗氧化活性。结果显示,银杏花粉粗提物具有清除DPPH自由基活性,对DPPH的半抑制率(IC50值)为1.32 mg/m L,乙酸乙酯相和正丁醇相的IC50值为0.46、0.84 mg/m L,分别是粗提物的34.85%和63.64%。可见,乙酸乙酯相的清除DPPH活性最高。进一步研究显示,乙酸乙酯相富集了6种主要银杏花粉黄酮,而且均具有清除DPPH活性。经纯化和结构鉴定分别为山奈酚-3,4’-双-O-β-D-葡萄糖苷(1)、山奈酚-3-O-β-D-葡萄糖基-7-O-α-L-鼠李糖苷(2)、山奈酚-3-O-β-D-葡萄糖苷(3)、山奈酚-3-O-α-L-鼠李糖苷(4)、柚皮素(5)和山奈酚(6)。其清除DPPH自由基的活性由高到低依次为:化合物6化合物2化合物3化合物4化合物1化合物5。其中山奈酚清除DPPH的IC50值为0.017 mg/m L,与维生素C及芦丁相比没有显著性差异(P0.05)。上述研究为银杏花粉功能成分的深入研究奠定了基础。     

Partial purification,characterization, and thermal and high-pressure inactivation of pectin methylesterase from carrots (Daucus carrota L.)

Pectin methylesterase (PME) from carrots (Daucus carrota L.) was extracted and purified by affinity chromatography on a CNBr-Sepharose 4B-PME inhibitor column. A single protein and PME activity peak was obtained. A biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters of carrot PME was performed. In a second step, the thermal and high-pressure stability of the enzyme was studied. Isothermal and combined isothermal-isobaric inactivation of purified carrot PME could be described by a fractional-conversion model.     

Isolation and purification of lutein from the microalga Chlorella vulgaris by extraction after saponification

A simple and efficient method for the isolation and purification of lutein from the microalga Chlorella vulgaris was developed. Crude lutein was obtained by extraction with dichloromethane from the microalga after saponification. Partition values of lutein in the two-phase system of ethanol-water-dichloromethane at different ratios were measured by HPLC so as to assist the determination of an appropriate condition for washing water-soluble impurities in the crude lutein. Partition values of lutein in another two-phase system of ethanol-water-hexane at different ratios were also measured by HPLC for determining the condition for removing fat-soluble impurities. The water-soluble impurities in the crude lutein were removed by washing with 30% aqueous ethanol, and the fat-soluble impurities were removed by extraction with hexane. The final purity of lutein obtained was 90-98%, and the yield was 85-91%.     

Multielement composition of wines and their precursors including provenance soil and their potentialities as fingerprints of wine origin

The influence of the provenance soil and vinification process on the wine multielemental composition was investigated. For this purpose, two different vineyards from the Douro wine district, Portugal, were selected. Monovarietal grapes from a 10 year old vineyard were used to produce a red table wine, in a very modern winery. Polyvarietal grapes from a 60-70 year old vineyard were used to produce a red fortified wine, similar to Port, through a traditional vinification process. The multielement compositions (Al, As, B, Ba, Be, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ga, Hf, Li, Mn, Mo, Nb, Ni, Pb, Rb, Sb, Sc, Sr, Ti, Th, Tl, U, V, W, Y, Zn, Zr, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu) of soil, grape juices (prepared in the laboratory), and samples collected in the different steps of each winemaking process were measured. Inductively coupled plasma mass spectrometry was used, after suitable pretreatment of the samples (by UV irradiation for liquid samples and high-pressure microwave digestion for soil). Both vinification processes influenced the multielement composition of the wines. Most of the elements presented similar or even lower concentrations in the wine as compared to that observed in the respective grape juice, probably as a result of precipitation or coprecipitation with suspended particles during fermentation and/or wine aging. Evidence of effective contamination during grape pressing, fermentation, and/or fining of wines (depending on the element) was observed for Cd, Cr, Cu, Fe, Ni, Pb, V, and Zn in the fortified wine and Al, Cr, Fe, Ni, Pb, and V in the table wine. Nevertheless, significant correlations were obtained between the multielement composition of the wine and the respective grape juice (R = 0.997 and 0.979 for the fortified and table wines, respectively, n = 31, P < 0.01), as well as between that in the wine (median of the two studied wines) and the provenance soil (R = 0.994, n = 19, P < 0.01), for the set of elements determined in common in the different types of samples. These results are promising concerning the usefulness of the elemental patterns of both soil and wine as fingerprints of the origin of the studied wines. Nevertheless, more wines from the same and other wine districts must be studied in order to consolidate this conclusion. The multielement compositions of the studied wines were compared with those of wines of different characteristics and origins, as well as with the respective legal threshold limit values, when available. Relatively low metal levels, below their threshold limit values, were found in all cases.     

Rapid purification of fumonisins and their hydrolysis products with solid-phase extraction columns

Fumonisins B(3) and B(4) (FB(3) and FB(4)) were recovered from the 50:50 acetonitrile/water extract of corn cultures of a strain of Fusarium moniliforme that does not make FB(1) or FB(2) by stirring the extract with IRA-68, a weak anion-exchange resin. The fumonisins were desorbed with 5% acetic acid in the same solvent. After dilution with water, the desorbed fumonisins were separated into FB(3) (FB(3) and FA(3)) and FB(4) (FB(4), FC(4), and FA(4)) fractions with a tC(18) solid-phase extraction (SPE) cartridge. The FB(3) fraction was then separated into FB(3) and FA(3) by using an NH(2) SPE cartridge and eluting with 5% acetic acid and increasing amounts of acetonitrile in water. Finally, FB(1) and FA(3) were hydrolyzed with calcium hydroxide. After recovery from the reaction mixture using a tC(18) cartridge, the hydrolyzed and partially hydrolyzed analogues were separated and the unreacted fumonisins recovered by using an NH(2) cartridge, initially in the normal-phase mode with increasing amounts of water in acetonitrile and then in the reversed-phase mode after the addition of 5% acetic acid to the solvent and eluting in the reverse order.     

Partial purification of latent polyphenol oxidase from peach (Prunus persica L. Cv. Catherina). Molecular properties and kinetic characterization of soluble and membrane-bound forms

This paper analyzes the kinetic and structural characteristics of polyphenol oxidase (PPO) from peach cv. Catherina. The PPO was obtained in a latent state in both the soluble and membrane-bound forms, and both forms were activated by acid shock and the detergent SDS. Plant defense is the main function assigned to PPO, which would be activated by the acid environment resulting from tissue damage. On the other hand, it has been suggested that, physiologically, the role played by SDS may be fulfilled by lipids. Native isoelectric focusing identified two acid isoforms of pI 5.7 and 5.8 for the soluble form and one isoform with pI 5.7 for the membrane-bound form. A partially denaturing SDS-PAGE revealed two very close bands of activity in both cases, but the Western blot performed on a totally denaturing SDS-PAGE, using polyclonal antibodies against bean PPO, revealed a single band in the membrane-bound fraction with a molecular mass of 60 kDa.     

Characterization and purification of glycosaminoglycans from crude biological samples

Chondroitin sulfate (CS) is a glycosaminoglycan derived from cartilage and commonly used to treat osteoarthritis, psoriasis, and other conditions. The dimethylmethylene blue (DMMB) assay has been used often to measure glycosaminoglycan levels in relatively pure samples. In this study, we verified the accuracy of the DMMB assay in measuring CS levels in unpurified extract from bovine trachea and shark cartilage, despite potential interference from salts, proteins, and DNA. We found that the glycosaminoglycan signal obtained was due to CS and not to other glycosaminoglycan species. This was confirmed using fluorophore-assisted carbohydrate electrophoresis, which also revealed that the majority of the CS was monosulfated at the C4 or C6 position. Finally, we used anion-exchange chromatography to purify the bovine extract and obtained complete recovery of the glycosaminoglycans, with no contaminating protein. The results of this study should be very useful for future purification and analysis of this common supplement.     

Extraction and purification of squalene from amaranthus grain.

Grain amaranth has been suggested as an alternative to marine animals as a natural source of squalene. Oil contents, squalene contents, and fatty acid profiles were determined in 11 genotypes of four grain amaranth species. Although the oil contents of grain amaranth were low (from 5.1% in Amaranthus tricolor to 7.7% in Amaranthus cruentus) as compared to other oil-containing grains, high concentrations of squalene were found in total lipids, ranging from 3.6% in Amaranthus hypochondriacus to 6.1% in A. tricolor. The major fatty acids in Amaranthus oil consisted of palmitic acid (19.1-23.4%), oleic acid (18.7-38.9%), and linoleic acid (36.7-55.9%). A high degree of unsaturation was observed in Amaranthus oils, with S/U ratios of 0.26-0.32. A method to isolate and purify the squalene from Amaranthus oil was developed. After the saponification of K112, the squalene content increased from 4.2% in the crude oil to 43.3% in the unsaponifiables by the removal of the saponifiables. The unsaponifiables were fractionated by silica gel column chromatography to get highly purified squalene. The squalene purity in certain fractions was as high as 98%. Combining the fractions rich in squalene gave a 94% squalene concentrate, with a yield of 90%. The structure of squalene in the purified sample was confirmed by comparison of its ultraviolet spectrum with a standard and from its nuclear magnetic resonance spectra.     

牛骨蛋白与多糖的逐级提取及分离方法研究

为确定牛骨中蛋白质和多糖的提取及分离方法，该文在分析骨胶原蛋白和多糖存在状态的基础上，分别利用不同溶剂探讨了牛骨中蛋白质与多糖的逐级提取方法。首先碱性蛋白、酸性蛋白分别被0.6 mol/L KCl和0.5 mol/L K-P(K₂HPO₄/KH₂PO₄)溶液提取出来；0.1 mol/L HCl脱钙时溶解出酸性、中性及碱性蛋白，再经过NaOH中和后，酸性蛋白随羟基磷灰石沉淀、中性及碱性蛋白存留在上清液中；其他在胶原纤维上的有机物被0.5 mol/L NaCl和1% Na₂CO₃分别提取出来。各级提取有机物用SDS-PAGE电泳以及Stains All染色法、阿里新兰染色和CBB染色进行检测。利用DEAE-葡聚糖凝胶离子交换树脂有效地将碱性蛋白、酸性蛋白和多糖分离精制。     

Simple method of isolation and purification of alpha-galactosides from legumes

A simple method for the isolation and purification of alpha-galactosides, raffinose family oligosaccharides (RFOs), from legumes has been developed. The method includes (i) imbibition of seeds, (ii) extraction with 50% ethanol, (iii) precipitation of RFOs, (iv) purification of RFOs on diatomaceous earth and charcoal, and (v) cation-exchange chromatography. The described method allows one to obtain high purity RFO preparations (90% for lentil and 80% for pea seeds, determined by HPLC-RI analysis) in the form of white, fine powder. Yields of alpha-galactosides isolated from 100 g of seeds of lentil and pea were 5.6 and 4.3 g, respectively.     

Chemometric characterization of fruit juices from Spanish cultivars according to their phenolic compound contents: I. Citrus fruits

The data set composed by phenolic compound profiles of 83 Citrus juices (determined by HPLC-DAD-MS/MS) was evaluated by chemometrics to differentiate them according to Citrus species (sweet orange, tangerine, lemon, and grapefruit). Cluster analysis (CA) and principal component analysis (PCA) showed natural sample grouping among Citrus species and even the Citrus subclass. Most of the information contained in the full data set can be captured if only 15 phenolic compounds (concentration ≥10 mg/L), which can be quantified with fast and accurate methods in real samples, are introduced in the models; a good classification which allows the confirmation of the authenticity of juices is achieved by linear discriminant analysis. Using this reduced data set, fast and routine methods have been developed for predicting the percentage of grapefruit in adulterated sweet orange juices using principal component regression (PCR) and partial least-squares regression (PLS). The PLS model has provided suitable estimation errors.     

Purification and Partial Characterization of an Endoxylanase Inhibitor from Barley

Hordeum vulgare L. xylanase inhibitor (HVXI), an endoxylanase inhibitor with a protein structure, was purified to homogeneity from barley (Hordeum vulgare L.). HVXI is a nonglycosylated monomeric protein, with a molecular weight of ≈40,000 and a pI ≥ 9.3. Although it inhibits different endoxylanases to a varying degree, the activities of an α‐L‐arabinofuranosidase and a β‐d ‐xylosidase were not inhibited. Apparently, HVXI occurs in two molecular forms. These characteristics and the N‐terminal sequences of the composing polypeptides show that HVXI is homologous with Triticum aestivum L. xylanase inhibitor I, an endoxylanase inhibitor from wheat flour.