Isolation and analysis of a polymeric thearubigin fraction from tea

A brown thearubigin fraction was isolated from a black tea liquor, using chromatography on a column of Solka-Floc cellulose. The paper chromatograms of the fraction resembled the chromatograms of thearubigin fractions produced in the past. The substance contained no protein or caffeine, and was free of flavonol glycosides. Reversed phase HPLC showed the fraction to be a mixture of polymers, the chromatograms showing a convex broad band. Infra-red spectroscopy showed the substance to be phenolic, and ¹³C NMR showed it was a flavanol polymer with various linkages to the proanthocyanidin polymers. The NMR spectra in this paper are the first NMR spectra of polymeric thearubigins. The brown fraction was designated the theafulvin fraction.     

Degradation kinetics of grape skin and seed proanthocyanidins in a model wine system

Catechin (monomer), purified grape skin proanthocyanidin (polymer), and purified grape seed proanthocyanidin underwent monitored accelerated oxidation under continuous oxygenation and UV light, at a constant 20 °C. Compounds were dissolved in model wine solutions with (and without) catechol. Solutions were examined and then contrasted by absorbance measurements, phloroglucinolysis, and subsequent HPLC analysis. Oxidation of these monomers and polymers revealed significant colour changes (measurable increase in colour density). The presence of catechol increased the half-life of catechin, but the opposite was observed for total skin and seed proanthocyanidins. Skin and seed proanthocyanidin degradation half-life decreased with the addition of catechol. In general, based on second order rate reactions, total subunits of seed proanthocyanidin solutions degraded faster than that of skin proanthocyanidin solutions. As expected, there were decreases of measurable phenolics in both monomer and polymer solutions. Under the study conditions, flavanol monomer and polymer oxidation was chiefly dependant upon initial solution concentration.     

Identification of the major flavonoids from pericarp tissues of lychee fruit in relation to their antioxidant activities

Large amount of polyphenolic compounds with strong antioxidant activity was present in the pericarp of harvested lychee fruits. Flavonoids were extracted with 85% ethanol:15% HCl from lychee fruit pericarp tissues. Most of the lychee flavonoids were partitioned into the ethyl acetate fraction. Three major components of the ethyl acetate fraction were obtained by reverse phase high-performance liquid chromatography and determined to be flavanol by their ultraviolet/visible spectra. Furthermore, these three components were identified as proanthocyanidin B4, proanthocyanidin B2 and epicatechin by nuclear magnetic resonance and mass spectrometry. The ethyl acetate fraction, proanthocyanidin B4, proanthocyanidin B2 and epicatechin exhibited a good antioxidant capability. The hydroxyl radical and superoxide anion scavenging activities of proanthocyanidin B2 was greater than those of proanthocyanidin B4 and epicatechin, while the epicatechin had the highest DPPH scavenging activity     

葡萄籽原花青素的纯化与结构研究

将大孔树脂HP-2MGL 和交联葡聚糖凝胶SephadexLH-20 用于葡萄籽原花青素的纯化。HP-2MGL 可除去蛋白、多糖、高聚物等杂质,获得原花青素含量为96.5% 的GSPP3。 SephadexLH-20 能够进一步纯化GSPP3,获得低聚原花青素GSPP3-SP1 和GSPP3-SP2。经HPLC 柱分离,GSPP3-SP1 有4 个峰,GSPP3-SP2 有7 个峰。GSPP3-SP1 和GSPP3-SP2 质谱分析发现,含有大量m/z287、m/z289、m/z290、m/z408、m/z426、m/z577、m/z578、m/z580 等分子离子及碎片质谱信息,证明是由儿茶素和表儿茶素构成的原花青素二聚体的同分异构体。激光光散射与GPC 联用测定原花青素粗提物GSP 的重均相对分子质量为4.15 × 104,多分散系数为1.65;GSPP3 的重均相对分子质量为4.26 × 103,平均聚合度为13,多分散系数为1.56,相对分子质量分布曲线呈对称分布,以高聚合度原花青素为主;葡萄籽原花青素纯化分级级分GSPP3-SP 的重均相对分子质量为1.89 × 103,平均聚合度为6,多分散系数为1.45,以低聚合度原花青素为主。     

葡萄籽低聚原花青素化学成分及抗氧化研究

对葡萄籽低聚原花青素中的原花青素B2进行了分离纯化,并将葡萄籽低聚原花青素(opc’s)、原花青素B2对比白藜芦醇、红酒提取物、苹果多酚、VC、VE和水溶性V(ETrolox)的抗氧化活性进行了研究。结果表明,原花青素B2和opc’s对DPPH自由基和羟自由基都具有较好的清除活性。对DPPH自由基的清除作用与VE相当,其IC50分别为1.20μg/mL和1.39μg/mL,明显强于VC和Trolox;原花青素B2和opc’s对羟自由基的抑制率明显高于VC、VE和Trolox。opc’s经过层析分离纯化后得到的原花青素B2清除DPPH自由基的效果有所提高,但对于羟自由基的清除活性提高不明显。     

采用高效液相色谱法分析荔枝酒中的酚类物质

选用乙酸乙酯作为荔枝酒酚类化合物的萃取溶剂,通过对HPLC洗脱条件的摸索,建立了HPLC同时分离荔枝酒中40多种组分的反相色谱条件。结合标准品保留时间比对和LC-MS-MS分析,准确定性了其中的(-)-表儿茶素、原花青素B2、没食子酸和芦丁等4种酚类物质,并在此基础上建立了这4种酚类化合物的HPLC定量测定方法,回收率均在85%以上。该技术为进一步研究荔枝酒的褐变问题提供了有效的分析手段。     

Degradation of anthocyanins from blood orange juices

Summary The degradation of anthocyanic pigments of fresh and industrial juices from blood oranges were studied during storage periods of a few days at −18 °C and 12 months at 4 °C in nitrogen. The change of anthocyanin pigments was followed by HPLC at 520 nm while the coloured polymeric pigments were fractionated by chromatography and characterized by their transformation indices and their reactivity with NaHSO₃. The results show that the process of polymerization is accompanied by production of chemical indicators of sugar and ascorbic acid degradation (furfural and hydroxymethylfurfural). It seems that the resulting polymeric compounds have structures different from those of the compounds formed during wine processing. Reactions between anthocyanic pigments and the intermediates of degradation of sugar and ascorbic acid in an acidic environment are the main causes of formation of these polymers.     

利用带光电二极管阵列检测的反相HPLC对ECG,EGCG和红茶中的氧化产物的分析研究

利用反相ＨＰＬＣ分析对ＥＣＧ和ＥＧＣＧ的氧化产物的色谱进行了比较。实验中有少量的ＥＧＣＧ在所设计的氧化体系中，由ＥＣＧ经所选择的化学氧化条件氧化形成。所以ＥＣＧ的氧化可能是先产生ＥＧＣＧ，随后再与ＥＧＣＧ共同被氧化或ＥＧＣＧ单独被氧化来继续其反应。用于分析洗脱红茶及多酚类的氧化产物的较为理想的ＨＰＬＣ洗脱梯度应为：试剂Ａ１％柠檬酸，自９２％至５０％，而相应的试剂Ｂ乙腈，则由８％在设计的区间内升至５０％。根据对高聚合氧化产物的重点分析，在这种洗脱梯度下仅用１５分钟便可以全部洗脱出叫做茶灰素的组分。相关研究表明多酚类的氧化反应即使在很低的温度下亦会自发地进行。     

Free amino acids and volatile compounds in vinegars obtained from different types of substrate

The present work reports on the free amino acids and volatile compounds in vinegars obtained from different types of raw materials (cider, white wine and red wine). Based on the amino acid contents of three types of vinegar and of the substrates from which they were obtained, the profile of the nitrogen‐supplying compounds and their uptake for the acetic acid bacteria were very similar. The most important amino acid in terms of supply and uptake was found to be L ‐leucine. L ‐Proline proved also important in the wine vinegars, however it was not depleted as L ‐leucine. The different acetification reactions involved were found to yield acetoin, and plus 1,1‐diethoxyethane in the wine vinegars. Copyright © 2004 Society of Chemical Industry     

Mass spectrometric evidence for the formation of pigmented polymers in red wine

The polymeric materials (wine tannin) were isolated from a three-year-old Pinot Noir using Toyopearl TSK HW 40-F size exclusion chromatography. The wine tannin was analysed by reversed-phase and gel permeation high performance liquid chromatography, UV-Vis spectrophotometry and mass spectrometry. Analysis by gel permeation chromatography indicated that the isolate was in large part polymeric, and also indicated the presence of pigmented material monitored at a wavelength of 520 nm. Acid-catalysis in the presence of phloroglucinol indicated that the wine tannin contained 45% w/w known proanthocyanidin material. The wine tannins gave a complicated mass spectrum featuring a large number of intense signals derived from proanthocyanidins and, to a much lesser extent, a series of ions corresponding to masses consistent with direct condensation products of an anthocyanin with proanthocyanidins. The degree of polymerisation by mass spectrometry was detected to be as high as an octamer composed of an anthocyanin combined with a proanthocyanidin heptamer. Based upon current evidence, the anthocyanin was proposed to be bound from the C-6/8 or C-4 positions of the anthocyanin to the respective electrophilic or nucleophilic centre of the proanthocyanidins. Only the species with the C-6/8 position of the anthocyanin linked with the proanthocyanidins seemed to be pigmented. Under acid-catalysed conditions, an increase in malvidin-3-glucoside was observed, suggesting that the non-pigmented form is possibly converted to the pigmented flavylium form. This is the first mass spectrometric evidence confirming the existence of pigmented polymers with an extension to octamers in wine.     

炭黑氨基柱净化HPLC-ICP-MS测定黄酒中砷形态及含量

建立了一种同时测定黄酒中亚砷酸根、砷酸根、砷甜菜碱、一甲基砷酸和二甲基砷酸等5种砷形态的高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)分析方法。黄酒经炭黑氨基柱净化,体积分数为10%甲醇水溶液洗脱,采用Hamilton PRP-XIO0阴离子交换色谱柱(4.1 mm×250 mm,10 μm),60 mmol/L硝酸铵梯度洗脱,高效液相色谱分离,电感耦合等离子体质谱进行定性和定量分析。在0.5～20.0 μg/L范围内各砷形态线性良好,相关系数R均＞0.999;加标回收率85%～92%,相对标准偏差(RSD)1.4%～5.2%;5种砷形态的定量限均为0.10 μg/L,检出限均为0.03 μg/L。利用该方法对不同年份、不同企业及不同类型的黄酒进行了检测,该方法适用于黄酒中砷残留的分析检测。     

A putative glucan synthase gene dps detected in exopolysaccharide-producing Pediococcus damnosus and Oenococcus oeni strains isolated from wine and cider

Some lactic acid bacteria can induce viscosity in wine, beer and cider by production of exopolysaccharides (EPS). A polymerase chain reaction (PCR) assay was previously described for the detection of ropy Pediococcus damnosus strains in wine [J. Appl. Microbiol. 90 (2001) 535]. The primers used in that study, PF5 and PF6, are investigated in addition to new primers which broaden the range of spoiling agents detectable by PCR. Primers PF1 and PF8 allow the amplification of DNA from ropy P. damnosus strains isolated from wine, as was observed with PF5 and PF6. In addition, PF1 and PF8, unlike PF5 and PF6, are able to generate an amplicon using template DNA from a ropy P. damnosus strain isolated from cider and a ropy Oenococcus oeni strain isolated from champagne. Two different ropy Lactobacillus species were also isolated, but their DNA was not amplified using primers PF1 and PF8. The new primers PF1 and PF8 were chosen from the sequence of gene dps, a putative glucan synthase gene, found across all the ropy P. damnosus strains isolated, from both wine or cider, and also in a ropy O. oeni strain. To our knowledge, this is the first time that an EPS-producing O. oeni strain is described. Glucan biosynthesis was assessed by agglutination tests done with Streptococcus pneumoniae type 37-specific antibodies, which specifically detect glucan-producing cells. The results show that there is a direct correlation between glucan production and detection of gene dps. Therefore, Dps is considered a candidate for the glucan synthase enzyme responsible for EPS production by ropy strains of P. damnosus and O. oeni.     

Influence of winemaking techniques on proanthocyanidin extraction in Monastrell wines from four different areas

The effect of two low pre-fermentative temperature techniques (cold soak and must freezing with dry ice) using two macerating enzymes was studied during the vinification of Monastrell wines from four different areas (Cañada del Judío, Cañada de Albatana, Montealegre and Chaparral) belonging to two Denomination of Origins (Jumilla and Bullas), to assess their influence on wine proanthocyanidin concentration and composition. When the control wines were compared, Chaparral wine showed the lowest proanthocyanidin content, together with the lowest mDP and the highest degree of galloylation in its proanthocyanidins, suggesting that the most of the proanthocyanidins in wine had been extracted from the grape seeds. However, Cañada de Albatana wines showed the highest proanthocyanidin content. Different results were found in the four areas when the oenological treatments were applied to the grapes. In Cañada de Albatana wines, no treatment had any effect, while in the case of Montealegre wines, the cold treatments led to a higher proanthocyanidin content compared with the control wines. In Cañada del Judío and Chaparral wines, all the treatments increased the proanthocyanidin content, except when galactoside was used in Chaparral wines, when the results were even lower than for the control wines. Discriminant analyses were applied to check whether the different areas influenced the wine proanthocyanidin profile. The results showed a clear separation between the four plots, suggesting that the proanthocyanidin wine composition was related to the zone where grapes were cultivated.     

沙棘籽原花色素组成和结构的研究

采用高效液相色谱-电喷雾串联质谱技术分析鉴定沙棘籽原花色素单体和寡聚体组分,采用Sephadex LH-20柱色谱按聚合度大小对沙棘籽多聚体组分进行分级,并通过苄硫醇酸降解反应(简称硫解)对各分级组分的组成结构特征进行分析.共检测到4种单体和8种二聚体:4种单体分别为儿茶素、表儿茶素、棓儿茶素和表棓儿茶素;二聚体包括3种原花青素二聚体、1种原飞燕草素二聚体和4种混合二聚体.并根据ESI提供的质谱信息,分析确定了混合二聚体组成单元的连接顺序.棓儿茶素是构成多聚体各分级组分末端单元的主要组成成分,棓儿茶素和表棓儿茶素(合并计算)是构成各分级组分延伸单元的主要组成成分;平均聚合度为9.1、13.2和17.0,原飞燕草素比例为69.2%、84.6%、87.4%的三个分级组分是多聚体的主要成分,占多聚体总量的74.7%.     

Mass spectrometry fragmentation pattern of coloured flavanol‐anthocyanin and anthocyanin‐flavanol derivatives in aged red wines of Rioja

Background and Aims: During wine ageing, a great variety of reactions take place, resulting in an immense variety of products whose structure sometimes remains unknown. The aim of this work is the study of different fragmentation patterns of flavanol‐anthocyanin derivatives formed along the wine ageing; these patterns are useful for elucidating the different structures of these compounds and other new related ones. Methods and Results: Several wines from the Protected Denomination of Origin Rioja have been studied by an analytical method that combines column chromatography and high‐performance liquid chromatography with diode array and mass and tandem mass spectrometric detections. Thirty‐five coloured flavanol‐anthocyanin compounds formed by direct reaction or by acetaldehyde‐mediated condensation have been identified. For direct reaction derivatives, two different fragmentation patterns (one of them not previously reported) have been observed depending on the position of flavanol in the coloured derivative. Several compounds have been identified in aged wines for the first time to the authors' knowledge, like (+)‐gallocatechin‐cyanidin‐3‐glucoside and (+)‐catechin‐cyanidin‐3‐glucoside Conclusions: The developed analytical procedure has allowed the identification of some compounds for the first time, and two different fragmentation patterns have been observed depending on the position of flavanol in the pigment. Significance of the Study: The establishment of different fragmentation patterns allows the structural elucidation of unknown compounds.     

高聚原花青素降解技术研究进展

原花青素是一类广泛存在于自然界中的黄烷-3-醇类化合物,由于大多数从自然界获得的原花青素为带有苯环或长碳链的高聚原花青素,导致其生理活性较弱.通过降解,可以增强原花青素的生理活性以及应用范围.因此,高聚原花青素降解技术成为国内外研究的热点.本文详细整理了国内外有关原花青素高聚体降解技术的文献报道,对化学、生物以及物理降...     

Reaction Between Malvidin 3-Glucoside and (+)-Catechin in Model Solutions Containing Different Aldehydes

ABSTRACT: The contribution of the aldehyde composition of wine spirit to the color changes in Port red wine was studied in model solutions. Malvidin 3-glucoside was shown to be very reactive towards catechin in the presence of different aldehydes: acetaldehyde, isovaleraldehyde, benzaldehyde, propionaldehyde, isobutyraldehyde, formaldehyde, and 2-methylbutyraldehyde. LC/MS data confirmed the formation of oligomeric pigments resulting from the reaction between the anthocyanin and the flavanol (colored products) and between 2 flavanol units (colorless products) mediated by each aldehyde assayed. The UV-visible spectra of the colored pigments showed a λ_max bathochromically shifted relatively to the λ_max of original anthocyanins. All samples revealed a "blueing" and "darkening" color effects using the CIELAB system.     

Wine tartrate stabilization by different levels of cation exchange resin treatments: impact on chemical composition,phenolic profile and organoleptic properties of red wines

The aim of this work was to evaluate the impact of cation exchange resin, on tartrate stabilization, chemical composition, general phenolic composition, individual anthocyanin and different proanthocyanidin fraction content of a red wine made from a mixture of three Portuguese red grape varieties. Additionally, the influence of this tartrate stabilization process on organolpetic properties was another aim of this work. The cation exchanged red wines were mixed in different proportions with the untreated red wine. The results showed that the application of a cation exchange resin used was useful to adjust the pH and total acidity and revealed a good capacity to retain potassium and calcium. In addition, the cation exchange resin treatment caused a significant decrease in the contents of individual anthocyanins well as in the different fractions of proanthocyanidins (especially in monomeric fraction). Finally, considering the organoleptic properties, the most highly evaluated wine, especially considering taste parameters, was the control wine. These results confirm that cation exchange is an interesting in red winemaking to improve tartaric stability, but at same time it is important analyze potential effects on phenolic content and organoleptic properties of red wines.     

原汁苹果醋酸发酵饮料的研究

试验以浓缩苹果汁为原料,分别采用酵母1450、醋酸菌株AM2进行酒精发酵及醋酸发酵,对醋酸发酵进行不同酒精度及不同糖度影响实验。结果认为,较高的酒精度和糖度对醋酸发酵有抑制作用,在酒精度3%,糖度7.5%的条件下进行醋酸发酵,可制得风味理想的原汁苹果醋酸发酵饮料。     

Molecular cloning, heterologous expression, and characterization of Ornithine decarboxylase from Oenococcus oeni

Ornithine decarboxylase (ODC) is responsible for the production of putrescine, the major biogenic amine found in wine. Oenococcus oeni is the most important lactic acid bacterium in the winemaking process and is involved in malolactic fermentation. We report here the characterization of ODC from an O. oeni strain isolated from wine. Screening of 263 strains isolated from wine and cider from all over the world revealed that the presence of the odc gene appears to be strain specific in O. oeni. After cloning, heterologous expression in Escherichia coli, and characterization, the enzyme was found to have a molecular mass of 85 kDa and a pI of 6.2 and revealed maximal activity at pH 5.5 and an optimum temperature of 35°C. Kinetic studies showed that O. oeni ODC is specific for L-ornithine with a K(m) value of 1 mM and a V(max) of 0.57 U·mg(-1). The hypothesis that cadaverine, which results from lysine decarboxylation, may be linked to putrescine production is not valid since O. oeni ODC cannot decarboxylate L-lysine. As no lysine decarboxylase was detected in any of the O. oeni genomes sequenced, cadaverine synthesis may result from another metabolic pathway. This work is the first characterization of an ODC from a lactic acid bacterium isolated from a fermented product.