Liquid chromatography-electrospray tandem mass spectrometry of cis-resveratrol and trans-resveratrol: development, validation, and application of the method to red wine, grape, and winemaking byproducts

The application of liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) was investigated for the analysis of trans-resveratrol in red wine, grape skin, grape pomace, and winemaking byproducts. Chromatographic elution performed under isocratic reversed-phase conditions using a C18 narrow-bore LC column allowed retention times lower than 12 min to be obtained. Qualitative analysis was performed in negative-ion (NI) full-scan and product-ion scan acquisition modes, whereas method validation in terms of linearity, detection limits, accuracy, and robustness was carried out under NI selected reaction monitoring conditions. The matrix-matched detection limit was calculated in the low parts per billion range (10 microg/L). Results of the application of the method to red wine, grape, and winemaking byproduct samples were compared with those obtained using an LC-UV/DAD technique. Determination of trans-resveratrol in the samples investigated showed that the highest concentration was found in red wine, whereas wine made from grape pomace contained the lowest content.     

Study of wine tannin oligomers by on-line liquid chromatography electrospray ionization mass spectrometry.

Thiolysis of a wine tannin fraction yielded trihydroxylated flavanol units (as previously observed in grape skins) in addition to the well-known procyanidins (dihydroxylated units), usually described in the literature for grape condensed tannins. To determine how they occur in condensed tannins, the wine fraction was analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry. Thus, various series of ion peaks containing a variable number of trihydroxylated units were detected as monocharged ions from dimers up to pentamers. From pentamers, oligomers were found as doubly charged ions. Heptamer species corresponded to the highest mass detected. These results showed that wine condensed tannins consist of, besides procyanidins, mixed tri- and dihydroxylated flavanol units and also of pure trihydroxylated flavanol units. These new data should be taken into account to interpret organoleptic properties of wines.     

The wide diversity of structurally similar wine proteins

In the present work, single grape variety wines, Moscatel and Arinto, were used. Analysis by denaturing polyacrylamide gel electrophoresis of the wine proteins revealed the presence of only a few polypeptides ranging in molecular mass from 15 to 30 kDa. However, a more detailed examination of the whole protein fraction, by a combination of techniques, showed that these wines contain a very large number (many tens and, possibly, many more) of distinct polypeptides, exhibiting similar molecular masses but different electrical charges. The results obtained using highly specific antibodies and N-terminal sequencing indicate that there is structural similarity among most of the wine polypeptides. These observations can be explained by the existence of a common precursor to most or all of the wine proteins, which could generate all of the detected polypeptides by limited proteolysis. Comparison of the N-terminal sequences of the polypeptides isolated from Moscatel wine with proteins from other sources revealed a high degree of homology to pathogenesis-related proteins.     

(E)-1-(2,3,6-trimethylphenyl)buta-1,3-diene: a potent grape-derived odorant in wine

(E)-1-(2,3,6-Trimethylphenyl)buta-1,3-diene (TPB) was identified as a potent odorant in acid hydrolysates of crude glycoconjugate fractions isolated from grapes and grape vine leaves. TPB was also identified in a Semillon wine, using gas chromatography/mass spectrometry, by co-injection with an authentic sample. TPB had an aroma detection threshold of 40 ng/L in a neutral white wine and the concentration of TPB in four out of five white wines analyzed ranged from 50 to 210 ng/L.     

Isolation and structures of oligomeric wine pigments by bisulfite-mediated ion-exchange chromatography.

Methods have been developed that are based on cation exchange chromatography in the absence and presence of excess bisulfite for the isolation of wine pigments from Australian red wine and grape marc extract. The pigments were identified using HPLC and electrospray ionization mass spectrometry. The mass spectral data indicate that these pigments are C4-substituted anthocyanins with a tetracyclic structure. The pigments form a series of closely related oligomeric pigments which include those previously described in the literature, such as pigment A and vitisin A, as well as some newly identified pigments.     

Quantification of selected aroma-active compounds in Pinot noir wines from different grape maturities

Effect of grape maturity on aroma-active compounds in Pinot noir wine was investigated using stir bar sorptive extraction-gas chromatograph-mass spectrometry (SBSE-GC-MS). High correlation coefficient (> 0.95) and low standard deviation (< 10%) were obtained for all aroma-active compounds of interest. Two vintages of Pinot noir wines with three different grape maturities each were analyzed. Statistical analysis showed that both grape maturity and growing year significantly affected the aroma composition of the final wine. Analysis of wine samples from the same vintage indicated that grape maturity could affect aroma compounds in different ways, based on their biochemical formation in the wines. For most short-chain fatty acid esters, there were no obvious trends with grape maturity, however, the concentrations of ethyl 2-methylpropanoate and ethyl 3-methylbutanoate consistently decreased with grape maturity. The decreasing trend was also observed for other esters including ethyl cinnamate, ethyl dihydroxycinnamate, and ethyl anthranilate, with the exception of ethyl vanillate, while C13 norisoprenoids, monoterpenes, and guaiacols had increasing trends with grape maturation.     

Use of electrospray mass spectrometry for mass determination of grape (Vitis vinifera) juice pathogenesis-related proteins: a potential tool for varietal differentiation

Methods based on liquid chromatography-mass spectrometry (LC-MS) and protein trap mass spectrometry (trap-MS) were developed to determine the complement of pathogenesis-related (PR) proteins in grape juice. Trap-MS was superior to LC-MS in terms of simplicity, rapidity, and sensitivity. Proteins with a wide range of masses (13--33 kDa) were found in the juices of 19 different varieties of grape (Vitis vinifera) and were identified as mostly PR-5 type (thaumatin-like) and PR-3 type (chitinases) proteins. Although the PR proteins in juices of grapes are highly conserved, small consistent differences in molecular masses were noted when otherwise identical proteins were compared from different varieties. These differences persisted through different harvest years and in fruits grown in different Australian locations. With the definition of four different masses for PR-5 proteins (range = 21,239--21,272 Da) and nine different masses of PR-3 proteins (range = 25,330--25,631 Da) and using statistical analysis, the methods developed could be used for varietal differentiation of grapes grown in several South Australian locations on the basis of the PR protein composition of the juice. It remains to be seen whether this technology can be extended to grapes grown worldwide and to wine and other fruit-derived products to assist with label integrity to the benefit of consumers.     

Metabolic influence of Botrytis cinerea infection in champagne base wine

Botrytis cinerea infection of grape berries leads to changes in the chemical composition of grape and the corresponding wine and, thus, affects wine quality. The metabolic effect of Botrytis infection in Champagne base wine was investigated through a (1)H NMR-based metabolomic approach. Isoleucine, leucine, threonine, valine, arginine, proline, glutamine, γ-aminobutyric acid (GABA), succinate, malate, citrate, tartarate, fructose, glucose, oligosaccharides, amino acid derivatives, 2,3-butanediol, acetate, glycerol, tyrosine, 2-phenylethanol, trigonelline, and phenylpropanoids in a grape must and wine were identified by (1)H NMR spectroscopy and contributed to metabolic differentiations between healthy and botrytized wines by using multivariate statistical analysis such as principal component analysis (PCA). Lowered levels of glycerol, 2,3-butanediol, succinate, tyrosine, valine derivative, and phenylpropanoids but higher levels of oligosaccharides in the botrytized wines were main discriminant metabolites, demonstrating that Botrytis infection of grape caused the fermentative retardation during alcoholic fermentation because the main metabolites responsible for the differentiation are fermentative products. Moreover, higher levels of several oligosaccharides in the botrytized wines also indicated the less fermentative behavior of yeast in the botrytized wines. This study highlights a metabolomic approach for better understanding of the comprehensive metabolic influences of Botrytis infection of grape berries in Champagne wines.     

An LC-MS method for analyzing total resveratrol in grape juice, cranberry juice, and in wine.

Resveratrol is an antioxidant found in grapes, grape products, and some other botanical sources with antiinflammatory and anticancer properties. In grapes and wine, it occurs both as free resveratrol and piceid, the 3beta-glucoside of resveratrol. Here we report a liquid chromatography-mass spectrometry method to analyze total resveratrol (including free resveratrol and resveratrol from piceid) in fruit products and wine. Samples were extracted using methanol, enzymatically hydrolyzed, and analyzed using reversed phase HPLC with positive ion atmospheric pressure chemical ionization (APCI) mass spectrometric detection. Following APCI, the abundance of protonated molecules was recorded using selected ion monitoring (SIM) of m/z 229. An external standard curve was used for quantitation, which showed a linear range of 0.52-2260 pmol of trans-resveratrol injected on-column with a correlation coefficient 0.9999. The coefficient of variance of the response factor over the same concentration range was determined to be 5.8%, and the intra-assay coefficient of variance was determined to be 4.2% (n = 7). The limit of quantitation, defined as signal-to-noise 10:1, was determined to be 0.31 pmol injected on-column. The extraction efficiency of the method was determined to be 92%. The stability of resveratrol under different conditions was also examined. For example, resveratrol was stable for up to 5 days at 4 degrees C in the dark but was not stable at room temperature without protection from light. Resveratrol was detected in grape, cranberry, and wine samples. Concentrations ranged from 1.56 to 1042 nmol/g in Concord grape products, and from 8.63 to 24.84 micromol/L in Italian red wine. The concentrations of resveratrol were silmilar in cranberry and grape juice at 1.07 and 1.56 nmol/g, respectively.     

Polysaccharide profile and content during the vinification and aging of Tempranillo red wines

Passing from must to wine produced a loss of low-molecular-weight grape structural glucosyl polysaccharides, and an important gain in yeast mannoproteins (MP) and grape-derived arabinogalactan proteins (AGP), and rhamnogalacturonans-II (RG-II). AGP were more easily extracted than RG-II, and small quantities of RG-II monomers and galacturonans were detected. Postmaceration produced a reduction in all grape polysaccharide families, particularly acute in AGP. The reduction of polysaccharides during malolactic fermentation only affected grape AGP, and MP were continuously liberated during the entire vinification process. Wine oak and bottle aging was associated with a relative stability of the polysaccharide families. AGP were thus the majority polysaccharides in young wines but, contrary to what may be thought, structural glucosyl oligosaccharides dominated in musts and MP in aged wines. Precipitation of polysaccharides was noticeable during vinification, and it mainly affected high-molecular-weight AGP and MP. Hydrolytic phenomena affected the balance of wine polysaccharides during late maceration-fermentation.     

Screening for potential pigments derived from anthocyanins in red wine using nanoelectrospray tandem mass spectrometry

Red wine extracts were screened for potential wine pigments derived from anthocyanins, using a combination of nanoelectrospray tandem mass spectrometry techniques. Fourteen aglycons were considered to be of anthocyanidin origin on the basis of their MS/MS spectra. The proposed structures of the aglycons were anthocyanidin C-4 substituted with vinyl linkage between C-4 and the hydroxy group at C-5. The anthocyanidin derivatives identified in the wine extracts were vinyl, vinylmethyl, vinylformic acid, 4-vinylphenol, 4-vinylguaiacol, and vinylcatechin adducts of malvidin as well as vinylformic acid and 4-vinylphenol adducts of peonidin and petunidin. The presence of vinyl alcohol, 4-vinylcatechol, and 4-vinylsyringol adducts of malvidin was also proposed.     

Influence of wine pectic polysaccharides on the interactions between condensed tannins and salivary proteins

Alpha-amylase, a major human salivary protein, and IB8c, a representative of the proline-rich proteins, were obtained by isolation from saliva and by solid-phase synthesis, respectively. The interactions between these proteins and condensed tannins isolated from grape seeds were studied at different protein and tannin concentrations by measuring their aggregation. Pectic polysaccharides were isolated from wine, and their effect on protein tannin aggregation was assessed. The results presented in this study showed that the most acidic fractions of arabinogalactan proteins have the ability to inhibit the formation of aggregates between the grape seed tannins and the two different salivary proteins. Rhamnogalacturonan II has the same ability toward alpha-amylase but not IB8c under the conditions of the present study. Polysaccharides show effects at concentrations at which they are present in wine, which could mean an influence in wine astringency. The interaction between condensed tannins and alpha-amylase is differently affected by ionic strength when compared with IB8c.     

Red wine making by immobilized cells and influence on volatile composition

Red wine making using yeast cells immobilized in two types of raisin berries, at various temperatures (6-30 degrees C), was studied. A modification of the batch bioreactor was used to separate the grape skins used for color extraction from the biocatalyst and the fermenting grape must. The evaluation of the immobilized biocatalysts was made on terms of productivity and organoleptic quality, including color intensity and formation of volatiles. The immobilized cells were found capable of low-temperature wine making, producing red wines containing more than 11% v/v alcohol in 8 days at 6 degrees C. The quality of wines was examined by gas chromatography (GC) and GC-MS analysis and sensory evaluation. Higher alcohol concentrations were decreased, and ethyl acetate concentrations increased by the drop of temperature. Many esters, alcohols, carbonyls, and miscellaneous compounds were identified in wines produced by immobilized cells, revealing no significant qualitative differences as compared to wines produced by free cells. The sensory evaluation showed that the best red wine was produced at 6 degrees C.     

Novel preparation method of template DNAs from wine for PCR to differentiate grape (Vitis vinifera L.) cultivar

Template DNAs were extracted from wine and purified for use as samples for PCR to differentiate grape cultivars. It has been pointed out that the authentication of grape material by PCR using wine as a material is very difficult. The problems are (1) decomposition of DNAs during fermentation; (2) contamination of DNAs from microorganisms such as yeast; (3) interference of DNA extraction by polysaccharides and polypeptides in the beverages; and (4) coexistence of PCR inhibitors, such as polyphenols. For this study was developed a novel preparation method of template DNA from wine to differentiate grape cultivars using PCR by (1) lyophilizing and pulverizing the fermented beverage to concentrate the DNAs; (2) decomposition of polysaccharides and proteins so as not to inhibit DNA extraction using heat-resistant amylase and proteinase K without DNA damage by endogenous DNase; and (3) separation of the template DNAs for PCR from PCR inhibitors, such as polyphenols, by purification using 70% EtOH extraction and isopropyl alcohol precipitation. To prevent the amplification of microorganisms' DNAs during PCR, suitable PCR primers closely related to the specific plant DNAs, such as chloroplast DNA and mitochondrial DNA, were selected. The sequences of the amplified DNAs by PCR were ascertained to be the same as those of grape materials.     

Immunodetection of proteins from grapes and yeast in a white wine

The objective of this study was to analyze the origin of proteins of a Chardonnay wine. Three various polyclonal antibodies raised against must, yeast, and bacteria proteins were produced. For microorganisms, only the secreted macromolecules were used. To this end, yeast and bacteria were cultured in a model medium under conditions close to those of winemaking. Results obtained using these specific antibodies indicate that most of the wine proteins came from grapes and many of them were glycoproteins. Some proteins of this Chardonnay wine came from the yeast; they were released during the alcoholic fermentation and consisted of high molecular weight mannoproteins. In contrast, no bacteria proteins were detected in this Chardonnay wine.     

优选非酿酒酵母胞外酶增香酿造干白葡萄酒效果

为了研究非酿酒酵母胞外酶促进葡萄酒发酵产香的效果,该文在爱格丽葡萄汁酒精发酵12 h后,分别添加优选胶红酵母(Rhodotorula mucilaginosa,RM)胞外酶液,优选发酵毕赤酵母(Pichia fermentans,PF)胞外酶液和商业糖苷酶制剂(almondβ-glucosidase,AG)(10 mU/mL),以不添加酶制剂的酿酒酵母纯发酵为对照,发酵过程中每24 h取样,采用HS-SPME-GC/MS(headspace solid-phase microextraction-gas chromatography/mass spectrometry)监测挥发性成分的生成变化。葡萄酒含糖量低于2 g/L时终止发酵,低温满罐密封储存,次年4月,进行葡萄酒香气仪器和感官量化分析。胞外酶液共测得6种风味酶活性,其中RM酶液中的β-D-葡萄糖苷酶、α-L-鼠李糖苷酶、β-D-木糖苷酶活性均显著(P<0.05)高于PF中的,而α-L-阿拉伯糖苷酶和酯酶在PF酶液中活性更高(P<0.05)。发酵过程中,胞外酶处理显著促进了(P<0.05)品种香气和发酵香气物质的生成,其中RM酶液显著提高了萜烯类物质和苯乙基类化合物含量,其作用效果分别比商业酶处理高41.7%和31.8%,PF酶液显著促进了发酵香气化合物的生成,尤其是脂肪酸乙酯含量约为对照的2倍。酒样感官分析结果显示,两株酵母的胞外酶处理表现出各自增香酿造的优势,其中RM酶液促进温带酸果类香气的效果突出,PF酶液显著提升了柑橘类香气。研究结果为酵母风味酶应用于葡萄酒的增香酿造提供了理论和实践指导。