Grape Proteins Research Articles

Optimized techniques for the extraction of grape allergens appropriate for in vivo and in vitro testing and diagnosis

Standardized allergen extracts are needed for diagnosis and therapy purposes. For grapes, standardization is hampered by low protein and high tannin and pectin concentrations. The aim of the current study was to develop an optimized method for the extraction of grape proteins and possibly extend this to other fruits. Several existing or modified extraction methods were compared by means of protein concentration determination, SDS-PAGE, immunoblotting and radioallergosorbent test (RAST). An optimized extraction protocol was obtained in which we combined a high concentration of plant tissue, a concentrated, enriched and neutral buffer able to remove sugars and keep proteins soluble and a bivalent buffer for pectin removal. Both the quantitative (protein concentration) and qualitative parameters (SDS-PAGE protein patterns and IgE reactivity) were compared to standard protocols and commercial extracts used as diagnostic tools in the clinical practice. This method proved to be the most efficient mainly compared to the standard Björksten protocol in extracting the low molecular weight proteins, including the major grape allergen (lipid transfer protein, Vit v 1). It proved to be an easy, low cost and reproducible method proposed to prepare grape extracts that could replace the commercially available ones, used for diagnosis and possibly extend the method to other fruits especially in extracting LTPs.

An alternative method for purification of a major thaumatin‐like grape protein (VVTL1) responsible for haze formation in white wine

AbstractA new laboratory‐based method for obtaining gram quantities of a principal heat‐unstable thaumatin‐like protein (VVTL1), in wine is presented. Separation and purification from grape juice was achieved by cation exchange and ultra‐filtration. Purity was confirmed by HPLC, sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), gel permeation chromatography (GPC) and high‐sensitivity amino acid analysis. This alternative method is more rapid and compact and can efficiently produce much larger quantities (>100×) than the current laboratory technique. Copyright © 2007 Curtin University of Technology and John Wiley & Sons, Ltd.

Reducing haziness in white wine by overexpression of Saccharomyces cerevisiae genes YOL155c and YDR055w

Grape proteins aggregate in white wine to form haze. A novel method to prevent haze in wine is the use of haze protective factors (Hpfs), specific mannoproteins from Saccharomyces cerevisiae, which reduce the particle size of the aggregated proteins. Hpf1p was isolated from white wine and Hpf2p from a synthetic grape juice fermentation. Putative structural genes, YOL155c and YDR055w, for these proteins were identified from partial amino acid sequences of Hpf1p and Hpf2p, respectively. YOL155c also has a homologue, YIL169c, in S. cerevisiae. Comparison of the partial amino acid sequence of deglycosylated-Hpf2p with the deduced protein sequence of YDR055w, confirmed five of the 15 potential N-linked glycosylation sites in this sequence were occupied. Methylation analysis of the carbohydrate moieties of Hpf2p indicated that this protein contained both N- and O-linked mannose chains. Material from fermentation supernatant of deletion strains had significantly less activity than the wild type. Moreover, YOL155c and YIL169c overexpressing strains and a strain overexpressing 6xHis-tagged Hpf2p produced greater haze protective activity than the wild type strains. A storage trial demonstrated the short to midterm stability of 6xHis-tagged Hpf2p in wine.

Protein characterization of Roditis Greek grape variety and Sauvignon blanc and changes in certain nitrogen compounds during alcoholic fermentation

<p style="text-align: justify;">Must and wine samples of the Greek grape variety Roditis and the French one Sauvignon blanc were analysed in order to obtain further knowledge of the protein profile of Roditis and to watch the evolution of grape proteins during the alcoholic fermentation of Roditis and Sauvignon blanc musts. For these purposes protein samples were isolated from must and wine samples by ammonium sulphate precipitation and subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS - PAGE). Eleven and nine bands with molecular weights between 11,1 and 64,4 kDa were detected on the electrophoregramms of Roditis and Sauvignon blanc must and wine samples respectively, using Coomassie Brillant Blue R-250 and silver staining methods. Two protein fractions of must and wine samples with molecular weights of 64,4 kDa and 34,4 kDa were identified as being glycoproteins in the profile of the Greek grape variety, according to the Periodic acid - silver staining, while only one must and wine fraction of 64,4 kDa had positively react with this stain, as far as it concerns Sauvignon blanc. None of the low molecular weight protein fractions found to be responsible for haze formation. A modified (Bradford) dye - binding procedure was used for the determination of musts and wines soluble proteins. Free amino nitrogen and the contents of neutral and acidic polysaccharides in the protein fractions after chromatography on Sephadex G - 25, were also analyzed.</p>

Evidence for Protein Degradation byBotrytis cinereaand Relationships with Alteration of Synthetic Wine Foaming Properties

Botrytis cinerea is an important fungal pathogen particularly dreaded in the cool climate vineyard. It is responsible for important damage, especially the decrease in foamability of sparkling wines, such as Champagne. Different studies have shown that proteins are largely involved in the stabilization of Champagne foam despite their low concentration. Other works demonstrated changes in the electrophoretic characteristics of must proteins originating from botrytized grapes, although the cause of such alterations was never explained. In the first part of this study, results showed the release by B. cinerea of 3.5 mg/L total proteins in a synthetic liquid medium. Among these proteins, the presence of a protease activity on bovine serum albumin (BSA) and must proteins was demonstrated by using a colorimetric method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the model wine, the Bradford method showed a BSA loss of 66% after 24 h and a loss of 96% after 120 h. In the same model wine, the soluble must protein concentration decreased by 35% after 1 week and by 53% after 2 weeks while the control showed no protein loss. B. cinerea proteases were then able to degrade BSA and must proteins and were above all active at must and wine pH and in the presence of ethanol and SO(2). The second part of this work was dedicated to the relationship between the presence of B. cinerea proteases and its effects on the synthetic wine foaming properties. The addition of a B. cinerea culture medium (1/33 v/v) to the synthetic wine containing 21 mg/L soluble grape proteins induced a decrease in foamability by 60% after 1 week. For BSA in the model wine, the foamability decreased by 32% after 24 h and by 95% after 120 h, as shown by the colorimetric method. These experiments demonstrate for the first time the relationship between B. cinerea protease activity and the decrease in wine foaming properties.

Characterization of Major Stable Proteins in Chardonnay Wine

Chardonnay wine produced in the conventional manner contained 69.8 to 108mg/L soluble proteins including nondialyzable polypeptides ; with an average of 87.2mg/L. Proteins were separated by precipitation with ammonium sulfate, and fractionated into two fractions, F1 (mainly invertase and proteoglycans) and F2 (glycoproteins), by Sephadex G-100 column chromatography. The fractions were further separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) [isoelectric focusing and SDS-polyacrylamide gel electrophoresis]. More than 160 and 150 protein spots were detected on the 2-D PAGE maps of F1 and F2, respectively. The major proteins or polypeptides were determined to be fruit proteins, such as thaumatin- and osmotin-like proteins, invertase, lipid transfer protein (LTP), and their hydrolysis products. This is first report on the existence of LTP (or its hydrolysis product) and the hydrolysis products of major grape proteins (invertase, osmotin-like protein, and thaumatin-like protein) in wine.

Profiling of Soluble Proteins in Wine by Nano-High-Performance Liquid Chromatography/Tandem Mass Spectrometry

Wine proteins play an important role in a wine's quality as they affect taste, clarity, and stability. To enhance our understanding of the proteins in wine, nano-high-performance liquid chromatography (HPLC)/tandem mass spectrometry was used to profile soluble proteins in wine. Twenty proteins were identified from a Sauvignon Blanc wine including five proteins derived from the grape, 12 from yeast, two from bacteria, and one from fungi. The findings are somewhat peculiar at first glance, but reasonable explanations can account for the results. The grape proteins identified are less in number, which may be due to the availability of an incomplete database and possibly bentonite fining. The relatively large number of identified yeast proteins may be due to their complete protein database. The identified bacterial and fungal proteins could possibly be attributed to sources in the vineyard including natural infections and improper handling during harvest. The use of nano-HPLC/tandem mass spectrometry is an important tool for identifying wine proteins and understanding how they affect its characteristics.

Osmotin and Thaumatin from Grape: A Putative General Defense Mechanism Against Pathogenic Fungi

ABSTRACT Little information is available concerning the expression of pathogenesis-related (PR) proteins in grapevine (Vitis vinifera) and their effect properties on the major fungal pathogens of grape. A systematic study was performed on the effect of total or individual grape proteins on mycelial growth, spore germination, and germ tube growth of Uncinula necator, Phomopsis viticola, and Botrytis cinerea. Two proteins, identified as PR proteins by immunological methods and by N-terminal sequencing as osmotin and thaumatin-like protein, exhibited strong antifungal activities in vitro, blocking the growth of Phomopsis viticola and Botrytis cinerea mycelia. In addition, they inhibited spore germination and germ tube growth of U. necator, Phomopsis viticola, and Botrytis cinerea. The presence of both proteins displayed a synergistic effect. The expression of osmotin and thaumatin-like protein was induced in grapevine leaves and berries infected with U. necator and Phomopsis viticola. Thaumatin previously was thought to occur exclusively in berries. Immunoblot analyses revealed the accumulation of the two PR proteins in infected leaves and berries, supporting a role in vivo in increasing the resistance of grapevine to fungal attack.

Effects ofBotrytiscinereaInfection on the Must Protein Electrophoretic Characteristics

Qualitative effects of Botrytis cinerea infection on a must protein fraction were studied by comparing the electrophoretic patterns of musts obtained from healthy grapes or from grapes highly infected by B. cinerea. Evidence was obtained that proteins secreted by B. cinerea can degrade grape proteins. Most of the proteins present in the healthy must, between 20 and 30 kDa and a major glycoprotein at 62/64 kDa, disappeared in the infected must. Moreover an immunochemical technique, using polyclonal antibodies raised against B. cinerea-secreted proteins, was developed to specifically detect proteins originating from B. cinerea in the infected must. This is of particular interest when considering that some of the grape proteins participate in the foaming properties of Champagne wines. Keywords: Protein; must; Botrytis cinerea; antibodies; electrophoresis; proteolytic activity

Glycoproteins: Characterization in a Hybrid Grape Variety (Muscat Bailey A) Juice, Fermenting Must, and Resultant Red Wine

Elucidation of the differences in properties between unstable and soluble proteins will assist in developing effective fining of wine and in breeding of grape varieties with fewer unstable proteins. Soluble proteins from juice, must pressed just after fermentation on skins, and from red wines were isolated by ammonium sulfate precipitation and fractionated by Sephadex G-100 followed by high performance liquid chromatography. At least 17, 18, and 22 protein fractions were obtained from the juice, fermenting must, and red wine samples, respectively. All of the fractions had sugars in the range of 0.3% to 33.3% of total glycoproteins (the sum of the total sugars and amino acids found in the hydrosolates). Most of the glycoproteins had less than 10% sugars. The glycoproteins differed from grape proteins, generally being rich in the amino acids aspartic acid, glycine, threonine, glutamic acid, and alanine and in the sugars arabinose, rhamnose, galactose, and glucose. Their isoelectric points and molecular weights ranged from 3.7 to 4.9 and form 12.1 to 30.8 K daltons, respectively. Differences in the HPLC patterns and in the chemical and physical properties of the protein fractions obtained revealed the presence of several individual glycoproteins, their concentrations, and occurrence of specific glycoproteins at each stage of vinification. The proportion of glycoprotein sugars decreased in the order juice proteins &gt; must proteins &gt; wine proteins — <i>i.e.</i>, degradation or modification of the sugar portions of the glycoproteins occurred during vinification. The origin of wine glycoproteins suggests roles in aging reactions and other wine characteristics.

The Proteins of Muscadine Grapes

ABSTRACTThe concentrations, molecular weights and isoelectric points of proteins present in muscadine grape cultivars were determined. The total protein contents of these grapes, are consistent with values reported in literature for V. vinifera grape proteins. Molecular weights of most of the protein fractions were, however, relatively high when compared with those of other grape cultivars. Isoelectric’ points of muscadine proteins ranged between pH 5.6 and pH 7.6.

A Review and Some Studies on Grape Protein

The investigation, still incomplete, indicates that wine protein is of rather complex structure. The Voit-N, when multiplied by 6.25, can be used to calculate the "crude protein" of wine but not the "wine protein." It is very probable that the Voit precipitate includes the "soluble" grape proteins, which may be identical to its water-insoluble fraction. The water-soluble constituents are suspected to be low-molecular-weight polypeptides and proteids. The proteins consist of different nitrogen bodies (globulins) that can be separated electrophoretically. So far, 17 amino acids have been identified with certainty as building blocks of these nitrogen bodies. If is not yet known how 5 of these are distributed among the electrophoretically separated fractions. Nor is it known in what order they are connected and how many peptide chains we are dealing with. Also unknown is the temperature coefficient for wine protein denaturation. In practice, heat treatment of wines and must leads quickly to protein stability, while bentonite treatment results in a protein-free wine.