Immunochemical and mass spectrometry detection of residual proteins in gluten fined red wine

Recently, wheat gluten has been proposed as technological adjuvant in order to clarify wines. However, the possibility that residual gluten proteins remain in treated wines cannot be excluded, representing a hazard for wheat allergic or celiac disease patients. In this work, commercial wheat glutens, in both partially hydrolyzed (GBS-P51) and nonhydrolyzed (Gluvital 21000) forms, were used as fining agents in red wine at different concentrations. Beside immunoenzymatic analyses using anti-gliadin, anti-prolamin antibodies and pooled sera of wheat allergic patients, a method based on liquid chromatography coupled to mass spectrometry has been proposed to detect residues of gluten proteins. Residual gluten proteins were detected by anti-prolamin antibodies, anti-gliadin antibodies and sera-IgE only in the wine treated with GBS-P51 at concentration 50, 150, and 300 g/hL, respectively, whereas no residual proteins were detected by these systems in the wine treated with Gluvital 21000. In contrast liquid chromatography-mass spectrometry analyses allowed the detection of proteins in red wines fined down to 1 g/hL of Gluvital 21000 and GBS-P51. Our results indicate that MS methods are superior to immunochemical methods in detecting gluten proteins in wines and that adverse reactions against gluten treated wines cannot be excluded.     

Clarification of Muscat musts using wheat proteins and the flotation technique

The bovine spongiform encephalopathy (BSE, mad cow) crisis has led some wine-makers to question gelatin as a fining agent and to reject the use of these animal proteins. The search for a substitute for gelatin was begun by comparing vegetable proteins (particularly gluten) with gelatin fining treatments. Clairette de Die (French-controlled appellation) is a sparkling sweet wine. The alcoholic fermentation begins in a tank, continues in a crown-cap bottle, and stops before the complete consumption of sugar. All particles present in the bottle have to be removed before corking, and it is important to have a must as clear as possible. This study concerned the clarifying of a Muscat must, treated with pectinases, using a very efficient flotation technique. The must is fined with bentonite/silica/protein (fish gelatin, wheat gluten, or lupin isolate) to induce flocculation and then pressurized (6 bar). After depressurization, microbubbles cling to flocculates and climb to the top of the flotation tank (this flotation foam is clarified using a rotary filter). At laboratory scale, gluten (20 g/hL) and gelatin (10 g/hL) (each combined with bentonite and silica gel) gave turbidities of 50 and 35 NTU, respectively (6.5 and 4.1% of that of the nontreated must). The Muscat must was also clarified by static settling. The turbidity decreased by 86% for the gluten/bentonite fining and by 60% for the gelatin/bentonite fining. Visually, gluten flocculation takes longer to occur and flocculates sedimentation is longer than with gelati, but the removal of insoluble particles is more complete and leads to lower turbidities. At an industrial scale, gluten (20 g/hL) and gelatin (10 g/hL) (each combined with bentonite and silica gel) gave turbidities of 60 and 48 NTU, respectively. Turbidities measured in the tanks 14 h after the flotation showed a better efficiency for the wheat gluten (24 NTU) compared to gelatin (28 NTU). This is explained by the static settling that completed the clarifying effect of the flotation. The last experiment showed comparable efficiencies for wheat gluten and lupin proteins. BSE caused a situation of crisis (in Europe particularly) leading the public and wine-makers to lose their confidence in the use of gelatin as fining agent and to reject animal proteins in general. It is proposed here that vegetable proteins could efficiently replace gelatin.     

Removal of ochratoxin A in red wines by means of adsorption treatments with commercial fining agents

The presence in wine of the fungal metabolite, ochratoxin A (OTA), represents a serious risk for consumer health. A variety of fining agents, including activated carbon, silica gel, potassium caseinate, egg albumin, and gelatin, was evaluated in relation to their abilities to remove OTA in fortified wines. Freundlich adsorption isotherms were used to model the adsorption behavior between ochratoxin A and the fining agent. Potassium caseinate and activated carbon were found to be the best fining agents that could be used to remove OTA in wine. Potassium caseinate removed up to 82% of OTA when used at 150 g/hL, whereas activated carbon showed the highest specific adsorption capacity due to a high surface area per mass and low adsorption of total polyphenols.     

Determination of the grape invertase content (using PTA-ELISA) following various fining treatments versus changes in the total protein content of wine. relationships with wine foamability

Proteins have proven to play a major role in the stabilization of foam in Champagne wines despite their low concentration that ranges from 4 to 20 mg/L. The aim of this study was to evaluate the effect of fining on total protein and grape invertase contents of champenois base wines and their foaming properties. Data showed that fining and especially the use of bentonite at doses ranging from 10 to 50 g/hL leads to a significant decrease in the total protein content of wines together with that of the grape invertase content, with such a decrease being very detrimental to the foaming properties of the treated wines in terms of foam height (HM) and foam stability (HS). Only a slight decrease in the total protein content, in the grape invertase concentration, and in the foam quality of wines was observed when using casein (10 and 20 g/hL) or bentonite combined with casein (both at 20 g/hL). Our study thus clearly establishes the good correlation existing between the wine protein concentration and its foaming properties. A remarkable correlation was observed between the decrease in the grape invertase content and the total protein content of wines, following bentonite treatments, suggesting that the grape invertase (which represents at least 10-20% of the wine proteins) follows a similar behavior upon fining to other proteins of Champagne wines, despite the high molecular mass and the highly glycosylated structure of this particular protein. Moreover, the decrease in total protein and grape invertase contents of wine after fining with bentonite was found to be correlated with a decrease in the foaming properties of the corresponding wines (with respectively R(2) = 0.89 and 0.95).     

Influence of lysozyme treatments on champagne base wine foaming properties

The objective of this study was to estimate the effect of lysozyme on the foaming characteristics of Champagne base wine. Lysozyme additions were made to the musts and also to the wines before and after bentonite or charcoal treatments, which remove endogenous proteins. Treatments with bentonite diminished foamability and foam stability of wines, whatever the dose (30 or 80 g/hL) and variety [Chardonnay, -28%; Pinot noir, -20% (at 30 g/hL)]. An addition of lysozyme in must raised Pinot noir wine foamability by 21%, whereas the difference is hardly perceptible for Chardonnay wine (+3%). Pinot noir and Chardonnay wines, originating from lysozyme-treated musts, in addition to bentonite treatment on the wine, presented higher foamability than wines treated only with bentonite. Lysozyme was removed (91-100%) by the bentonite treatment. Then, it was not responsible for the increase in foamability but seemed to have a protective effect on the wine proteins. When wines were initially treated with bentonite (150 g/hL) and then enriched with 80 g/hL lysozyme, this enzyme was not able to restore foaming properties. Treatments with charcoal always diminished foamability. The average increase in foamability due to an addition of lysozyme after charcoal treatment (80 g/hL) was 23%. Results showed a real positive effect of lysozyme on foam stability when wines have to be treated with charcoal (+25% and +56% for the Pinot noir wine and the Chardonnay wine, respectively, at 30 g/hL).     

Investigation of the allergenic potential of wines fined with various proteinogenic fining agents by ELISA

Hidden allergens are a common problem in food safety that has been known for many years. This is why the European Parliament adopted Directive 2003/89/EC amending 2000/13/EC. In addition to specific ingredients, Directive 2003/89/EC also requests the declaration of specific products that were used in the production and could be a risk for allergic individuals. This also includes the declaration of fining agents and lysozyme used in wines. In fact, it could be assumed that fining agents would be almost completely removed during the manufacturing process; however, until now there has been no necessity to analyze wine for these fining agents. By applying enzyme-linked immunosorbent assay (ELISA), residuals of fining agent proteins and the stabilizer lysozyme were investigated in various German wines. The results showed no detectable amounts of fining agents in wines, except for dried egg white and lysozyme, both derived from hen's egg white. For those products, adverse reactions against treated wines could not be excluded.     

Impact of protein size distribution on gluten thermal reactivity and functional properties

Wheat gluten structure was modified in different ways: Disulfide bonds were reduced by sulfitolysis, or protein chains were enzymatically hydrolyzed at three different degrees of proteolysis. A kinetic study of the thermal reactivity of the modified glutens showed that gluten aggregation kinetic was slowed in consequence to the shift of gluten size distribution toward smaller proteins. In contrary to sulfitolysis, proteolysis also affected the gluten reactivity potential because of the formation of numerous nonreactive species. Moreover, the thermally induced browning reaction was greatly enhanced by proteolysis, which increased the amount of free amine residues, substrates of the Maillard reaction. On the contrary, a whitening effect was observed for reduced gluten with bisulfite. Proteolysis was also found to decrease plasticized gluten viscosity, to increase gluten-based materials water solubility, and to enhance gluten adhesiveness properties but to reduce its mechanical performance. Sulfitolysis was considered as a possible way of extending gluten processability by extrusion or injection molding, whereas proteolysis was found to confer enhanced gluten stickiness that suggests new potential end uses of gluten in the pressure sensitive adhesives domain.     

Biochemical Analysis and Rheological Properties of Gluten Modified by Transglutaminase

A transglutaminase from Streptoverticillium sp. was used to create new covalent intermolecular cross‐links between proteins in gluten. This modification induced drastic changes in its physicochemical properties as well as in its rheological behavior. To understand these changes, we characterized the gluten extractability in acetic acid and identified the proteins of supernatant and pellet by immunoblotting using antibodies specific for each prolamin class. The proportion of soluble proteins decreased drastically after transglutaminase treatment due to the formation of large insoluble polymers as shown by SDS‐PAGE. Among the constitutive proteins of gluten, the high molecular weight glutenin subunits were the most affected in the transglutaminase reaction. The rheological behavior of gluten after 18 hr of incubation with transglutaminase was studied in shear by dynamic measurements over 10^‐3 – 10¹ Hz frequency range and by creep and recovery tests. The behavior of treated glutens remained that of a transient network, but the viscoelastic response was shifted toward shorter times and the steady‐state viscosity was greatly increased. The enzymatic treatment caused a considerable reinforcement of the network. The modified glutens were also less sensitive to thermal processing than unmodified glutens, as shown by a lower amplitude of variation of storage modulus G′ with temperature after enzymatic treatment.     

Quantitative and Qualitative Role of Added Gluten on White Salted Noodles

A commercial gluten and glutens isolated from four soft and four hard wheat flours were incorporated into a hard and a soft white flour by replacement to directly determine the quantitative and qualitative role of gluten proteins in making noodles. Gluten incorporation (6%) decreased water absorption of noodle dough by 3%, shortened the length of the dough sheet by 15 and 18%, and increased the thickness of the dough sheet by 18 and 20% in soft and hard wheat flour, respectively. Noodles imbibed less water and imbibed water more slowly during cooking with gluten incorporation, which resulted in a 3‐min increase in cooking time for both soft and hard wheat noodles. Despite the extended cooking time of 3 min, noodles incorporated with 6% gluten exhibited decreases in cooking loss by 15% in soft wheat. In hard wheat flour, cooking loss of noodles was lowest with 2% incorporation of gluten. Tensile strength of fresh and cooked noodles, as well as hardness of cooked noodles, increased linearly with increase in gluten incorporation, regardless of cooking time and storage time after cooking. While hardness of cooked noodles either increased or showed no changes during storage for 4 hr, tensile strength of noodles decreased. There were large variations in hardness and tensile strength of cooked noodles incorporated with glutens isolated from eight different flours. Noodles incorporated with soft wheat glutens exhibited greater hardness and tensile strength than noodles with hard wheat glutens. Tensile strength of cooked noodles incorporated with eight different glutens negatively correlated with SDS sedimentation volume of wheat flours from which the glutens were isolated.     

Yeasts used as fining treatment to correct browning in white wines

White wine was subjected to several fining treatments using baker's yeast at concentrations of 0.5, 1, 2, 3, 4, and 5 g/L. At all these concentration levels, the yeasts decreased the color of the wine in different degrees. The wine samples treated with the higher yeast concentration were subjected to analysis of phenolic compounds by HPLC and found to exhibit significantly decreased contents of vanillic, syringic and c-coutaric acids, and procyanidins B2 and B4, and colored compounds eluted at high retention times. The efficiency of the yeast-based fining treatment (1 g/L) was compared with traditional treatments such as those involving the use of activated charcoal or PVPP, which were employed at the usual concentrations in Sherry winemaking. This yeast treatment was found to provide results similar to those of the activated charcoal treatment in terms of A(420). Likewise, significant differences in the degree of retention of various phenols were observed among the three treatments compared. Finally, the wine samples obtained with the different treatments were subjected to a sensory panel. All the wines were found to exhibit improved color, aroma, and flavor with respect to the untreated samples, although the treatment using yeast at 1 g/L provided the best results in terms of aroma.     

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.     

Characterization of Functional Properties of Gluten Proteins in Spelt Cultivars Using Rheological and Quality Factor Measurements

Wet glutens of 27 European spelt (Triticum aestivum ssp. spelta (L.) Thell.) cultivars were examined using fundamental rheological methods (oscillatory and creep tests) in conjunction with the determination of moisture contents of these glutens and the wet gluten contents of the flours. Furthermore, SDS sedimentation volumes were determined. A special baking test for spelt was developed that encompassed the characteristic elements used in the production of traditional German spelt speciality breads. Various significant correlations between gluten properties and baking results were found for three sets of spelt cultivars obtained from different demographic locations and years of harvest. Furthermore, the relationship between baking results (response) and gluten properties (predictors) could be modeled quite well with the help of multiple linear regression analysis. Radar charts used to profile the gluten properties of a particular cultivar showed a great amount of diversity within the spelt material, but there were also similarities between several cultivars. The differences between spelt cultivars should be taken into account when characterizing spelt in general terms or when comparing spelt and modern wheat.     

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.     

Relationships Between Gluten Rheological Properties and Hearth Loaf Characteristics

The rheological properties of fresh gluten in small amplitude oscillation in shear (SAOS) and creep recovery after short application of stress was related to the hearth breadbaking performance of wheat flours using the multivariate statistics partial least squares (PLS) regression. The picture was completed by dough mixing and extensional properties, flour protein size distribution determined by SE‐HPLC, and high molecular weight glutenin subunit (HMW‐GS) composition. The sample set comprised 20 wheat cultivars grown at two different levels of nitrogen fertilizer in one location. Flours yielding stiffer and more elastic glutens, with higher elastic and viscous moduli (G′ and G″) and lower tan δ values in SAOS, gave doughs that were better able to retain their shape during proving and baking, resulting in breads of high form ratios. Creep recovery measurements after short application of stress showed that glutens from flours of good breadmaking quality had high relative elastic recovery. The nitrogen fertilizer level affected the protein size distribution by an increase in monomeric proteins (gliadins), which gave glutens of higher tan δ and flatter bread loaves (lower form ratio).     

Properties of deamidated gluten films enzymatically cross-linked

Films were prepared at neutral pH from deamidated gluten by casting with or without enzymatic treatment by transglutaminase in the presence of various concentrations of diamines added to the film-forming solution. Variation in the glycerol/deamidated gluten ratio from 0.2 to 0.5 had a major effect on the film mechanical properties, which is characteristic of a plasticizing effect. A ratio of 0.35, producing a tensile strength of 1.14 +/- 0.12 MPa and an elongation at break of 376 +/- 62%, was chosen for most of the enzymatic modifications. The action of transglutaminase with or without the addition of external diamines induced a simultaneous increase in tensile strength and elongation at break of the films but tended to decrease the contact angle between the film surface and a water droplet. The presence of diamines in the film solution affected the elongation at break more than the tensile strength of the films. These diamines, able to react at their two extremities, probably acted as spacers between gluten proteins. The decrease in solubility was related to the formation of high molecular weight polymers in the film. The film properties were unaffected by the type of diamine added as secondary substrate in the transglutaminase reaction.     

Aging, free radicals, and antioxidants in wheat seeds

Free radical oxidative attack is considered a major cause of disruption and deteriorative changes observed in aged seeds. Antioxidant defense mechanisms may remove potentially damaging molecular species, and carotenoids may act as radical scavengers. The content of lutein, the major carotenoid in wheat seeds, was determined in the flours. It showed a rapid decrease during seed aging. In addition, the content of free radicals in glutens made from flours of wheat seeds after long-term storage was studied. The concentration of radicals appeared to be age dependent, because the highest content of radicals was detected between 13 and 15 years of aging over 36 years of storage. Specific spin labeling of the sulfhydryl groups of gluten proteins enabled comparative EPR studies of the rigidity of the protein chains. A progressive stiffening of polymeric gluten with seed storage was found.     

Wheat gluten phenolic acids: occurrence and fate upon mixing

Ferulic acid (FA, 4.9-17.7 microg/100 mg), sinapic acid (SA, 1.4-3.5 microg/100 mg), and traces of p-coumaric acid and vanillic acid were detected after saponification of six wheat glutens from industrial and pilot-scale origins. FA and SA occurred mostly as soluble-bound and insoluble-bound forms according to their extractability by acetone/methanol/water (7:7:6, v/v/v). The major part of FA (50-95%) was found in the unextractable fraction, whereas SA was mostly extractable (64-85%). The carbohydrate contents of the glutens were determined also after acid hydrolysis. The highest levels of glucose, arabinoxylan, and FA were obtained from the unextractable fractions of the pilot-scale extracted glutens, probably in relation with a lower efficiency of washing during extraction compared to industrial processes. On the other hand, SA compounds were in similar concentrations in all samples, suggesting their involvement in specific interactions during gluten protein agglomeration. Saponification of the soluble-bound phenolic acids released mainly glucose, whereas a beta-glucosidase treatment had no effect. FA and SA extractability, especially that of soluble-bound ones, decreased strongly in overmixed gluten/water doughs. These low molecular weight conjugates of phenolic acids could be involved in the dough breakdown phenomenon.     

Residues of spiroxamine in grapes following field application and their fate from vine to wine

Dissipation of the fungicide spiroxamine in grapes of two vine varieties, Roditis and Cabernet Sauvignon, exposed to field treatments was evaluated. Vines of a grape vineyard located in central Greece were sprayed once or twice with a commercial formulation of the fungicide at 30 g a.i./hL. Residues in grapes, must, and wine were determined by gas chromatography/IT-MS after extraction with cyclohexane-dichloromethane (9:1), with a limit of quantitation 0.02 mg/kg in grapes and 0.012 mg/kg in wine. Under field conditions, spiroxamine dissipation on grapes was faster during the first 2 weeks and then slower to the sixth week. About 7 days after application, half of the initial spiroxamine concentration remained on the grapes; the respective proportion at 42 days was about 10%. At 14 and 35 days, residues were lower than 0.44 and 0.22 mg/kg, respectively, values below the maximum residue levels set by the European Union (1 mg/kg). Spiroxamine residues transferred from grapes into the must and through the vinification process into the wine were also studied. Mean transfer factors of 0.26 and 0.55 were found from grapes into wine for the wines obtained without maceration and with maceration, respectively. Residues in wine, prepared from grapes with a spiroxamine content of 0.11-0.20 mg/kg, varied from <0.026 to 0.09 mg/kg. Spiroxamine diastereomer B was found to dissipate slower than diastereomer A in the field as well as during the vinification process.     

Rapid Assessment and Prediction of Wheat and Gluten Baking Quality With the 2‐g Direct Drive Mixograph Using Multivariate Statistical Analysis

A computerized 2‐g direct drive mixograph was used to study the mixing characteristics of flours milled from a range of breadmaking cultivars obtained from five separate locations around the UK, providing 54 flour samples. Fifteen parameters were extracted from each mixograph trace using the Mixsmart software program and correlated with baking volume using partial least squares multiple regression statistical analysis to give a prediction of baking volume. Location had a considerable influence on the prediction of baking volume. Excellent predictions of baking volume were obtained from flours from individual locations (R² = 0.805–0.995), but predictions based on all cultivars without discriminating locations were poor. When mixograph and baking volume data for each cultivar were averaged over all five locations, a very high correlation was obtained (R² = 0.999). Preparation of flour samples using rapid, small‐scale milling procedures (Brabender Quadrumat Jr. mill and Perten 3100 hammer mill) did not have any adverse effect on prediction of baking volume. Mixograph parameters obtained from six commercial glutens of varying quality gave good correlations with test baking volumes, based on 6% gluten addition to a control flour.