Haze-active Proteins Research Articles

A Minimally Invasive Approach for Preventing White Wine Protein Haze by Early Enzymatic Treatment.

Protein stability in bottled white wine is an essential organoleptic property considered by consumers. In this paper, the effectiveness of an early enzymatic treatment was investigated by adding a food-grade microbial protease at two different stages of winemaking: (i) at cold settling, for a short-term and low temperature (10 °C) action prior to alcoholic fermentation (AF); (ii) at yeast inoculum, for a long-lasting and medium temperature (18 °C) action during AF. The results reveal that protease sufficiently preserved its catalytic activity at both operational conditions: 10 °C (during cold settling) and 18 °C (during AF). Furthermore, protease addition (dosage 50–150 μL/L) raised the alcoholic fermentation rate. The treatment at yeast inoculum (dosage 50 μL/L) had a remarkable effect in preventing haze formation, as revealed by its impact on protein instability and haze-active proteins. This minimally invasive, time and resource-saving enzymatic treatment, integrated into the winemaking process, could produce stable white wine without affecting color quality and phenol content.

Haze in Beer: Its Formation and Alleviating Strategies, from a Protein-Polyphenol Complex Angle.

Beer is one of the oldest and most widely consumed alcoholic beverages. Haze formation in beer is a serious quality problem, as it largely shortens the shelf life and flavor of beer. This paper reviews the factors affecting haze formation and strategies for reducing haze. Haze formation is mainly associated with specific chemical components in malt barley grains, such as proteins. The main factor causing haze formation is a cross-linking of haze active (HA) proteins and HA polyphenols. Many HA proteins and their editing genes or loci have been identified by proteomics and quantitative trait locus (QTL) analysis, respectively. Although some technical approaches have been available for reducing haze formation in beer, including silica and polyvinylpolypyrrolidone (PVPP) adsorbent treatments, the cost of beer production will increase and some flavor will be lost due to reduced relevant polyphenols and proteins. Therefore, breeding the malt barley cultivar with lower HA protein and/or HA polyphenols is the most efficient approach for controlling haze formation. Owing to the completion of barley whole genome sequencing and the rapid development of modern molecular breeding technology, several candidate genes controlling haze formation have been identified, providing a new solution for reducing beer haze.

Characterization of recombinant GRIP32 as a novel haze protein for protein-polyphenol haze models and prevention of haze formation with polysaccharides in the models

Haze-active proteins are an important factor in haze formation in wine and clear beverages. The aim of this study was to understand the role of haze proteins from pland origin, which are rich in basic amino acids from the plants, in the haze formation involving proteins and polyphenols, as well as to develop strategies to prevent haze formation. The grape ripening-related protein-like (GRIP) containing 12.50% histidine, 28.13% glycine, 16.41% glutamic acid and 1.56% arginine was screened as a potential haze protein from grape by bioinformatics, and recombinantly expressed, purified and characterized. The recombinant GRIP32 (rGRIP32) could be effectively removed by the adsorbents (i.e. bentonite and kaolin), or hydrolyzed by proteases (i.e. papain, neutral protease, and alkaline protease). Procyanidins (PCs) or epigallocatechin gallate (EGCG) were proved to form a haze with rGRIP32. Protein-polyphenol haze models were established using rGRIP32 at 25 °C and pH 5.0. Polysaccharides including pectin, xanthan gum, and guar gum were shown to suppress the haze caused by rGRIP32. The microscale thermophoresis (MST) assay provided new evidences for the binding of proteins and polyphenols to form haze, and demonstrated that the mechanisms of polysaccharide-based haze prevention include competitive binding of polyphenols and formation of ternary complexes with them.

Proteome-Based Analysis of Colloidal Instability Enables the Detection of Haze-Active Proteins in Beer.

Colloidal haze is a serious quality defect of bright beers that considerably reduces their shelf life and is thought to be triggered by hordeins, a class of proline-rich barley proteins. In this work, the proteomes of fresh and old beers were investigated in bottled pilsners and compared to the protein inventory of haze to identify specific haze-active proteins. Haze isolates dissolved in rehydration buffer contained high concentrations of proteins and sugars but provided protein gels with weak spot signals. Consequently, a treatment for the chemical deglycation with trifluoromethanesulfonic acid was applied, which resulted in the identification of protein Z4, LTP1, CMb, CMe, pUP13, 3a, and Bwiph as constituents of the haze proteome. Because only one hordein was detectable and the proline content in haze hydrolysates was lower than those of barley prolamins, our results suggest that this class of proteins is of minor importance for haze development.

Identification of two key genes controlling chill haze stability of beer in barley (Hordeum vulgare L).

BackgroundIn bright beer, haze formation is a serious quality problem, degrading beer quality and reducing its shelf life. The quality of barley (Hordeum vulgare L) malt, as the main raw material for beer brewing, largely affects the colloidal stability of beer.ResultsIn this study, the genetic mechanism of the factors affecting beer haze stability in barley was studied. Quantitative trait loci (QTL) analysis of alcohol chill haze (ACH) in beer was carried out using a Franklin/Yerong double haploid (DH) population. One QTL, named as qACH, was detected for ACH, and it was located on the position of about 108 cM in chromosome 4H and can explain about 20 % of the phenotypic variation. Two key haze active proteins, BATI-CMb and BATI-CMd were identified by proteomics analysis. Bioinformatics analysis showed that BATI-CMb and BATI-CMd had the same position as qACH in the chromosome. It may be deduced that BATI-CMb and BATI-CMd are candidate genes for qACH, controlling colloidal stability of beer. Polymorphism comparison between Yerong and Franklin in the nucleotide and amino acid sequence of BATI-CMb and BATI-CMd detected the corresponding gene specific markers, which could be used in marker-assisted selection for malt barley breeding.ConclusionsWe identified a novel QTL, qACH controlling chill haze of beer, and two key haze active proteins, BATI-CMb and BATI-CMd. And further analysis showed that BATI-CMb and BATI-CMd might be the candidate genes associated with beer chill haze.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1683-1) contains supplementary material, which is available to authorized users.

Identification of QTLs associated with haze active proteins in barley

Haze formation in beer is a trait closely related to beer quality and it is largely affected by the haze active proteins (HAPs) in barley (Hordeum vulgare L.). Up to date, little is known about the genetics of HAPs and relevant genes. In this study, we obtained the beer samples from a Franklin/Yerong double haploid (DH) population and the two parents using micro-malting and micro-brewing, and determined tannin-related HAPs. It was found that there was a wide difference in HAPs among all lines of the DH population, and Yerong had a higher HAPs content than Franklin. Quantitative trait locus (QTL) analysis identified five QTLs associated with HAPs in beer, being located on chromosomes 1HS, 5HL and 6HS, respectively. The loci QHAP1.FrYe-1H and QHAP2.FrYe-1H were overlapped in the short arm of chromosome 1H, and they controlled HAP1 and HAP2, respectively. Moreover, the candidate genes were also predicted based on published whole barley genome sequence and corresponding gene annotations.

Polysaccharides influence on the interaction between tannic acid and haze active proteins in beer

Colloidal instability in beer is mainly caused by interactions between proteins and polyphenols. These two combine producing a visible haze that reduces the physical shelf life of beer. The haze active proteins (HAPs) react with tannic acid (TA); therefore, this reaction provides a way to determine HAP concentrations in beer. Beers also contain a number of constituents that may influence the protein–polyphenol haze formation. We used a response surface methodology to predict the influence of total polysaccharides (TPS) and proteins on beer haze. Experiments were carried out using the Central Composite Design (CCD) methodology. Samples of beer were prepared with variable concentrations of TPS and proteins. TPS concentrations ranged between 1.34 and 2.23gL−1 and proteins concentrations between 0.11 and 0.18gL−1. Results show that the increase in turbidity in response to TPS addition was similar to that in response to protein addition. Our regression analyses indicate a significant dependency and correlation between the observed values and the predicted response values (R2=97.87% and R2[adj]=95.75%). Furthermore, these values indicate that our experimental model can explain 95.75% of the total variation. Therefore, using TA as an indicator of the interaction of TPS with proteins, as commonly done, can lead to considerable errors, since the polysaccharides also react with TA, and this reaction actually causes a considerable increase in turbidity.

INVESTIGATION OF HORDEINS DURING BREWING AND THEIR INFLUENCE ON BEER HAZE BY PROTEOME ANALYSIS

ABSTRACT Colloidal storage haze is one of the principal indicators of beer quality. In this study, the changes of malt hordeins during the brewing process and the roles of malt hordeins in the formation of beer haze were investigated using two-dimensional electrophoresis (2-DE). It was found that most of malt hordeins disappeared during the brewing process and only the few faint hordein spots (about eight spots) were detected in beer haze. Moreover, these spots were found in wort protein and beer haze protein. The complex 2-DE patterns of malt hordein, wort and beer haze displayed a relationship with some chemical modifications during the brewing. These spots were analyzed and identified as B and γ3 hordein and storage protein (Hordeum vulgare) by mass spectrometry. Our results suggested that hordein might be minor haze-active proteins in beer but was a critical factor in the formation of beer haze. PRACTICAL APPLICATION The formation of haze is a serious quality problem that limits the storage life of beer products, an issue that has become increasingly important to brewers. It is conventionally accepted that proline-rich hordeins play a major role in the formation of haze. In this study, it was found that most of the hordeins disappeared and that small amounts of B hordein and γ3 hordein from the malt in beer haze were detected by proteomics analysis. Moreover, this study demonstrated that hordeins in beer were not a key contributor to beer haze formation. To our knowledge, to date, this result in the existing literature of similar studies has not been reported. This study laid the theoretical foundation for further investigations to determine the mechanism of the formation of beer haze. An improved understanding of the impact of brewing on hordeins could potentially provide further scope for optimizing beer quality.

INVESTIGATION OF THE RELATIONSHIP OF MALT PROTEIN AND BEER HAZE BY PROTEOME ANALYSIS

ABSTRACT The changes in malt proteins during the brewing process and the roles of malt proteins in the formation of beer haze were investigated in this study. Coomassie blue stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) revealed that proteins with molecular weights of ∼40, 25–29 and 6.5–17 kDa were the major components of beer haze protein, originating primarily from malt water-soluble protein, partly from hordein. Two-dimensional electrophoresis and mass spectrometry analysis highlighted malt protein modification occurring during the brewing process. It was found that most of the malt hordeins disappeared during the brewing process, except B and γ3 hordein and storage protein (Hordeum vulgare). The results also suggested that the heat- and proteolysis-stable hydrophobic proteins such as barley trypsin inhibitor CMe protein, germin E (H. vulgare) and protein Z might be the important haze-promoting proteins, and that B and γ3 hordein might be the minor haze-active proteins in beer but were a critical factor in the formation of beer haze. PRACTICAL APPLICATIONS Haze stability is of great importance to brewers as it is the first characteristics by which a consumer judges the quality of their beer. It is conventionally accepted that proline-rich hordeins from malt play a major role in the formation of haze. In this study, it was found that B and γ3 hordein might be the minor haze-active proteins, but the heat- and proteolysis-stable hydrophobic proteins such as barley trypsin inhibitor CMe (BTI-CMe) protein, germin E (Hordeum vulgare) and protein Z might be the important haze-promoting proteins. This study made a further confirmation on this issue that nonhordein proteins are regarded as haze-active and laid the theoretical foundation for further investigations to determine the mechanism of the formation of beer haze. An improved understanding of the impact of brewing on malt proteins could potentially provide further scope for optimizing beer quality.

The Effects of Hop-α-Acids and Proline-Specific Endoprotease (PSEP) Treatments on the Foam Quality of Beer

Influences on foam stability and cling were compared by brewing trials investigating beer hopping rate, hopping type and modification of beer protein composition by the inclusion of a proline specific protease (PSEP). The comparison of the NIBEM, Rudin and lacing foam assessment methods with the level of hopping demonstrated the superiority of hydrogenated hop alpha-acids with respect to foam stability and particularly lacing. In addition, the NIBEM and Rudin foam analysis tests appear to respond relatively similarly with respect to hopping rate and hop type, with the NIBEM being somewhat more responsive in terms of foam stability measurments. The PSEP trials suggested that protein composition may only have a subtle effect on foam stability. Although more specific to haze active proteins, PSEP treatment in the small and pilot scale trials generally, but not always, resulted in a minor reduction in foam stability. This effect was not observed in 20 hL pilot and industrial scale beer productions. It was verified that both NIBEM and Rudin were positively influenced by increased levels of foam positive proteins. Although both foam tests were responsive to hopping rate and type, it is suggested that the Rudin foam test is somewhat biased towards foam positive proteins, particularly albuminous foam positive proteins (LTP1 and protein Z4), while in comparison the NIBEM foam test appears somewhat biased towards hordein foam positive proteins.

Dead-End Microfiltration of Rough Nonalcoholic Beer by Different Polymeric Membranes

Clarification of rough nonalcoholic beer using microfiltration as an alternative to conventional filtration with filter aids presents scientific and technical challenges for the brewing industry. An experimental pilot plant was used to evaluate the permeability and selectivity of polymeric membranes in the clarification process. Cellulose acetate (CA) with pore sizes of 0.2, 0.45, 0.8, and 1.2 μm, together with cellulose nitrate (CN), nylon (NY), and polytetrafluoroethylene (PTFE) with a pore size of 0.45 μm, were used at transmembrane pressures (TMP) of 1.0 and 2.0 bar. The data corroborated that the flux values of the CA, CN, and NY membranes were almost the same and reduced drastically, whereas PTFE was not permeable at 1.0 bar and its flux at 2.0 bar increased at the beginning and decreased after reaching a maximum. At both TMP, the CN membrane displayed the highest rejection of suspended particles and haze-active proteins, while retention of polyphenols and color were highest for the NY membrane. The lower selectivity of the PTFE membrane can be attributed to its hydrophobicity and less interaction with the hydrophilic haze-active proteins. The results confirmed that by increasing the pore size of CA membranes the separation factor of the sensitive proteins and suspended particles decreased and permeability increased.

The Effect of Beer pH on Colloidal Stability and Stabilization--A Review and Recent Findings

pH has a pronounced effect on the amount of haze produced in both model systems and beer from the same amounts of haze-active (HA) proteins and HA polyphenols. The greatest haze intensity is seen slightly above pH 4 and varies slightly with alcohol content. This pH promotes removal of HA material during maturation, resulting in better beer colloidal stability. Fining with HA proteins (gelatin or isinglass) or HA polyphenols (e.g., tannic acid) is similarly affected by pH, with the greatest effectiveness near pH 4.3. Factors that influence beer pH are reviewed for their effects on colloidal stability. Under low adsorbent dose conditions, both silica and PVPP are slightly less effective at the pH at which proteins bind polyphenols most strongly. This appears to be due to competition between a stronger protein�polyphenol interaction and binding of HA material to an adsorbent.

Identification of novel haze-active beer proteins by proteome analysis

Colloidal haze reduces beer quality considerably. Four haze samples were analyzed by two-dimensional gel electrophoresis (2DE) in order to identify haze-active proteins. Several protein spots were observed in all of the four haze samples. Using mass spectrometry analysis followed by a database search identified these spots as barley dimeric alpha-amylase inhibitor (BDAI-1), CMb component of tetrameric alpha-amylase inhibitor (CMb) and trypsin inhibitor CMe precursor (CMe). These proteins were considered to be haze-active. Since haze-active proteins are adsorbed by silica gel in the beer filtration process, we eluted proteins adsorbed onto silica gel (PAS) and identified their species. These major PAS were identified as protein Z4, protein Z7 and trypsin/amylase inhibitor pUP13 (TAI), rather than BDAI-1, CMb and CMe. Furthermore, we analyzed proline compositions in the beer proteins, PAS and the haze proteins. Consequently, we found that the proline compositions of PAS were higher (ca. 20 mol%) than those in the beer proteins (ca. 10 mol%), although those of the haze-active proteins such as BDAI-1, CMb and CMe were 6.6–8.7 mol%. Our results suggest that BDAI-1, CMb and CMe are not predominant haze-active proteins, but growth factors of beer colloidal haze.

Turbidimetric Titration of a Haze-Active Polyphenol in Beer

At least two commercial instruments that perform automated turbidimetric titrations are available for determining haze-active (HA) constituents in beer. They have advantages over single-point measurements due to the interactions between beer HA polyphenols, HA proteins, and other beer constituents. Analysis of HA polyphenols is particularly difficult due to their low concentration in beer. Manual turbidimetric titrations with soluble polyvinylpyrrolidone (PVP) were carried out in model systems with a HA polyphenol (tannic acid [TA]), a non-HA polyphenol (epicatechin), a HA protein (gliadin), and a non-HA protein (lysozyme). Increasing concentrations of TA resulted in higher turbidity peaks at higher titrant volumes. Essentially no haze was seen with epicatechin or lysozyme. Gliadin solutions had an initial modest haze that decreased with increasing titrant. The effects of adding fixed amounts of epicatechin, lysozyme, or gliadin to TA were examined. In the presence of epicatechin or lysozyme, the titration endpoints were shifted higher. The endpoints with mixtures of gliadin and TA were similar to those found with TA alone. PVP titration also was applied to samples of unchillproofed beer treated with different amounts of silica or polyvinylpolypyrrolidone and showed responses of the expected magnitudes.

On the Mechanisms of Adsorbent Interactions with Haze-Active Proteins and Polyphenols

Solutions of gliadin and catechin were prepared in pH 4.2 alcoholic buffers. The effects of the hydrogen bond acceptor N,N-dimethylformamide (DMF), the nonpolar solvent dioxane, and sodium chloride (NaCl) on removal of catechin by treatment with polyvinyl polypyrrolidone (PVPP) and on removal of gliadin by silica adsorption were examined. DMF and dioxane greatly inhibited removal of the polyphenol by PVPP, whereas NaCl considerably increased it. It was concluded that PVPP attaches to polyphenols through a combination of hydrogen and hydrophobic bonding. DMF and dioxane also greatly inhibited removal of the protein by silica, whereas NaCl increased it. It was concluded that silica attaches to proteins through a combination of hydrogen and hydrophobic bonding. Polysarcosine, like polyproline, has a tertiary amine structure that also forms haze with polyphenols and was removed by silica. The mechanisms of attachment of haze-active constituents to silica and PVPP are similar to those involved in forming protein-polyphenol haze.

Comparison of Methods for Degassing Beer for Analysis

Replicate beer samples were degassed using ASBC Method Beer-1D (rotary shaker) and the Innlab decarbonyzer. Assessments of alcohol and real extract (RE) were made using an Anton Paar beer analyzer. The results for foam-active (FA) and haze-active (HA) proteins also were compared. RE and alcohol results were significantly lower (P < 0.001) with the decarbonyzer than with the Beer-1D method, indicating more vigorous removal of CO2 and other volatile compounds. The results for HA and FA proteins were not significantly different between the two methods.

Chemical Composition of Clarified Bayberry (Myrica rubraSieb.et Zucc.) Juice Sediment

Clarified bayberry juice turned hazy upon storage at 25 degrees C for 6 months, and the chemical composition of centrifugally separated sediment was analyzed. Bayberry juice haze was mainly protein-tannin haze. The lyophilized sediment contained 20.4 +/- 4.3% of protein, 70.2 +/- 2.6% of total polyphenols, 7.2% of monosaccharides, and 6.7 +/- 0.6% of ash. Amino acid analyses and molecular weight distribution estimation indicated that bayberry proteins were haze-active proteins with a molecular weight less than 8 kDa. Gallic acid, quercetin hexoside, quercetin deoxyhexoside, and quercetin were found in the methanol-dissolved sample, while gallic acid, protocatechuic acid, cyanidin, ellagic acid, and quercetin were detected in the acid-hydrolyzed sample. Ellagic acid was the dominant individual phenolic (9.9 +/- 0.19 g/100 g dry weight, 55.3% of the total amount) in the sediment. Monosaccharides of rhamnose, arabinose, mannose, glucose, and galactose in the sediment were most probably the glycoside moieties of the anthocyanins, flavonols, and elllagitannins. Metal ions of calcium, magnesium, potassium, iron, and copper also indicated the heterogeneous characteristics of the sediment.

Comparison of Methods for Measuring Protein in Beer

Mixtures containing various combinations of haze-active (HA) and non-HA proteins (water soluble hordein and lysozyme, respectively) and HA and non-HA polyphenols (tannic acid and epicatechin) were prepared in buffer model systems. A battery of protein methods (Bradford Coomassie brilliant blue dye binding method [CBB], HA [sensitive] protein, the Bicinchoninic acid [BCA] method, and absorbance at 280 nm) were applied to each sample and the results were compared. The various methods gave quite different responses to the different test compounds. With concentrations of the various substances roughly equivalent to those found in beer, absorbance at 280 nm and BCA suffered strong interference from the polyphenols. The CBB and HA protein methods, on the other hand, responded very little to polyphenols. CBB response was much greater to lysozyme than to hordein. Tannic acid haze induction responded only to HA protein, but exhibited some nonlinearity. Results obtained when these methods are applied to beer must be interpreted with caution.

Electrophoretic method for the identification of a haze-active protein in grape seeds.

A simple, fast, and more selective approach is presented in this study for the identification of haze-active proteins. Grape seed proteins were unfolded by 1% SDS and then interacted with different amounts of tannin at 4 degrees C, followed by gel electrophoresis. It was found that the intensity of the band at 45 kDa was decreased as tannins increased. The amino acid composition of this isolated 45-kDa protein was higher in proline (9.49%) than the average proline content of total grape seed proteins (4.85%). To verify the selectivity of the proposed method, a globular protein (bovine serum albumin, BSA) and a proline-rich protein (gelatin) were selected and used in the model system. As expected, gelatin was removed as it reacted with the increasing added tannins, whereas BSA did not. These results showed that it is possible to identify haze-active proteins by modulating the accessibility of protein to tannins, suggesting this new method can be used by the beverage industry to trouble-shoot haze problems and for quality control.

Application of Immunological Methods to Differentiate between Foam-Positive and Haze-Active Proteins Originating from Malt

Beer foam and storage haze stability are of critical importance to brewers because they are among the first characteristics by which a consumer judges the quality of his or her beer. Key malt-derived, foam-positive proteins, including protein Z4, lipid transfer protein 1 (LTP1), and some members of the hordein storage protein family, interact principally with hop acids to stabilize foam. Silica-mediated beer stabilization procedures remove haze-active protein with high levels of proline (>30%) and glutamine (>30%), suggestive of a hordein origin, without reducing foam stability. Polyclonal antibodies to protein fractions of enriched beer foam proteins and proteins extracted from silica used for beer stabilization were developed. The antibodies were found to measure different malt-derived proteins. The antibodies were subsequently used to select foam-positive and haze-active hordein-recognizing monoclonal antibodies, which enabled the development of enzyme-linked immunosorbent assays (ELISAs). The ELISAs and immunochemical procedures were applied to predict beer foam and haze quality from malt used to brew beer by using pilot- (50 L) and small-scale (0.6–0.8 L) brewing procedures. It is anticipated that the immunochemical methods may be applied to select and manipulate malt for brewing to improve beer foam stability while reducing the likelihood of storage haze formation.