Haze-active Protein Research Articles

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.

Absorbability of Active Carbon Influencing the Haze in Clarified Apple Juice

The relationship of adsorption characteristic about active carbon and the haze in clarified apple juice was investigated. Particle size and addition and adsorption temperature of active carbon were studied.0.18mm particle size with the biggest specific surface area and 0.075mm particle size with the widest microspore distribution of active carbon lowered the turbidity and contents of haze active protein and that of haze active phenolics. Contents of haze active protein and that of haze active phenolics in apple juice were decreased by increasing the adding amounts of active carbon. High temperature of adsorption showed the positive relationship with volume of haze active phenolic and the negative relationship with volume of haze active protein. Particle size of active carbon with 0.18mm or 0.075mm and addition of 15g/kg and adsorption temperature with 50°C are chosen in reducing the haze in apple juice. Absorbability of active carbon has great effects on the haze of apple juice.

INFLUENCE OF ENZYMOLYSIS‐ADSORPTION INTERACTION ON THE SECONDARY HAZE FORMATION IN APPLE JUICE

ABSTRACTThe secondary haze formation in clarified apple juice produced under enzymolysis and adsorption processing conditions was studied in order to control the haze formation by monitoring the processing conditions of enzymolysis and adsorption. The turbidity and content of haze‐active protein and content of haze‐active phenolics of the apple juice were monitored at intervals. Haze formation could be prevented if optimum enzymatic time and suitable time of adding activated charcoal are adopted. Enzymatic time contributed to the adsorption and the time of adding activated charcoal during the enzymatic treatment also had effects on the enzymolysis. Varying the time used for enzymolysis of haze had a marked effect on the turbidity and content of haze active protein of apple juice and had no effects on the content of haze‐active polyphenols. Adsorptive treatment given prior to complete enzymolysis did not appear to prevent the haze formation. Adsorptive treatment had more effects on the turbidity and the content of haze active protein and that of haze active polyphenols.PRACTICAL APPLICATIONSThe processes of enzymolysis and adsorption can be used for the production of clarified apple juice to control the haze formation. The quality of clarified apple juice with enzymatic treatment combining with adsorptive treatment by activated charcoal is much superior to the product using the convectional method. If activated charcoal is added to the enzymatic process at optimum time, the enzymolysis and adsorption can be used successfully for the production of clarified apple juice.

Allelic Diversity of a Beer Haze Active Protein Gene in Cultivated and Tibetan Wild Barley and Development of Allelic Specific Markers

The formation of haze is a serious quality problem in beer production. It has been shown that the use of silica elute (SE)-ve malt (absence of molecular weight (MW) ∼14000 Da) for brewing can improve haze stability in the resultant beer, and the protein was identified as a barley trypsin inhibitor of the chloroform/methanol type (BTI-CMe). The objectives of this study were to determine (1) the allelic diversity of the gene controlling BTI-CMe in cultivated and Tibetan wild barley and (2) allele-specific (AS) markers for screening SE protein type. A survey of 172 Tibetan annual wild barley accessions and 71 cultivated barley genotypes was conducted, and 104 wild accessions and 35 cultivated genotypes were identified as SE+ve and 68 wild accessions and 36 cultivated genotypes as SE-ve. The allelic diversity of the gene controlling BTI-CMe was investigated by cloning, alignment, and association analysis. It was found that there were significant differences between the SE+ve and SE-ve types in single-nucleotide polymorphisms at 234 (SNP(234)), SNP(313), and SNP(385.) Furthermore, two sets of AS markers were developed to screen SE protein type based on SNP(313). AS-PCR had results very similar to those obtained by immunoblot method. Mapping analysis showed that the gene controlling the MW∼14 kDa band was located on the short arm of chromosome 3H, at the position of marker BPB-0527 (33.302 cM) in the Franklin/Yerong DH population.

Effect of ultrafiltration and fining adsorbents on the clarification of green tea

Ultrafiltration, silica gel and chitosan treatment methods were investigated to study the clarification effect on the green tea infusion. The results showed that ultrafiltration reduced the protein and polyphenol content, as well as color of the tea infusion. The silica gel and chitosan treatment were effective methods to clarify green tea infusion; they decreased the haze-active protein significantly, without much effect on the polyphenol. The silica gel and chitosan treatment also reduced the proline content by 18.65% and 23.79%, and histidine amino acid by 90.97%, and 90.31%, respectively. After the silica and chitosan treatment, the protein did not decrease significantly compared to the raw (untreated) infusion, while ultrafiltration treatment reduced the protein content significantly from 11.36 to 5.85 mg/100 mL with the 10 kDa membrane. Transmittance of silica gel and chitosan treated infusion was above 95.0% and 99.0%, respectively; and decreased slowly under prolonged storage time. The chitosan and silica treatment decreased the greenness and yellowness of the green tea infusion.

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.

The identification of a barley haze active protein that influences beer haze stability: The genetic basis of a barley malt haze active protein

In bright beers, the formation of haze is a serious quality problem, which places limitations on the storage life of the product. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) immunoblot analysis using an antiserum that was raised against a silica eluate (SE) protein fraction (obtained from silica gel, used for the colloidal stabilisation of beer), detected a range of protein bands in barley, malt, beer and haze. A polymorphism was observed in which some barley varieties contained a molecular weight (MW) ∼12,000 band (SE +ve) while in other varieties this band was absent (SE −ve). A survey of 219 Australian and international barley varieties, including a comprehensive selection of current and past malting varieties, identified 181 varieties as SE +ve, and 38 varieties as SE −ve. Previous pilot brewing trials demonstrated that SE −ve varieties are desirable as the beer brewed from the malt of these varieties formed less haze after accelerated ageing than beers brewed using SE +ve malt varieties. The genetic basis for the absence of the SE protein was conferred by a recessive allele at a single locus. Interval mapping analysis showed that the MW ∼12,000 band mapped to the short arm of chromosome 3H.

The identification of a barley haze active protein that influences beer haze stability: Cloning and characterisation of the barley SE protein as a barley trypsin inhibitor of the chloroform/methanol type

Previous pilot brewing trials have demonstrated that in the absence of a molecular weight (MW) ∼12,000 (barley silica eluate (SE) protein), the beer brewed from the malt of these SE −ve varieties formed less haze after accelerated ageing than beers brewed using SE +ve malt varieties. The previously described SE protein was characterised using comparative two-dimensional (2-D) gel electrophoresis immunoblots of barley seed extracts from both SE +ve and SE −ve varieties. The SE protein spot identified was excised and its partial sequence determined, after in-gel cleavage using trypsin and separation of the resulting fragments by reversed-phase high performance liquid chromatography (HPLC). N-terminal sequence analysis of the tryptic peptides from SE +ve and SE −ve varieties identified the SE protein as the barley trypsin inhibitor-CMe precursor (BTI-CMe). The mature BTI-CMe protein is 13.3 kDa and its functional gene is located on chromosome 3H. Cloning of the BTI-CMe protein demonstrated that both SE −ve and SE +ve barley varieties contain a BTI-CMe protein family member that is similar but has a consistently different sequence, primarily in the last 30 amino acid residues of their C-termini.

The Effect of Beer pH on Colloidal Stabilization with Adsorbents

Solutions of the haze-active (HA) protein gliadin were prepared in buffer at different pH's and treated with silica. Analysis using the bicinchoninic acid and HA protein methods produced similar patterns, with greater protein removal near pH 3.5 and 5.0. Portions of an unchillproofed beer were adjusted to different pH's, treated with silica, and subjected to HA and foam-active (FA) protein analyses. The HA protein results showed greater protein removal near pH 3.8 and least removal near pH 4.4. FA protein was hardly affected, although slightly more was removed near pH 4.4. Tannic acid (TA) and catechin were each combined with soluble polyvinylpyrrolidone in buffer solutions with different pH's, and turbidity was determined after incubation. TA formed maximum haze near pH 4, while catechin formed more haze above pH 5. Portions of unchillproofed beer were adjusted in pH and treated with polyvinylpolypyrrolidone (PVPP). The treatment pH had essentially no effect on total polyphenol removal. HA polyphenols were removed to a greater extent near pH 3.8 and 4.6, with lower effectiveness at intermediate pH's. The colloidal stabilization performance of both silica and PVPP was better at pH's near the extremes of the beer pH range. This may account for difficulties encountered in stabilizing some beers.

Comparison of Methods for Measuring Polyphenols in Beer

Mixtures containing various combinations of haze-active (HA) and non-HA polyphenols (tannic acid and epicatechin, respectively) and HA and non-HA proteins (water soluble hordein and lysozyme, respectively) were prepared in buffer model systems. A battery of polyphenol methods (the Folin-Ciocalteu method, the international method for total polyphenols in beer, and the HA polyphenol method) were applied to each sample and the results were compared. The various methods gave quite different responses to the different test compounds. The best measure of the polyphenols involved in astringency and haze formation was obtained with an improved HA polyphenol method. Results of this method are, however, likely to be affected by endogenous HA protein.

Haze formation in beverages

The most frequent cause of hazes in beverages is due to protein–polyphenol interaction. Proteins that bind polyphenols contain proline, and the more proline, the greater the haze-forming activity. Polyphenols that cause haze have at least two sites that can bind to proteins; this enables them to cross-link proteins together and results in insoluble particles that scatter light. Protein–polyphenol interaction is non-covalent and, at least initially, reversible. The size of haze particles and the haze intensity are both affected by the ratio of haze-active (HA) polyphenol to HA protein. Alcohol content and pH also exert strong influences. Particle size in a model system does not vary smoothly as conditions change; rather shifts in the proportions of particles of a few discrete sizes are seen. Haze development occurs over time in packaged products. In clear beverages a treatment (fining, adsorption or ultrafiltration) is normally applied to reduce the concentration of either HA protein or HA polyphenol in order to delay the onset of haze formation beyond the intended product shelf life. A nephelometer observation gives a good indication of the amount of haze people can perceive.

Haze Formation in Model Beer Systems

The interaction of a haze-active protein (gliadin) and a haze-active polyphenol (tannic acid) was studied in a model beer system in order to investigate the principle mechanisms of haze formation at low temperatures. Low concentrations (g/L) of tannic acid, high concentrations of gliadin, and comparatively high temperatures lead to maximum haze values. When considered on a molar basis, the greatest haze levels are displayed at an approximate 1:1 equivalence of polyphenol and protein. The greater part of haze formation was completed within 0.5 h, irrespective of the concentration of gliadin, the concentration of tannic acid, and the temperature of the model solution.

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.

Characterization of haze-active proteins in apple juice.

The nature of the haze-active protein (HAP) in apple juice was investigated. Heat treatment removed protein indiscriminately while polyvinylpolypyrrolidone (PVPP) treatment was fairly specific for proteins of 15 and 28 kDa. Presumably, the PVPP bound to polyphenols, which in turn were complexed with protein. Three candidate apple HAPs were isolated. Two were extracted from juice with acetone and fractionated by hydrophobic interaction chromatography and solid phase extraction with C18 (HAP I) or SAX (HAP II) material. Hydroxyproline-rich protein was extracted from apple tissue (HAP III). The order of haze formation with tannic acid was gliadin > HAP III > HAP II > HAP I > bovine serum albumin, which shows increasing haze formation with increasing proline content. The sizes of HAP I, II, and III were 28, 15, and 12 kDa; the first two corresponded to the sizes of proteins removed by PVPP treatment and are involved in juice haze formation.

Beer Fluorescence and the Isolation, Characterisation and Silica Adsorption of Haze-Active Beer Proteins

The fluorescence spectra and lifetimes of diluted beer have been explored and found not to report on protein removal either by silica or tannic acid, nor polyphenol uptake by PVPP. Comparing the fluorescence spectra of beer with that of tea and hops, it seems that proteins, complex polyphenols and iso-α-acids can contribute to the intrinsic fluorescence of beer, although the contribution from polyphenols must be minimal since treatment with PVPP does not dramatically change the background fluorescence. To eliminate the problem of background fluorescence haze-active protein was isolated. Steady-state and time-resolved fluorescence techniques were used to characterise these and to monitor their uptake by different silica gels as a function of pH. Heat treated large pore volume, small surface area silicas were the more effective adsorbers for the proteins under study, with pH 4 being optimum. Using both intrinsic amino acid fluorescence and the extrinsic fluorophore fluorescamine, the time-resolved fluorescence anisotropy has been measured and the radius of the isolated haze protein found to be ∼ 35 A. Comparisons have been made with proteins of known size and structure such as human and bovine serum albumins (HSA and BSA).

Development of a Method for Assessing Haze-Active Protein in Beer by Dye Binding

Fifteen candidate dyes were combined with the haze-active (HA) protein gliadin in a search for one that would produce a chromatic shift and form the basis of an analytical method for HA protein in beer. Two dyes, alizarin red S and nuclear fast red, produced shifts in the UV region, and two, bromopyrogallol red (BPR) and plasmocorinth B, produced shifts in the visible range. The responses of these four dyes to proteins with a range of haze-forming activity were compared. BPR was selected for method development. A statistical experiment design and response surface modeling were used to determine optimum conditions. BPR produced a linear response to gliadin in buffer, but it suffered interference in the beer matrix. Pretreatment of beer with a C18 cartridge and centrifugal ultrafiltration removed the interference. Measurements of HA protein via the BPR method and by haze induction with tannic acid were performed on unchillproofed beer samples treated with varying levels of silica gel, bentonite, and polyvinylpolypyrrolidone. The results showed agreement in the patterns but some differences in the levels. It appears that the BPR method gives a different perspective on beer haze stability that should not be influenced by the level of endogenous polyphenol.

Apple Cultivar and Maturity Affect Haze‐Active Protein and Haze‐Active Polyphenol Concentrations in Juice

ABSTRACT:Juices were prepared from single apple cultivars in 3 crop years. The levels of haze‐active (HA) protein and HA polyphenol were much more similar in 1997 and 1998 than in 1996. Fuji had by far the highest HA protein while Spartan, Jonagold, and Monroe were intermediate. HA polyphenol levels were less consistent, but Empire, Liberty, and Monroe were low, and Jonamac, Jonagold, Golden Delicious, Cortland, and Ida Red were intermediate. The highest HA polyphenol was found in Rome. During fruit maturation, HA protein tended to increase while HA polyphenol declined. This led to a distinct tendency toward a lower HA polyphenol/HA protein ratio with time.

Effect of Protein-Polyphenol Ratio on the Size of Haze Particles

Various concentrations of the haze-active (HA) protein gliadin and the HA polyphenol tannic acid were combined in pH 4.5 buffer. The resulting hazes were measured by light scattering, and their particle size distribution patterns were examined with a laser diffraction particle size analyzer. The shape of the haze intensity response generally followed that seen in previous experiments, but haze intensity was noticeably greater with two particular gliadin-tannic acid ratios (2:1 and 5:1, w/w) than with other combinations. The size of the particles varied with the protein-polyphenol ratio, as previously hypothesized. In general, larger particles were seen with intermediate ratios, while both higher and lower proportions resulted in smaller particles. The data expressed as volume percent were highly quantized—essentially, only three particle sizes were seen. The proportions of these varied as the amounts of protein and polyphenol changed. The contributions of particles of each size to the haze intensity in different samples were estimated. The results suggested that the particles that contributed the most haze were typically in a narrow band, with diameters in the range of 10–20 μm. Variations in particle size induced by normal brewing practices are expected to have an impact on fining and filtration performance.