The chemical components in malt associated with haze formation in beer

In bright beer, haze formation is a serious quality problem, which reduces beer storage and shelf life. In this study, haze‐active proteins, alcohol chill haze formation ability, α‐amylase activity, the contents of total polyphenol, protein and its fractions and amino acids were analysed using 23 barley accessions to investigate the relationship between the quality components in the malt and the haze character in beer. The results showed that there were largely genotypic variations for all examined traits among the 23 barley accessions. However, there was no significant correlation between the haze character and α‐amylase activity. All haze characteristics were significantly and positively correlated with total protein content, albumin, globulin and the hordein content, as well as the glutamic acid (glutamine), proline and phenylalanine content, and were not correlated with total polyphenols. A model describing the relationship between the chill haze in the beer and the protein content in the malt was developed. Copyright © 2016 The Institute of Brewing & Distilling     

Beer Polypeptides and Silica Gel Part I. Polypeptides Involved in Haze Formation

Beer contains approximately 500 mg/L protein depending on the brewing procedures employed. This protein is in the form of polypeptides, the majority of which lie within the 10–40 kD size range. Some of these polypeptides are responsible for causing colloidal haze, others enhance foam stability and the remainder appear to have no function in beer except to contribute to mouth‐feel. The polypeptides responsible for haze formation are those that can combine with polyphenols to produce a visible cloudy haze. This is undesirable as it can have a negative effect on the beer's shelf life. One way to reduce this effect is to remove these polypeptides using silica gels. It is important that this removal is selective, and the desirable foam enhancing polypeptides are not removed. Data will be presented to show that beer polypeptides are glycosylated and that silica preferentially adsorbs glycoproteins, particularly those with protein components rich in the amino acid proline. The molecular size and composition of glycoproteins recovered from untreated beer, cooked adjunct, silica exposed to beer and beer aged for one year are presented. Glycoproteins involved in foam, and the apparently functionless polypeptides, will be discussed in a subsequent paper.     

THE REACTIONS BETWEEN POLYPHENOLS AND ALDEHYDES AND THE INFLUENCE OF ACETALDEHYDE ON HAZE FORMATION IN BEER

The reactions and interactions between proteins and polyphenols are, among other phenomena, responsible for haze formation in beer. The participation of aldehydes in the polymerisation of polyphenols is considered. The formation of phenolic Baeyer-type condensation products containing phenolic residues linked by CH₃CH-bridges through reaction with acetaldehyde is possible at the pH of beer (4.0). These and other reactions with acetaldehyde in beer participate in beer haze formation.     

THE USE OF 14C-LABELLED POLYPHENOLS TO STUDY HAZE FORMATION IN BEER

Haze formation in beer is claimed to involve the gradual polymerization of polyphenols and their subsequent reaction with proteins to form insoluble complexes. The mechanism of haze formation has now been studied using ¹⁴C-labelled epicatechin and dimeric catechin. Epicatechin does not polymerize to form dimeric or trimeric polyphenols when beer is stored and is only incorporated into beer haze to a small extent. Some of the epicatechin combines with nitrogen-containing compounds during storage to form soluble complexes. However, dimeric catechin does not form soluble complexes with nitrogen-containing compounds and instead there is a substantial incorporation of this dimer into beer haze. Dimeric polyphenols are important haze precursors which form insoluble complexes with one or more of the polypeptides of beer, probably by an oxidative coupling mechanism.     

Protein changes during malting and brewing with focus on haze and foam formation: a review

Beer is a complex mixture of over 450 constituents and, in addition, it contains macromolecules such as proteins, nucleic acids, polysaccharides, and lipids. In beer, several different protein groups, originating from barley, barley malt, and yeast, are known to influence beer quality. Some of them play a role in foam formation and mouthfeel, and others are known to form haze and have to be precipitated to guarantee haze stability, since turbidity gives a first visual impression of the quality of beer to the consumer. These proteins are derived from the malt used and are influenced, modified, and aggregated throughout the whole malting and brewing process. During malting, barley storage proteins are partially degraded by proteinases into amino acids and peptides that are critical for obtaining high-quality malt and therefore high-quality wort and beer. During mashing, proteins are solubilized and transferred into the produced wort. Throughout wort boiling proteins are glycated and coagulated being possible to separate those coagulated proteins from the wort as hot trub. In fermentation and maturation process, proteins aggregate as well, because of low pH, and can be separated. The understanding of beer protein also requires knowledge about the barley cultivar characteristics on barley/malt proteins, hordeins, protein Z, and LTP1. This review summarizes the protein composition and functions and the changes of malt proteins in beer during the malting and brewing process. Also methods for protein identification are described.     

EFFECT OF VARIOUS POLYPHENOLS ON THE RATE OF HAZE FORMATION IN BEER

The effect of the addition of a range of monomeric, dimeric and polymeric polyphenols to beer on the rate of haze formation has been examined. Monomeric polyphenols had no significant effect on haze formation. The addition to beer of dimeric or polymeric polyphenols caused a large increase in the rate of haze formation.     

A Discussion of Polyphenols in Beer Physical and Flavour Stability

Generally referred to as polyphenols (PPs), beer flavonoids such as the flavan‐3‐ols and their condensed products, the proantho‐cyanidins, represent a class of readily oxidizable compounds capable of hindering or preventing the oxidation of other molecules present in beer. Flavan‐3‐ol and proanthocyanidin capacity to improve oxidative stability has been well established in other food systems, and thus these antioxidants have recently gained significant consideration as potential beer flavour modifiers and/or stabilizers. The duality of their presence in beer is that PPs complex with proteins in the beer matrix to form temporary and permanent hazes. Undesirable physical instability caused by PP‐protein interactions can be resolved via use of adsorptive resins such as polyvinylpyrrolidine. While there is no doubt that polyphenol removal increases beer shelf stability in terms of haze formation, the impact of polyphenol removal on beer flavour remains unresolved. This review discusses the sources, content and impact of polyphenol presence and removal on beer physical and flavour stability.     

Wheat Variety and Barley Malt Properties: Influence on Haze Intensity and Foam Stability of Wheat Beer

Laboratory wheat beers were brewed with different wheat varieties of different protein content (8.7–14.4%) and with five different barley malts, varying in degree of modification (soluble protein: 3.9–6.9%). In a first series of experiments, it was investigated whether wheat positively influences the foam stability, a major characteristic of wheat beers. NIBEM and Rudin (CO₂) foam analyses revealed that the effect of wheat on foam stability depended on the barley malt used for brewing. When using malt with high foaming potential, wheat exerts a negative influence. However, wheat added to over‐modified malt with less foam promoting factors, ameliorates beer foaming characteristics proving that wheat contains foam active compounds. In addition, Rudin (N₂) values suggested that wheat positively influences foam stability by decreasing liquid drainage, probably caused by a higher beer viscosity and/or a finer foam bubble size distribution. Furthermore, the haze in wheat beers, which is another important quality characteristic of these beers, was investigated. Permanent haze readings of the 40% wheat beers were lower than 1.5 EBC haze units. For 20% wheat beers, an inverse relation between the permanent haze (9.4–19.3 EBC haze units) and the protein content of the wheat was established. The barley malt used for brewing also influenced permanent haze readings. A positive correlation between the modification degree of the malt and the permanent haze intensity was found. It was concluded that the choice of raw materials for wheat beer brewing considerably influences the visual properties of the beer.     

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 Å. Comparisons have been made with proteins of known size and structure such as human and bovine serum albumins (HSA and BSA).     

HAZE- AND FOAM-FORMING SUBSTANCES IN BEER

A study of recent literature on haze- and foam-forming substances in beer suggests the following picture: Traces of true protein, apparently above 150,000 in molecular weight (MW), exist in beer. Apart from these, the main complex nitrogenous substances are present in compounds in the 5,000–70,000 MW region. Nevertheless, these are not proteins, but amino-acid complexes (proteoses) coupled with non-nitrogenous substances. As is well known, haze and haze precursors are proteoses coupled with polyphenols. These two species can be joined by hydrogen bonds to give compounds which are readily dissociated. However, a much firmer, covalent, linkage can be formed. This type of linkage arises from oxidation of polyphenols to quinones, which in turn can oxidize and couple with several groupings on proteoses. One of the most probable points of attack is the sulphydryl group of cystine, and the relatively high percentage of cystine in haze indicates that this occurs. It seems likely that this reaction, to form acidic complexes, occurs mainly in the initial stages of mashing and boiling. In beer, further coupling between pairs or triplets of polyphenolic or quinolic groupings can take the products into the very high molecular weight region of hazes. Correspondingly, the foam-enhancing proteoses tend to be lower in cystine content and to show less combination with polyphenols. However, they too are largely in the form of complexes and the evidence suggests that here the proteoses are mainly combined with glucose polymers, a reaction which takes place during kilning and possibly during boiling. These complexes are less acidic than the polyphenolic ones and tend to be rather lower in molecular weight. Suggestions arising from these concepts may be helpful in controlling the haze and foam potentialities of beers.     

Beer Polypeptides and Silica Gel Part II. Polypeptides Involved in Foam Formation

Beer contains approximately 500 mg/L protein depending on the brewing procedures employed. This protein is in the form of polypeptides, the majority of which lie within the 10–40 kD size range. Some of these polypeptides are responsible for causing colloidal haze, some enhance foam stability and the remainder appear to have no function in beer except to contribute to mouth‐feel. Those polypeptides involved in haze formation were described in a previous paper. To continue these studies, data is presented to show that foam polypeptides are highly glycosylated and that purified foam glycoprotein contains low levels of the amino acid proline. As silica preferentially adsorbs polypeptides rich in proline, it is unlikely to adsorb this material and damage foam stability. The molecular sizes and composition of glycoproteins recovered from untreated beer, purified foam and beer from which the foam component has been removed are presented. These fractions include the polypeptides responsible for foam stability and those that appear to have no role in physical stability.     

Preventing protein haze in bottled white wine

Slow denaturation of wine proteins is thought to lead to protein aggregation, flocculation into a hazy suspension and formation of precipitates. The majority of wine proteins responsible for haze are grape‐derived, have low isoelectric points and molecular weight. They are grape pathogenesis‐related (PR) proteins that are expressed throughout the ripening period post véraison, and are highly resistant to low pH and enzymatic or non‐enzymatic proteolysis. Protein levels in un‐fined white wine differ by variety and range up to 300 mg/L. Infection with some common grapevine pathogens or skin contact, such as occurs during transport of mechanically harvested fruit, results in enhanced concentrations of some PR proteins in juice and wine. Oenological control of protein instability is achieved through adsorption of wine proteins onto bentonite. The adsorption of proteins onto bentonite occurs within several minutes, suggesting that a continuous contacting process could be developed. The addition of proteolytic enzyme during short term heat exposure, to induce PR protein denaturation, showed promise as an alternative to bentonite fining. The addition of haze‐protective factors, yeast mannoproteins, to wines results in decreased particle size of haze, probably by competition with wine proteins for other non‐proteinaceous wine components required for the formation of large insoluble aggregations of protein. Other wine components likely to influence haze formation are ethanol concentration, pH, metal ions and phenolic compounds.     

Characterisation of the Colloidal Haze in Commercial and Pilot Scale Belgian White Beers

Hazes from four commercial Belgian white beers were studied. Proteins (7–74%) and polyphenols (1.1–7.7%) were the major components, although hazes with a high content of glucose polymers also occurred. The glucose polymers are mainly starch or degraded starch (9–65%) and only to a minor extent β‐D‐glucan (0‐0.5%). Arabinoxylans and metal ions (mainly calcium) were minor constituents. The hazes also contained arabinogalactan‐peptide in minor quantities due to the large level of wheat used in the grist. A high mannose content (0.9‐3.3%), originating from yeast cell wall mannans, was also found in the hazes. Characterisation of the haze of three pilot scale brews with different production processes showed that the composition of the hazes was strongly influenced by the process employed. Hazes can be enriched in proteins or starch/dextrins by changing the raw materials and/or the brewing process.     

Evaluating haze formation in flavoured lager beers using a range of forcing methods

Haze formation is a quality problem that can affect a wide range of drinks for a number of different reasons. Although well‐characterized in traditional beers, the emergence of novel beer‐based beverages has led to the potential for chemical interactions that can result in the precipitation of haze material. In this study, the haze formation in 19 commercially available flavoured lager beers was investigated. The chemical parameters of each of the drinks were measured and haze formation was induced through three forcing tests, developed for use in both the beer and wine industries, as well as using a novel forcing test, designed for this study. Several of the products were particularly susceptible to a number of haze forcing tests and the EBC temperature cycling and the Wine Forcing (70°C) tests were found to generate the most forced haze (measured at 90 and 13° scatter angles, respectively) in these products. There was no overall correlation between the drinks' chemical parameters and their susceptibility to haze formation. However, a weak, but statistically significant, correlation (p  = 0.044) was observed between the concentration of polyphenols in the products and the presence of haze measured by 90° scatter angle. Copyright © 2017 The Institute of Brewing & Distilling     

The Role of Malt and Hop Polyphenols in Beer Quality,Flavour and Haze Stability

Pilot‐scale brewing trials of a 12°P pale lager beer were conducted to look at the effect of a modified dose of hop and malt polyphenols on haze, flavour quality, and stability. Results confirmed that malt polyphenols, and particularly hop polyphenols, in the course of wort boiling, improved reducing activity values and the carbonyl content in fresh and stored beers. Hop polyphenols significantly increased reducing activity and decreased the formation of carbonyls (TBA value) in fresh and stored beer. Reduced content of malt polyphenols, combined with the use of hop CO₂ extract, caused an increase in the TBA value in beer. PVPP stabilized beers tended to be lower in reducing activity. Both malt and hop polyphenols affected the intensity of “harsh taste” in fresh beers and a significant influence from PVPP stabilization of beer was not observed. The staling degree of forced‐aged beers depended on the polyphenol content in the brewhouse. Both hop and malt polyphenols had a positive impact on flavour stability. PVPP treatment of beer had a positive effect on the flavour stability of heat‐aged beers. Polyphenols, especially hop polyphenols, slowed down flavour deterioration during the nine month storage period, but the primary effect was seen during the first four months of storage. Storage trials did not show any unambiguous effects for PVPP stabilization on beer flavour stability. Results confirmed the negative impact of malt and hop polyphenols on haze stability, and PVPP stabilization minimized differences in shelf life prediction values between beers prepared with the modified dose of polyphenols.     

啤酒中蛋白质及其测定方法研究进展

啤酒中蛋白质对啤酒品质的各方面有着很大的影响,尤其是对啤酒泡沫和浑浊的形成.本文在叙述啤酒泡沫和浑浊蛋白质性质的基础上探讨了啤酒中蛋白质的测定方法,给酿造者提供选择合适的方法来测定和控制啤酒中的蛋白质,以此来提高啤酒的品质.     

国外啤酒混浊及其预测方法

啤酒是一种成分复杂、稳定性不强的胶体溶液,在贮存过程中易产生混浊沉淀现象。啤酒中多种物质均能引起混浊,最觉的是多酚,蛋白质复合引起的混浊。对国外在混浊类型、蛋白多酚混浊、混浊敏感蛋白、混浊敏感多酚、蛋白多酚作用模式、保质期的新型方法６个方面的最新研究进行论述和介绍。     

Analysis of total polyphenols,bitterness and haze in pale and dark lager beers produced under different mashing and boiling conditions

Malting and brewing processes should be performed under process conditions in a way that minimizes beer bitterness, maximizes polyphenol content and reduces the amount of raw materials ending up in solution in the form of hazes, particles and precipitates. This work examined the influence of different mashing temperature conditions and boiling procedures on the total polyphenol content, bitterness and haze of pale and dark lager beers produced on an industrial scale. Two hop types (hop pellets and/or hop extract) and different hop varieties (Hallertau Magnum, Styrian Goldings, Saaz, Aurora and Sladek) were utilized with varying times of hop addition into the wort. Measurements of total polyphenols, colour, bitterness, alcohol content, CO₂ and pH were carried out on the beer samples. Results showed that pale lager beers had a lower total polyphenol concentration (110–179 mg/L) than dark beers (230–260 mg/L). Using hop extracts instead of hop pellets led to a lower total polyphenol concentration and to less beer foam creation. The change in the proteolytic temperature during mashing only had an influence on the total polyphenol content in the pale lager beer hopped with the pellets. Conducting proteolysis over a 20 min period led to a haze increase in all of the beers produced. In the dark beer, the haze was substantial after just 10 min at 52°C. Copyright © 2015 The Institute of Brewing & Distilling     

啤酒的非生物混浊和稳定化处理

该文介绍了啤酒形成非生物混浊和沉淀的机理，混浊活性蛋白质和混浊活性多酚相互作用的模式。文中还对啤酒中添加吸附剂、澄清剂和蛋白酶等稳定化处理方法的作用机理进行了探讨，比较了几种稳定化处理物质的特点。     

EUROPEAN BREWERY CONVENTION: HAZE AND FOAM GROUP. TOWARDS THE CHEMICAL PREDICTION OF SHELF LIFE

Rapid chemical methods are described for assesssing in beer the levels of “oxidizable polyphenols” and “sensitive proteins,” both of which influence non-biological shelf life. The method for oxidizable polyphenols depends on the formation of an insoluble complex with cinchonine sulphate. Freshly processed beers form only a slight haze on the addition of cinchonine sulphate, but the haze produced is greatly increased if the beers are previously oxidized. For beers stabilized by a process involving reduction of the polyphenol content, it is found that the rate of haze formation is then directly related to shelf life and is inversely proportional to it.