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.     

PHENOLIC CONSTITUENTS OF BEER AND BREWING MATERIALS. II. THE ROLE OF POLYPHENOLS IN THE FORMATION OF NON-BIOLOGICAL HAZE

Simple and polymeric polyphenols differ in the way in which they influence the development of chill and permanent hazes in bottled beer. The addition of simple anthocyanogens to beer causes a marked increase in the rate of haze formation whereas added polymeric materials induce immediate and intense turbidities. Beer anthocyanogens constitute a class of materials ranging in complexity from simple compounds to heterogeneous polymers which sometimes include polyphenolic units that are incapable of yielding anthocyanidins with acid. The extent to which the various anthocyanogens present in beer are involved in haze formation is not reflected by their contributions to the overall anthocyanogen value of the beer. Haze-inducing polymers which contain no anthocyanogen residues may also be present in beer. The nature and origin of polyphenolic substances in beer and the methods by which they may polymerize to give immediate haze precursors are discussed.     

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.     

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 ESTIMATION AND SIGNIFICANCE OF CATECHIN AND DIMERIC CATECHIN IN BEER

A gas-liquid chromatographic method for the estimation of monomeric and dimeric catechin in beer is described. This method has been used to estimate the concentrations of these compounds in a number of beers, which were found to contain 0·5 to 8·0 ppm of monomeric catechin and up to 22·0 ppm of dimeric catechin. Beers bottled with a high level of air in the headspace can have a long shelf-life providing the level of dimeric polyphenols is low. The shelf-lives of beers which contain high concentrations of dimers are very sensitive to the levels of air in the headspace. At the levels found, monomeric catechin has no significant effect on haze whether or not headspace air is present.     

Turbidity and Haze Formation in Beer — Insights and Overview

Beer is a complex mixture of over 450 constituents. In addition, it contains macromolecules such as proteins, nucleic acids, polysaccharides and lipids. Proteins influence the entire brewing process with regard to enzymes, which degrade starch, β‐glucans and proteins; with protein‐protein linkages that stabilize foam and are responsible for mouthfeel and flavour stability; and in combination with polyphenols, thought to form haze. With this complexity, problems in processability are as various as the constituents. Several substances in beer are responsible for haze formation. Organic components such as proteins, polyphenols and carbohydrates (α‐glucans, β‐glucans) are known to form haze. In addition, inorganic particles such as filter aids and label remains can cause increased turbidity. In this article only non‐microbiological induced hazes are described. Many studies have been conducted on the identification of haze and foam active components in beer. Hence the aim of this work was to survey the different possibilities of haze formation and for haze identification. A summary is provided on methods for haze identification including dyeing methods, microscopic analyses and size exclusion chromatography.     

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.     

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 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     

ANALYSIS COMMITTEE OF THE EUROPEAN BREWERY CONVENTION THE MEASUREMENT OF TOTAL POLYPHENOLS IN WORTS AND BEERS

A modification of the De Clerck & Jerumanis method for total polyphenols in beer is recommended for use in studying their relationship to haze in general and to the action of adsorbents in particular.     

啤酒中的多酚物质及其作用

啤酒中的多酚物质是一类重要化合物，其在啤酒的非生物稳定性、风味、保鲜、色泽、溶氧等方面起重要作用，近年来成为人们研究的热点。随着啤酒生产和市场竞争日趋激烈，生产企业要在竞争中立于不败之地，首要是提高啤酒质量，增强啤酒的稳定性，很多研究结果证实了多酚物质在啤酒中的作用。综述了啤酒中主要多酚物质的作用，检测方法的研究情况等。     

Prediction of Colloidal Stability of Highly Stabilized Beers by a Modified Chapon Tannoid Content Test

The tannoid content test, introduced by Chapon, is accepted as a recommended MEBAK analytical method for the detection of haze‐forming polyphenols in beer and other intermediates. Under certain conditions, the results can be used directly for predicting the colloidal stability of beer. Its disadvantage is that it does not provide detectable results for beers stabilized by high doses of polyvinylpolypyrrolidone (PVPP). A test modification is proposed that increases the test sensitivity and gives non‐zero results, even for highly stabilized samples that are not measurable by the standard test. The modification is based on adding saturated ammonium sulphate solution (SASS) to the sample to reach 9% saturation (0.5 mL of SASS to 5 mL sample volume) before the actual test performance. The results of the modified test can simply be converted to results obtained by the standard method. Tannoid content correlates linearly with the content of haze‐forming polyphenols and with chill haze produced after defined accelerated aging. In a set of beer samples of one brand having different degrees of stabilization, a high correlation was determined between test results and long‐term colloidal stability. The modified test can be used as a rapid and simple test for checking the stabilization process. It is particularly suitable for Pilsner type beer of long (more than 6 months) colloidal stability controlled by stabilization on the side of polyphenols.     

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     

IDENTIFICATION OF PROANTHOCYANIDINS IN BEER AND THEIR DIRECT MEASUREMENT WITH A DUAL ELECTRODE ELECTROCHEMICAL DETECTOR

A sensitive and reliable method was developed for the measurement of simple flavanols in beer by direct-injection High Performance Liquid Chromatography using dual-electrode electrochemical detection. When operated in the amperometric mode the detector was extremely sensitive for readily-oxidized substances such as polyphenols, so that sample preparation was minimal. The dimeric flavanols in a commercial beer were positively identified as procyanidin B3 and prodelphinidin B3. Tentative identifications for three trimeric proanthocyanidins were made also. Collectively, monomeric, dimeric and trimeric flavanols accounted for only about 4% of the colorimetrically determined total polyphenol content, and about 35% of the total flavanol content of a non-haze stabilized beer. The improved chromatographic method was applied to measuring the decreases in simple flavanols caused by treatment of beer with polyvinylpolypyrrolidone.     

CONTENTS OF SIMPLE,POLYMERIC AND COMPLEXED FLAVANOLS IN WORTS AND BEERS AND THEIR RELATIONSHIP TO HAZE FORMATION

The highest concentrations of complexed and polymeric flavanols were found in the early runnings from a small-scale mash tun. Formation of break during and after boiling was accompanied by the removal of flavanols from solution, especially the complexed and polymeric fractions. Variations in boiling procedure affected the amount of break formed, the amount of residual soluble flavanols and the colloidal stability of the resultant worts. Colloidal stability in beer was influenced by the contents of simple flavanols and the availability of air during storage. Dimeric and trimeric flavanols seemed especially susceptible to oxidation which implied an important role in haze formation. Flavanols found in commercial beers were mainly monomeric and complexed forms.     

THE INTRINSIC INFLUENCE OF CATECHINS AND PROCYANIDINS ON BEER HAZE FORMATION

Different concentrations of catechins and procyanidins were added to an all-malt Pilsner beer brewed from proanthocyanidin-free malt (ant 13·13 × Rupal) and tannin-free hop extract in order to evaluate their intrinsic role in beer haze formation, i.e. in the absence of malt and/or hop flavanoids. The molar tanning capacities of flavanoids depend upon their degree of polymerisation. Procyanidin B6 was much more haze active than procyanidin B3. Both immediate haze and formation of haze after 150 days of storage at room temperature was in general linearily related to the added concentration of phenolics.     

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。     

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.     

Application of a hop by‐product in brewing: reduction in the level of haze‐active prolamines and improved antioxidant properties of the beer

After supercritical CO₂ extraction of hops or formation of lupulin‐enriched pellets (type 45), polyphenol‐rich by‐products remain. These have some industrial and agricultural applications (preparation of polyphenol extracts and in the production of low‐bitterness beers). In this work, both materials were used in pilot‐scale brewing experiments for the production of beer enriched with polyphenols. Samples of beer made from these residues underwent analyses for haze‐active prolamines, total polyphenols, phenolic monomers, antioxidant capacity, bitterness and colour. An almost identical decrease in the content (from hopped wort to final beer) of the prolamines of approximately 30% was detected in standard beer as well as in the beer prepared from the hop residue (HR 1) after supercritical CO₂ extraction. In contrast, the level of prolamines was reduced by more than 50% in the beer following addition of the hop residue (HR 2) after production of lupulin enriched pellets. Both hop materials contributed to an increase in the antioxidant capacity of the final beer by 21.5 and 11.9%, respectively. Another part of this work was aimed at the investigation of colour and bitterness changes in beers made ??using hop by‐products. Addition of the material HR 2 into the wort caused an increase in colour and in bitterness of the matured beer by 20 and 11% rel., respectively. Copyright © 2014 The Institute of Brewing & Distilling     

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