INTERACTIONS OF PROTEOSES AND POLYPHENOLS IN WORTS,BEERS AND MODEL SYSTEMS

Four high-molecular weight proteose fractions from beer were precipitated to varying extents by four different tannin fractions, as shown turbidimetrically in a simple assay. According to this test the most acidic proteose fraction reacted least with all four tannins and the formation of insoluble proteose-tannin complexes was strongly dependent on pH. Moreover, tannins obtained from hops displayed greater tanning power with beer proteoses than did either a tannin isolated directly from barley or one prepared by aerial oxidation of barley prodelphinidin B3. The different reactivities of tannins to proteoses, however, did not correlate well with their reactivities to cinchonine sulphate (CS). Whereas the amounts of break precipitated on boiling a wort moderately were related to the amounts of hop tannins added, barley flavanols had little effect on break formation. In beer, about 50% of the proteoses were of low molecular weight (< 10,000 Daltons) and some were complexed with flavanols. In contrast, isolated fractions of high molecular weight beer proteoses (MW > 10,000 Daltons) were not associated with flavanols. The most acidic of these proteoses were the least stable in solution, being precipitated from beer by down-shifts in pH. The tanning power of unstabilised beer as measured by reaction with cinchonine sulphate was shown to arise from the cumulative effects of at least four different beer components, which should be considered when interpreting the effects of different haze-stabilisation treatments.     

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.     

THE NITROGENOUS COMPLEXES OF HAZE AND FOAM AND THEIR MEASUREMENT

Earlier work and a review of recent studies have led to the view that the complex nitrogenous substances of beers are all, to some extent, irreversibly co-complexed either with glucose polymers or with polyphenols, or with both. Where amino-groups are still free, there can, in addition, be reversible combination with complex acids in worts and beers. The proportions of these different types of co-complex are considered to have important effects on beer properties, so that methods for the measurement of the amounts present are regarded as essential. With this as objective, it has been found that the relatively unmodified “free-amino” nitrogenous complexes, as well as those co-complexed with polyphenols, can be precipitated by suitable acidic dyes. The resulting precipitates can then be dissolved to give a simple and rapid colorimetric measurement of the amounts of these complexes present. By using the specific reagent polyvinyl pyrrolidone (AT) to absorb and remove the polyphenolic co-complexes, the remaining “free-amino” nitrogenous complexes can be precipitated alone, so that separate measures of both types can be obtained. Using bromophenol blue as the dye, some precipitation, mainly of the higher molecular weight fractions, occurs around pH 5 and by progressively lowering the pH to 3, or by progressively increasing the dye concentration, larger and larger amounts are precipitated to give a spectrum of the nitrogenous complexes present. Using a method based on these findings, wide variations have been found in the amount of different complexes in different beers. Studies have been made of the selective migration of complexes into foam and of the changes occuring during increase of haze. The methods have also been used to study the changes produced by proteolytic enzymes and by silica gel absorption, as well as those produced by changes in the nitrogen content of malts used. In one stout, dye-precipitable complexes are present in very small amounts. It appears that only non-reactive “crypto-nitrogenous” co-complexes persist after the prolonged mashing period used: a rapid method of measurement has been found for these also.     

CHARACTERIZATION OF STABILIZED AND UNSTABILIZED BEERS

The preparation and properties of polyvinylpolypyrrolidone (PVPP) and silica gel and the mechanisms by which they stabilize beer are discussed. In stabilized beer, chill stability was associated with lower protein sensitivity to precipitation with tannins. Approximately 60% of the proteoses were of low molecular weight (<10,000 daltons). The most acidic of these proteoses were the least stable in solution, being precipitated from beer by cold treatment and adsorbents.     

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.     

MALTING AND BREWING WITH BARLEYS HAVING BLUE ALEURONES

Blue aleurones in barley are associated with elevated levels of polyphenolic materials such as anthocyanins and anthocyanidins. A rapid method has been developed for assessing the anthocyanin content of barleys, malts and worts. Malts were prepared from a range of barleys, some of which had intensely blue aleurones, while others were only slightly blue or showed no visible pigmentation. The malting quality of barley was not affected by aleurone colour and ales and lagers of sound flavour as well as acceptable analytical parameters were brewed from malts having pronounced blue aleurones. In some cases sweet worts prepared from blue aleurone malts had a slight pinkish tinge, but this disappeared during wort boiling, and beer colours were normal. Levels of anthocyanins in barley correlated with those in malt and in wort. However, the concentration of anthocyanins was unrelated to the amount of anthocyanogens or total polyphenols. High anthocyanin levels in either barley or beer had no deleterious effects on beer flavour or the rate of haze development.     

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.     

A SCIENTIFIC BASIS FOR BEER FOAM FORMATION AND CLING

The most complex types of nitrogen compound in beer (here termed “proteoses”) are, when not combined with polyphenols, highly surface active and hence concentrate in the foam. With unhopped beer, the foam is of the “liquid-viscous” type, which eventually drains and collapses leaving no residual solids. The iso-α-acids in hopped beers are also surface-active, so they too are concentrated in the foam. At the corresponding concentration in bulk solution the solubility limits of some isohumulates* are exceeded. Thus we believe that solids are similarly formed in the bubble films, so that these are reinforced and stiffened to give “cling”. One consequence of the increased concentration of iso-α-acids in the foam is that there can be precipitation of iron, nickel, cobalt and zinc isohumulates. This results in concentration of these metals in foam. The most significant finding is that the concentration of both proteoses and iso-α-acids in the foam exceeds the bulk solubility limits and results in the formation of proteose-isohumulate salts which make the main contribution to the solid reinforcement of bubble films. These findings make it possible to explain satisfactorily “lacing” or “cling” and also isohumulone losses (a) during boiling and trub separation, (b) during post-fermentation bittering, and (c) during any subsequent foam formation. An explanation is also provided why losses of bitterness and of head retention are linked. Consequently, linked recovery of both bitterness and head retention can occur, as has been found is some recent fermentation improvements, such as continuous fermentation.     

PROTEIN PRECIPITATION DURING WORT BOILING: QUALITY ASPECTS OF DIMINISHED WORT BOILING TIMES OF BREWS PREPARED FROM PROANTHOCYANIDIN-FREE OR REGULAR RAW MATERIALS

When using proanthocyanidin-free materials for the production of beer, a reduction of the wort boiling time can be considered. In worts prepared with regular malt and tannin-free hop extract there is a continuous precipitation of the malt flavanoids while in brews prepared from proanthocyanidin-free malt and regular hops there is a simultaneous extraction and removal of the hop flavanoids leading to constant levels of these hop flavanoids. The results also show that the level of Kjeldahl nitrogen in worts boiled with hops will be the same as that in worts boiled with n-hexane tannin-free hop extracts. These results and the fact that more protein precipitates in brews containing no malt or hop proanthocyanidins suggest that, unlike what is the case during the development of beer haze, polyphenols are not necessary for an effective protein precipitation during wort boiling.     

STUDIES ON HAZE PRECURSORS IN BEERS AND WORTS

Detailed studies have been carried out on the fractionation of worts and beers on Sephadex and Bio-gel columns with high degrees of cross-linking. On G.25 Sephadex, the excluded fraction contains much of the total protein in the form of complexes with polyphenols bound in such a way that they do not interact with the column material. Continued elution with aqueous solvents gives many low molecular weight wort components and in addition some protein-polyphenol complexes which are retarded by relatively weak adsorption on the column. These compounds have been further studied by rechromatography in urea to dissociate the complexes and after reaction with excess flavonoid. Since the adsorbed compounds interact with the column through hydrogen bonding of active phenolic bonds, it may be that these are also the compounds which cross-link with other protein complexes in the beer to build up haze particles. Molybdate and alkali can be used subsequently to elute the free tannin and flavonoid molecules which are strongly absorbed.     

Enrichment of xanthohumol in the brewing process

Xanthohumol (XN), a component of hops, is lost in significant quantities in the conventional brewing process. In commercial beers less than 0.2 mg XN/L are found. In order to increase the yield of XN in the brewing process, the parameters of XN recovery were studied. During wort boiling, XN is largely isomerised to isoxanthohumol. Further losses are owing to the precipitation and absorption of XN to yeast cells and haze particles and by filtration. The use of XN-enriched hop products combined with a late hop dosage during wort boiling proved to be effective in increasing the XN content in beer. The yield was further raised by a low-pitching rate and the abnegation of beer stabilisation. The use of dark malts had a positive effect on the XN recovery. Investigations of roasted malt extracts revealed several high-molecular substances that are able to form complexes with XN. These complexes proved to be stable in the brewing process. Depending on the addition of roasted malt or special XN-enriched roasted malt extracts, dark beers with more than 10 mg XN/L were achieved. Results obtained led to a brewing technology that produced on an industrial scale pale wheat beer with more than 1 mg XN/L.     

EUROPEAN BREWERY CONVENTION—HAZE AND FOAM GROUP ORIGIN OF CARBOHYDRATE IN BEER SEDIMENTS

Carbohydrate-containing sediments can be divided into two types: in the commoner type the polysaccharide is associated with protein and polyphenol, and the sediment is chemically related to haze; the second type of sediment is almost entirely carbohydrate and may give rise to a gel. An enzymic procedure has been developed which allows one to determine whether the carbohydrates are derived from an α-glucan (dextrin) or a β-glucan. Addition of ethanol (20%, v/v) to the beer precipitates a mixture of substances including high molecular weight α- and β-glucans. However, the polysaccharide which becomes associated with protein and polyphenol appears to be exclusively α-glucan. α-Glucans readily give cyclodextrins which are well known to form inclusion complexes with a wide variety of molecular types. It is suggested that inclusion complexes of this type are involved in protein-polyphenol-polysaccharide sediments and may modify the rate of polymerization reactions.     

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.     

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.     

应用新技术提高啤酒的胶体稳定性

引起啤酒胶体非稳定性混浊的主要物质为蛋白质和多酚物质。可采取在麦汁煮沸阶段加入胶体物质和在贮酒过程中加入硅胶，使啤酒中的高分子蛋白质沉淀而过滤除去。利用PVPP作为多酚吸附剂对啤酒进行二级过滤，可降低啤酒中的多酚物质，也可将PVPP同硅藻土、硅胶联用过滤，降低啤酒的多酚物质。     

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.     

Total Stabilisation of Beer in a Single Operation*

The total costs of lager beer stabilisation and clarification are high because of high capital investment, the production of solid and liquid wastes and additional handling. A single operation for the removal of sensitive proteins, sensitive polyphenols and radical initiators as metal ions reduces the costs substantially¹⁶. A gallotannin (GT)and a polyphenol absorber (PA) are added subsequently to the beer stream; the haze sensitive compounds are adsorbed and removed together via single centrifugation followed by a regeneration. The normal Kieselguhr filtration can be replaced by a Kieselguhr reduced filtration or membrane filtration^10,12. An extended shelf-life is obtained by an optimum ratio-gallotannin/polyphenol absorber, e.g. 6 g/10 g or by complete removal of sensitive proteins, metal ions and a little part of the flavanoids. The shelf-life or sensorial stability can be evaluated by chemiluminescence intensity and positively influenced with gallotannin. The high molecular weight gallotannin is completely natural (present in many herbs) and produces 100% residue-free beer.     

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。