PHENOLIC CONSTITUENTS OF BEER AND BREWING MATERIALS III. SIMPLE ANTHOCYANOGENS FROM BEER

By use of chromatography on a dextran gel, (+)-catechin and six simple anthocyanogens have been isolated from polyamide-beer adsorbates. Three of the anthocyanogens have been obtained in a homogeneous condition whilst two others appear to be mixtures of at least two compounds; a sixth, which is derived by dehydration between two others, was obtained contaminated by minute traces of these. Chromatographic and structural studies indicate that one anthocyanogen is probably a stereo-isomeric form of 5:7:3′:4′-tetrahydroxyflavan-3:4-diol possessing the epicatechin configuration at C₂ and C₃, another is a biflavan derived by dehydration between (+)-catechin and the flavandiol, and a third appears to be a triflavan derived from one molecule of the flavandiol and one of the biflavan. A further biflavan was isolated which yields delphinidin and (+)-catechin separately under appropriate acidic conditions. The remaining anthocyanogens are mixtures and contain, in addition to (+)-catechin-derived compounds, components which yield (+)-epicatechin with acid. Re-examination of the relevant adsorbates from beer indicates that Polycar removes, proportionally, more (+)-catechin and (+)-epicatechin from beer than does Nylon. A possible relationship between anthocyanogen structure and beer stability is discussed.     

PROCESSES FOR PRODUCING BEER WITH A LOW TENDENCY TO FORM HAZE

It was confirmed that wort from malt resteeped in a solution of formaldehyde (1000 mg./litre), had a low level of anthocyanogens. It was shown that beer brewed from this malt had a lesser tendency to form haze than beer brewed from a malt resteeped in water. Malt yielding wort with usefully reduced levels of anthocyanogens could be prepared by adding formaldehyde (500–1000 mg./litre), to the final steep, in an otherwise conventional malting sequence. The rapid rate of haze formation that occurred in beers to which formalin had been added was shown to be a useful, quick guide to their stability under different storage conditions. When hydrogen peroxide (100 mg./litre) mashing liquor was added to mashes it reduced the anthocyanogen levels of the wort. The beers prepared from treated mashes were remarkably slow to form haze. The effect was greatest when hydrogen peroxide was added in small increments throughout the mashing period. The other alterations in wort characteristics resulting from this process, including marginal increases in colour and decreases in fermentability, were small. In most trials the treatment did not significantly alter the flavour of the beer. Charcoal (Norit, N.K.), added to the mash (500 mg./kg. grist), or to the copper (500 mg./kg. grist), reduced the anthocyanogen contents of the worts; the final beers had greatly enhanced shelf-lives. Charcoal was most effective when spread over the surface of the mash at the start of sparging.     

CHEMICAL CONSTITUENTS OF NYLON-66 BEER ADSORBATE. I. PRELIMINARY INVESTIGATIONS AND ANTHOCYANOGEN CONTENTS

A short survey of the polyphenolic substances of interest in brewing research and practice is given and a simple but comprehensive classification of phenolics (“tannins”) is proposed. A preliminary investigation has been made of the chemical constituents of a Nylon-66 beer adsorbate from a Canadian ale. Different fractions were obtained from the Nylon resin beer adsorbate and its caustic desorbate by using various organic solvents for extraction; the extractive power and selectivity of different solvents are compared. The anthocyanogen content of various fractions was determined by five different methods, and the widely varying results obtained indicate the serious shortcomings of the methods at present available for anthocyanogen determination.     

EUROPEAN BREWERY CONVENTION—HAZE AND FOAM GROUP PHENOLIC CONSTITUENTS OF BEER AND BREWING MATERIALS IV. FURTHER OBSERVATIONS ON ANTHOCYANOGENS AND CATECHINS AS HAZE PRECURSORS IN BEER

An earlier supposition that catechins (flavan-3-ols) can favourably influence the stability of a beer by co-polymerizing with flavan-3: 4-diols has been experimentally justified. D-(+)-Catechin, however, is also a direct haze precursor, particularly of chill haze, and its effect is most marked under conditions of high air content when it polymerizes relatively rapidly by an oxidative mechanism. The haze-inducing behaviour of a biflavan anthocyanogen isolated from barley is a little different from that described previously for a very similar anthocyanogen (component 12) from beer. A synthetic 5:7:3′:4′-tetrahydroxyflavan-3:4-diol appears to differ only slightly as a haze precursor from its stereo-isomer, beer component 73. These findings are considered in terms of probable chemical structures. Traces of an anthocyanogen, identical on paper chromatograms with a synthetic 5:7:3′:4′-tetrahydroxyflavan-3:4-diol, and thus stereo-isomeric with beer component 73, are present both in polyamide-beer adsorbates and in barley.     

LOW PROANTHOCYANIDIN MALTS FOR PRODUCTION OF CHILLED AND FILTERED OR FINED BEER

Malts which conform to most commercial specifications can be prepared from Galant barley, which is low in anthocyanogens. However, the malting performance of this variety is only medium and considerable care is required during kilning in order to avoid excessive colour development. Beers brewed from Galant malt have greatly improved chill haze stability, although flavour stability is not significantly affected. Fining and filtration studies on rough beer and forcing tests for the development of non-biological haze in finished beer indicate that the use of Galant malt greatly reduces the extent of precipitation of particulate matter during brewing, conditioning and subsequent storage. This can result in considerable savings in the use of finings, and in longer filter runs since levels of addition of body feed can be reduced. It is suggested that the use of low anthocyanogen malts could, with some beers, reduce the necessity for chilling prior to and during filtration.     

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.     

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.     

USE OF FORMALDEHYDE FOR THE REDUCTION OF THE ANTHOCYANOGEN CONTENT OF WORT*,†

Reduction in wort anthocyanogen content is achieved effectively when formaldehyde is introduced to the mash at the beginning of the mashing operation. Concentrations of 50 p.p.m. of formaldehyde with respect to the malt (equivalent to about 5 p.p.m. with respect to the final wort volume) are effective in reducing the anthocyanogen content by about 30%. Treatments four times as strong reduce the anthocyanogen content by 60–70%, and the effect is accompanied by a fall of up to 10% in the soluble nitrogen content of the wort. It seems likely that a condensation reaction occurs between anthocyanogens and a nitrogenous fraction of the wort. Under the conditions quoted, formaldehyde residues in the finished beers are less than 0·2 p.p.m. and are no greater than those observed in corresponding untreated control beers. Beers produced from treated mashes exhibit great non-biological stability, but are quite normal in other respects. The effect of formaldehyde on the anthocyanogen contents of worts and beers is of substantial theoretical interest and it is probable that it will also have a commercial application.     

THE USE OF FORMALDEHYDE IN RESTEEPING

Small-scale malting trials showed that resteeping in a solution of formaldehyde was more uniformly effective than resteeping in water in reducing rootlet yield and the overall malting loss. The hot water extract, the fermentability of the wort, and the formol nitrogen were the same in worts from malts prepared by resteeping in water or formaldehyde solutions. However, cold water extract was reduced, as was the total soluble nitrogen, by the presence of formaldehyde. The anthocyanogen level in the wort was reduced by about 73% when formaldehyde was used in the resteep liquor. The residue of formaldehyde in the unboiled wort was about I p.p.m. It is considered that, in view of the improved resteeping performance and the expected increase in beer stability resulting from the reduction in anthocyanogens and wort nitrogen, the use of formaldehyde in resteeping is worth evaluating on a larger scale. In addition the use of formaldehyde in the resteep liquor prevented fouling by micro-organisms and resulted in a cleaner malt.     

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.     

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.     

PHENOLIC CONSTITUENTS OF BEER AND BREWING MATERIALS

Beer components removed by treatment with small quantities of Nylon 66 or Polyclar A.T. are composed of small amounts of large numbers of simple phenols and polyphenols derived from malt and hops together with a relatively large quantity of polymerized polyphenolic material associated with protein. Quercetin, kaempferol, isoquercitrin, rutin, (+)-catechin and (+)-epicatechin were identified paper chromatographically as were chlorogenic, neochlorogenic, p-hydroxybenzoic, vanillic, syringic, p-coumaric and ferulic acids. Glycosides and esters of p-hydroxybenzoic, protocatechuic, vanillic, syringic, caffeic, ferulic, sinapic and gentisic acids also occur in beer-polyamide absorbates. Nylon and A.T. remove what appear to be the same simple polyphenols from beer. Aggregates of protein with polymerized polyphenols show marked resemblances to hazes and probably represent precursors very close to beer turbidities. The aggregates, which contain methoxylated phenolic units in addition to anthocyanogen residues, are present in dialysable and non-dialysable forms.     

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.     

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.     

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.     

FLAVOUR AND HAZE STABILITY DIFFERENCES DUE TO HOP AND MALT TANNINS IN ALL-MALT PILSNER BEERS BREWED WITH PROANTHOCYANIDIN-FREE AND WITH REGULAR MALT

All-malt Pilsner beers were brewed with and without hop and malt proanthocyanidins by using regular malt and whole leaf hops as well as proanthocyanidin-free malt and n-hexane tannin-free hop extract. The different beers were analysed chemically and presented to an expert panel to detect possible differences in bitterness and astringency. The impact of proanthocyanidins on the formation of haze was demonstrated clearly. No differences between the beers were found in triangular tests although their levels of total polyphenolics and anthocyanogens differed by about 225 and 85 mg/litre. Statistical analysis of the paired comparison tests showed only very slight differences between beers with or without hop and malt proanthocyanidins. Thus, proanthocyanidin-free beer was considered less than slightly more bitter and astringent than regular beer and there was a very slight preference for the latter.     

SYSTEM OF WORT ANALYSIS. IV: THE ESTIMATION OF THE TRUE PROTEIN NITROGEN OF WORTS AND BEERS

A method is given in which wort or beer is heated with sufficient sodium nitrite and acetic acid to deaminate the nitrogen compounds present. On cooling a precipitate forms which, the experiments suggest, represents the true protein* present, since it appears to correspond to the albumin and globulin of barley extracts and to include the nitrogen compounds of wort which are coagulable by boiling. It is possible that the amount of this fraction bears a relation to non-biological haze, while a subsequent paper suggests an important effect on fermentation. The results indicate that boiling conditions play an important part in controlling the amount, as also may the amount of tannin in hops and fermentation conditions.     

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 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.     

CHEMICAL CONSTITUENTS OF NYLON-66 BEER ADSORBATE. IV. FLAVOUR EFFECTS

Phenolic concentrates, derived from Nylon-66 beer adsorbate, have been submitted to strong oxidative conditions involving refluxing in butanol and air. The changes in chemical composition of the concentrates were followed by various methods, including simultaneous determination of anthocyanogens and catechins (tanninogens). Flavour effects of the fresh concentrates, as well as of their oxidized mixtures, were examined in 5% aqueous ethanol and in beer. The influence of concentrates on the beer flavour was comparable with that of the fresh polyphenols, while the oxidized mixtures resembled in that respect the oxidized polyphenols. It was thereby proved that these concentrates retained their basic phenolic character in spite of the contaminants present, both before and after oxidation. Furthermore, the role of polyphenols in beer flavour was thus shown for the actual phenolic combinations occurring in beer.