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.     

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.     

Control of antioxidant beer activity by the mashing process

Beer is considered to be a good source of antioxidants. The composition and the quantity of the antioxidant compounds depend not only on the qualities of the raw materials, but also on the technology processes. Barley and malt represent the main source of antioxidant compounds in beer and the contribution of the hop antioxidants is lower. The influence of the mashing process on the antioxidant activity and polyphenol concentration is crucial. The antioxidant state of the sweet wort and the hopped wort are dependent on the technology processes and the raw materials used. The spontaneous sorption of polyphenols onto wort dregs and the polymerization of catechin and epicatechin lead to decreasing concentrations of individual polyphenols in the final beer. Two methods, based on electron spin resonance were used to determine antioxidant activity. These were the DPPH (2,2‐diphenyl‐1‐picryl‐hydrazyl) assay and the ‘lag time’ assay using free radical spin‐trapping agent PBN (N‐tert‐butyl‐α‐phenylnitrone). HPLC with CoulArray detection was used to measure the concentration of the individual polyphenols. This study focused on the antioxidant compounds and on the correlation of their concentrations with the values of total antioxidant activity depending on the mashing process. A good correlation was found between the decline in the concentrations of DPPH (expressed as ARA2) and concentrations of catechin and epicatechin (in sweet wort samples R² = 0.970, R² = 0.961, respectively, and in hopped wort samples R² = 0.949, R² = 0.956 respectively). Copyright © 2012 The Institute of Brewing & Distilling     

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.     

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 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 influence of various phenolic compounds on scavenging activity assessed by an enzymatic method

The effect of pure monomeric and dimeric procyanidins on superoxide anion radicals (O₂°⁻) was studied with an enzymatic method and their IC₅₀ was determined. A comparative study of the results suggested that the antiradical potential of these phenolic compounds closely depends on their stereochemical structure. Modification of the chiral carbon, C₃ involved in the oxidation mechanism induces differences in the scavenging action of (+)‐catechin (2R : 3S) or (−)‐epicatechin (2R : 3R). The stereochemical conformation of the interflavan linkage (axial or pseudo) according to the chemical nature of dimeric procyanidin units also influences the radical oxidation mechanism. The nature of the interflavan linkage between the two units in dimeric procyanidins (C₄–C₈ or C₄–C₆ linkage) is an important factor responsible for effectiveness of the behaviour polyphenols towards O₂°⁻. The extraction of oligomeric procyanidins in grape seeds enabled us to study the effect of polymerisation and galloylation on their scavenging activities. The results clearly showed that the molecular scavenging mechanism of these molecules is closely connected with their spatial conformation. © 1999 Society of Chemical Industry     

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.     

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.     

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.     

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     

The role of phenolic compounds during formation of turbidity in an aromatic bitter

An aromatic bitter produced from alcoholic extracts of botanicals were studied over 2½ years. The concentrations of gallic acid and the flavonoids catechin, epicatechin, eriodictyol, and taxifolin decreased during the first year of storage in the presence of oxygen, whereas the turbidity stayed below the limit for visible detection. After one year the turbidity increased linearly with time and the total concentration of phenolic compounds decreased. In contrast, the concentrations of eugenol and phenolic acids with two or one hydroxyl groups remained constant or increased, and these compounds are probably not involved in formation of turbidity. The oxidative properties of the phenolic compounds were evaluated in an assay based on iron-catalysed Fenton reactions and spin trapping of radicals. The fresh bitter and bitter stored for one year increased the amount of radicals detected in the assay, demonstrating an overall prooxidative effect of the phenolic compounds. It is concluded that easily oxidised phenolics in the bitter are converted into soluble intermediates that slowly react with proteins and carbohydrates forming haze and deposits.     

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。     

外源多酚氧化酶催化合成儿茶素二聚体氧化产物的研究进展

在红茶加工过程中,儿茶素通过多种氧化途径形成种类和构成都很复杂的氧化聚合产物,也赋予了红茶特有的色泽和口感。目前研究较多的儿茶素二聚体氧化产物主要是茶黄素类和聚酯型儿茶素。本文综述了近年来国内外在儿茶素二聚体氧化产物形成的反应途径、儿茶素酶性氧化中的外源多酚氧化酶及其与底物的特异性、儿茶素酶性氧化生成二聚体氧化产物及其生物活性、外源多酚氧化酶催化儿茶素氧化生成二聚体氧化产物在茶类加工中应用等方面的研究现状,旨在为进一步深入理解儿茶素酶性氧化机理提供参考,同时也为茶叶深加工提供应用科学理论依据。     

Identification of the Polyphenols in Barley and Beer by HPLC/MS and HPLC/Electrochemical Detection

Barley polyphenols were isolated using acetone/water (70/30) followed by Sephadex LH-20 chromatography. HPLC/Electrospray mass spectrometry showed the presence of proanthocyanidin dimers, trimers, tetramers, and pentamers. HPLC/electrochemical detection was used to survey the contents of these compounds in a range of barley varieties. Principal components analysis showed that the naked barley varieties Morrell, and to a lesser extent Namoi, were different from the other barley varieties. Beer polyphenol extracts were prepared using Sephadex LH-20 chromatography. A large number of proanthocyanidin dimers and trimers could be detected using HPLC/MS. HPLC/electrochemical detection was used to survey a range of beer types. Compounds detected included catechin, epicatechin, gallocatechin, epigallocatechin, a number of proanthocyanidin dimers and trimers, and a number of phenolic acids. The peaks removed by PVPP have been determined. Principal components analysis was used to identify those compounds which distinguished the beer types. The first principal component was due to prodelphinidin B3, catechin, gallocatechin and two unidentified compounds which were removed by PVPP and the second principal component was due to phenolic acids. Efforts to identify the compounds removed by PVPP have commenced.     

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.     

Hop proanthocyanidins for the fining of beer

Fining agents are used in the clarification of beers; they help to reduce the time required to sediment suspended yeast cells and ensure the clarity and colloidal stability of beer. Following an adventitious observation during dry‐hopping experiments, we identified a fining activity associated with Saaz hops. Extracts of hop cones were subsequently shown to have the capacity to flocculate yeast and result in their sedimentation. This activity has since been identified in extracts of many different hop varieties and, significantly in spent hops, the co‐product resulting from commercial extraction of hops with either CO₂ or ethanol. Here we illustrate the activity of the novel finings extracted from spent hops following CO₂ extraction of Galena hops. The sediments formed on fining were compact, relative to those obtained when commercial isinglass was used to fine the same beers. The hop extracts were also effective in reducing 90° haze in beers under conditions designed to mimic both cask ale (12 °C) and lager (4 °C) type applications. The compounds responsible for the fining activity appear to be large (30–100 kDa, or more) polyphenols. Analysis of the polyphenols using colourimetric tests indicated the presence of proanthocyanidins. On acidic hydrolysis these generated cyanidin, which would be derived from a polymer composed of catechin and epicatechin subunits. The presence of these materials in spent hops offers the possibility to develop commercial products, with desirable fining properties, from an existing co‐product stream. Furthermore, the finings are derived from a traditional ingredient of the brewing process. Copyright © 2015 The Institute of Brewing & Distilling     

Quantification of Catechin and Epicatechin in Foods by Enzymatic-Spectrophotometric Method with Tyrosinase

Catechin and epicatechin are important phenolic compounds found mainly in tea leaves, cocoa beans, and in the peels and seeds of fruits. The reliable detection of these compounds by spectrophotometry is limited by the interference with substances that absorb light at similar wavelengths; thus, an effective alternative is to detect the oxidation products of these compounds at more specific wavelengths. This study aimed to develop an analytical method to quantify total catechin and epicatechin in some food samples (cocoa beans, guarana powder, apples, and tea leaves) by enzymatic oxidation with tyrosinase. The samples were extracted in an ultrasonic bath and the purified extracts were oxidized with tyrosinase solution. The quinones formed in the reaction were detected by spectrophotometry. The method was selective and presented linearity between 1.21 and 7.26 mg g^?1, limit of detection of 0.48 mg g^?1, and limit of quantification of 1.61 mg g^?1. The accuracy of the proposed method was reasonable, with recoveries between 84.3 and 90.7% in the fortified matrices. High precision was observed, with low coefficients of variation for repeatability (3.17 to 3.68%) and intermediate precision (3.27%). In cocoa beans, the total catechin and epicatechin level was 1.65 mg g^?1 (3.58 mg g^?1). The successful application of the method was also demonstrated in guarana powder. Therefore, the method can be applicable for spectrophotometric analysis of catechin and epicatechin in samples containing above 1.61 mg g^?1 of these compounds.     

UPLC and Spectrophotometric Analysis of Polyphenols in Wines Available in the Polish Market

Ultra performance liquid chromatography–photodiode array detection (UPLC–PDA) was used to analyze five biologically active polyphenolic compounds (catechin, epicatechin, rutin, trans-resveratrol, and quercetin) in different types of wine available in the Polish market. The results showed that the major polyphenols in chosen wines were catechin and epicatechin, the minor polyphenol was trans-resveratrol. The use of UPLC system allowed a shorter run time of up to six times in comparison with the conventional HPLC system. Total polyphenols were analyzed according to the Folin-Ciocalteu method using gallic acid as a standard and the results were presented as gallic acid equivalents. The wines were also analyzed for anthocyanins content using malvidin-3,5-diglucoside as a reference material in spectrophotometric analysis.