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 Effect of Proteinase A on Foam‐Active Polypeptides During High and Low Gravity Fermentation

The ability of beer to produce good foam is influenced by the level of foam‐active polypeptides. Specific polypeptides with hydrophobic domains, such as Lipid Transfer Protein (LTP1), are important components of beer foam. Although, high gravity brewing is a commercially viable technique, it has the disadvantage of producing beer with less foam stability compared to lower gravity brewed counterparts. It is thought that proteinase A plays a key role in the degradation of these hydrophobic polypeptides responsible the beer foam stability. The object of this study was to compare and quantify the loss of hydrophobic polypeptides and specifically foam‐LTP1 during high gravity (20°Plato) and low gravity (12°Plato) wort fermentations and to evaluate the effect of proteinase A on these polypeptides. The losses of hydrophobic polypeptides and foam‐LTP1 were generally greater in high gravity brews. Furthermore, the results obtained suggest that proteinase A alters the hydrophobicity of these polypeptides rather than their molecular size. Approximately 20% of hydrophobic polypeptides and approximately 57% of foam‐LTP1 appeared to be proteinase A resistant. These differential losses of hydrophobic polypeptide and foam‐LTP1 could have implications for the foam stability of the finished product.     

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 Effects of Hop‐α‐Acids and Proline‐Specific Endoprotease (PSEP) Treatments on the Foam Quality of Beer

Influences on foam stability and cling were compared by brewing trials investigating beer hopping rate, hopping type and modification of beer protein composition by the inclusion of a proline specific protease (PSEP). The comparison of the NIBEM, Rudin and lacing foam assessment methods with the level of hopping demonstrated the superiority of hydrogenated hop α‐acids with respect to foam stability and particularly lacing. In addition, the NIBEM and Rudin foam analysis tests appear to respond relatively similarly with respect to hopping rate and hop type, with the NIBEM being somewhat more responsive in terms of foam stability measurments. The PSEP trials suggested that protein composition may only have a subtle effect on foam stability. Although more specific to haze active proteins, PSEP treatment in the small and pilot scale trials generally, but not always, resulted in a minor reduction in foam stability. This effect was not observed in 20 hL pilot and industrial scale beer productions. It was verified that both NIBEM and Rudin were positively influenced by increased levels of foam positive proteins. Although both foam tests were responsive to hopping rate and type, it is suggested that the Rudin foam test is somewhat biased towards foam positive proteins, particularly albuminous foam positive proteins (LTP1 and protein Z4), while in comparison the NIBEM foam test appears somewhat biased towards hordein foam positive proteins.     

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.     

啤酒中浑浊敏感蛋白的分离与鉴定

啤酒非生物稳定性是影响啤酒质量的重要指标之一,非生物浑浊主要是啤酒中的浑浊蛋白质与多酚结合体组成的,浑浊敏感蛋白质主要来自大麦醇溶蛋白,是一种富含脯氨酸的蛋白质.主要阐述了浑浊敏感蛋白质的分离,以及利用蛋白免疫印迹法(Immunobloting)鉴定啤酒中的浑浊敏感蛋白质,此外还研究了硅胶对啤酒的浑浊蛋白质的影响.     

The Relative Significance of Physics and Chemistry for Beer Foam Excellence: Theory and Practice

Bubble nucleation sites in beer glasses should be uniformly small to ensure that the foam produced comprises homogenously small bubbles with attendant reduced degrees of disproportionation. The latter phenomenon is also hindered by increased film thickness in foam, which in turn will be enhanced by increased localised viscosity, encouraged perhaps by polysaccharide‐polypeptide complexes. Erring on the “high end” of specification for carbon dioxide will promote replenishment of foam through beading. Nitrogen at a relatively low level (15–20 ppm) will hinder disproportionation with limited suppression of hop aroma and introduction of undesirable textural features. Hydrolysed hordein appears selectively to enter beer foams at the expense of the more foam‐stabilising albuminous polypeptides, suggesting that any future procedures that might selectively eliminate hydrolysed hordein components from beer would be expected not only to enhance haze stability but also foam, by eliminating species that preferentially enter into the bubble but have less foam‐stabilizing capability once they are there.     

125th Anniversary Review: The role of proteins in beer redox stability

Flavour stability is usually approached through inhibition of reactive oxygen species (ROS). It may be possible to suppress ROS, but never to entirely eliminate them in packaged beer. The role of proteins in ROS suppression seems to have been lost in the compliant acquiescence to supply haze‐free bright beer. Proteomics allows beer polypeptides to be finely resolved, identified and correlated with beer quality and stability. This has already produced a broader view of what stabilizes beer foam. No doubt it could do the same for beer stability and the broader roles that proteins, such as LTP1, can have in redox reactions and free radical suppression. Cysteine oxidation and reversibility is central to cellular signalling in biological systems. Thiol chemistry is also integral to beer redox stability. We can, and should, extrapolate the recent biological findings to the simple pleasure of creating a high‐quality beer. Copyright © 2012 The Institute of Brewing & Distilling     

The foaming of mixtures of albumin and hordein protein hydrolysates in model systems

Although hydrolysed albumin and hydrolysed hordein derived from barley are independently capable of stabilising foams in model beer solutions, when both are present together, the net foam stability is less than anticipated. In particular, it seems that hordein, even in an unhydrolysed state, interferes with the ability of albumin‐derived polypeptides to stabilise foam. It appears, therefore, that the observed foam stability of a product such as beer is not only dependent on the absolute level of individual foam‐stabilising polypeptides but also on the relative proportions of polypeptides derived from the albumin and hordein protein subsets. Copyright © 2004 Society of Chemical Industry     

The Loss of Hydrophobic Polypeptides during Fermentation and Conditioning of High Gravity and Low Gravity Brewed Beer

The object of this study was to investigate the loss of hydrophobic polypeptides, which are important for foam quality and stability in finished beer. Loss of hydrophobic polypeptide due to fermenter foaming occurs during transfer of fermented wort since a gradient of hydrophobic polypeptides towards the surface is created during fermentation. Due to higher polyphenol levels in high gravity (20°Plato) wort, more hydrophobic polypeptides are lost due to cold break (cold trub) precipitation compared to low gravity (12°Plato) wort. Another important factor affecting the loss of hydrophobic polypeptides could be proteinase A activity during fermentation, especially in high gravity fermentation where the yeast is exposed the higher stress. During high gravity fermentation, where osmotic pressures are higher, ethanol levels become greater, and nitrogen‐carbohydrate ratios are lower, more proteinase A is released by the yeast. This release of proteinase A into fermenting wort could have implications for the foam stability of the finished product.     

CAMBRIDGE PRIZE LECTURE BIOCHEMICAL APPROACHES TO BEER QUALITY

New techniques, approaches and concepts have been applied to the study of aspects of beer quality: colour, alcohol content, haze, foam and flavour. Particular emphasis is placed upon the methodology of alcohol determination; β-glucan, its role in restricting the recovery of fermentable sugars and how it can be dealt with; pseudo-hazes; foam proteins; dimethyl sulphide; and the role of radical forms of oxygen in causing flavour deterioration. A new nomenclature for describing the foaming properties of polypeptides is described and attention is drawn to the influence which methods for protein determination can have on results of foam studies.     

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.     

啤酒缓冲性与质量稳定性关系的初步研究

选取了十几种市售啤酒 ,研究了啤酒缓冲容量与啤酒质量稳定性之间的相关性。同时有针对性地提出了增加啤酒缓冲容量的措施 ,并对酿造过程中磷酸盐的主要来源进行了研究。研究结果表明 ,啤酒缓冲容量与啤酒泡持、浊度稳定性和老化速度之间存在着线性相关性。增加麦汁或啤酒中总磷酸盐浓度既是提高啤酒缓冲容量的有效措施 ,又是减缓啤酒老化速度的重要手段之一。     

Influence of high molecular weight polypeptides on the mouthfeel of commercial beer

Low malt beers have high sales volumes in Japan, but improving their mouthfeel, including softness, smoothness and decreasing astringency, is challenging because the compounds responsible remain unclear. In this study, beer was fractionated by preparative size‐exclusion chromatography, with the polypeptide and maltodextrin fractions purified using solid‐phase extraction and ion‐exchange resin. Sensory data from a spike test showed that the mouthfeel (softness, smoothness, and reduced astringency) of low malt beer was improved both by the degree of polymerisation (DP) of maltodextrins (DP of 2‐10; at increased concentration of 40 to 60%; P < 0.01) and by 10 ‐ 20 kilodalton (kDa) high molecular weight (HMW) polypeptide and 2‐3 kDa low molecular weight polypeptide fractions (at a 50% increase in concentration; P < 0.01). Furthermore, highly purified 10 to 20 kDa HMW polypeptides improved the softness and smoothness and decreased the astringency (at a 25% increase in concentration). This report is the first to provide experimental sensory data indicating that HMW polypeptides improve the mouthfeel of beer. Based on these findings, a new low malt beer was developed that showed significantly higher levels of the 10‐20 kDa HMW polypeptides with an overall improved mouthfeel. Mass spectrometric analysis of the 10 to 20 kDa proteins identified several unique foam positive proteins, including barley dimeric alpha‐amylase inhibitor‐1 and non‐specific lipid‐transfer protein 1. These 10‐20 kDa HMW proteins are likely to be responsible for the improved mouthfeel of beer. © 2020 Kirin Holdings Kabushik Kaisha Co. Ltd. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling     

采用复合诱变选育低分泌蛋白酶A啤酒酵母菌株的研究

啤酒酵母在发酵过程中分泌胞外的蛋白酶A是影响纯生啤酒泡沫稳定性的关键因素，本研究通过采用亚硝基胍（TNG）和甲基黄酸乙酯（EMS）复合诱变选育满足食品安全性的低蛋白酶A、发酵性能正常的优良啤酒酵母菌株，为解决纯生啤酒泡沫稳定性奠定基础。     

IMMUNOCHEMICAL STUDIES ON THE NITROGENOUS CONSTITUENTS OF BEER FOAM*

Samples of foam prepared by two different procedures were analysed using immunoelectrophoretic methods with anti-barley and anti-chill haze immune sera and their constituents were compared to those of beer (total and defoamed) and of chill-haze. Two, and probably three, constituents reacting with the immune sera have been detected in both foam preparations and it was shown that at least two of the constituents are identical with the main nitrogenous components of chill-haze. Defoamed beer contains the same substances but at a low concentration when compared on the basis of weights of dry matter. These observations may have some practical applications.     

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     

啤酒中疏水性蛋白质提取鉴定及其对于泡沫性能的影响的研究

利用蛋白疏水层析色谱法(HIC)从啤酒泡沫中分离疏水性蛋白,研究发现,HIC依次分离的蛋白组分表面疏水性增强的同时,泡沫稳定性随之增强,说明HIC分离得到强疏水性多肽对于啤酒泡沫稳定性具有较明显的积极作用,而弱疏水性多肽的影响效果不明湿.此外通过质谱(Mass Spectrometry)鉴定结果显示所得到的疏水性蛋白质为蛋白质乙,通过对啤酒酿造过程中的关键控制点的跟踪,确定了啤酒酿造过程中,发酵阶段疏水性蛋白质的损失最为严重,对啤酒泡沫质量的影响最大.     

POLYPEPTIDES WITH ENHANCED FOAM POTENTIAL

The ability of beer to produce a good foam is strongly influenced by the level of foam active polypeptides. It has previously been proposed that a means of ensuring an adequate level of such species is to add an exogenous preparation of foam active protein³. One such preparation, hydrolysed liquid egg white (HLEW), has been shown to impart a good foam to beer with substandard foam performance, without detriment to product quality. The foam active properties of this material are the subject of the work described here . HLEW was characterised by a combination of reverse phase chromatography and polyacrylamide gel electrophoresis to reveal a heterogeneous mixture of low molecular weight (2100–6000 daltons) relatively hydrophilic polypeptides. When this material was subjected to foaming and the foam positive and foam negative fractions similarly characterised, it was apparent that the foam positive fraction consisted almost exclusively of hydrophobic polypeptides. Accordingly, preparative reverse phase chromatography was used to isolate foam active fractions from the hydrophobic species present. These fractions were subsequently subjected to both functional and physical characterisation. It was apparent from a small-scale Rudin test that the foam activity per unit dry weight of protein was enhanced in the foam active factions; in one case an enhancement of 2 fold greater than the HLEW itself was observed. Moreover, upon addition to beer a positive effect on HRV was achieved with as little as 0.0025 mg protein/ml beer. Physical characterisation of the foam active material revealed the presence of tightly bound polypeptide aggregates which could only be separated by the use of protein denaturing agents.