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.     

FRACTIONATION OF SMALL PEPTIDES FROM BEER

Small peptides (MW<1000) were isolated from beer by hollow fibre ultrafiltration followed by column chromatography using the gel matrix Sephadex LH20. This simple procedure enabled the isolation of small peptides relatively free of other low molecular weight nitrogenous compounds. Analysis of the composition of subfractions from the small peptide fraction from pilot brewery beer and a commercial beer using reverse phase HPLC showed superior resolution of individual components compared to previously reported analytical techniques and demonstrated the presence of many peptide components. Acid hydrolysis and amino acid analysis of small peptide subfractions showed that the principal amino acid in each subfraction was glutamic acid/glutamine consistent with the release of peptides by proteolytic degradation of barley and malt proteins.     

Barley and Malt Proteins and Proteinases: II. The Purification and Characterisation of Five Malt Endoproteases,Using the Highly Degradable Barley Protein Fraction (HDBPF) Substrate

Five barley‐malt endoproteases have been purified using the highly degradable barley protein fraction (HDBPF) as the substrate for activity detection and measurement. The five purified endoproteases were identified as the most active and, hence, we believe the most important proteolytic enzymes during barley germination and malting. This was demonstrated by showing that the component of HDBPF, degraded in test tubes during the reaction to determine their activity, cannot be recovered from malt by extraction, indicating that this component has been degraded during germination and malting. These endoproteases differ in their specificities, pH and temperature optima, thermostability and ionic‐cationic behaviour. The gel filtration chromatographic‐profiles of the peptide products of these enzymes versus parallel profiles of beer peptides exhibit very close similarities.     

100%大麦啤酒酿造过程中老化Strecker醛的研究

分别采用上面发酵工艺与下面发酵工艺进行100%大麦啤酒及100%麦芽啤酒的酿制,并对其麦汁的氨基酸含量、老化Strecker醛、自由基以及新鲜啤酒中老化Strecker醛的含量等进行了对比分析。研究发现,就麦汁而言,100%大麦麦汁中老化Strecker醛的含量都明显低于100%麦芽麦汁;同样的麦汁,上面发酵方式还原Strecker醛的能力明显优于下面发酵方式。就啤酒而言,经酵母还原后,新鲜啤酒中的老化Strecker醛含量较麦汁含量低,且100%大麦啤酒中老化Strecker醛的含量低于100%麦芽啤酒中的含量。100%麦芽麦汁的自由基含量是100%大麦麦汁的近3倍。这都预示着100%大麦啤酒的风味稳定性（新鲜度）明显好于100%麦芽啤酒。     

Differences in protein content and foaming properties of cloudy beers based on wheat malt content

To investigate differences in protein content, all barley malt beer, wheat/barley malt beer and all wheat malt beer were brewed, and the protein during mashing, wort, fermentation and beer determined. It was shown that protein was mainly extracted during mashing and the protein rest phase, decreased in the early stages of fermentation and remained almost steady during wort boiling and cooling, in the middle and late stages of fermentation. By separating beer foam from beer, similar protein bands of 51.7, 40.0, 27.3, 14.8, 6.5 and < 6.5 kDa appeared in the three beers, defoamed beers and beer foams using the sodium dodecyl sulphate polyacrylamide gel electrophoresis. Quantitatively, protein bands of 6.5–14.8 and <6.5 kDa had the highest contents in the three beers. Unique bands at 34, 29.2, 23.0, 19.7 and 17.7 kDa were found in beer, defoamed beer and beer foam from wheat beer and all‐wheat malt beer, respectively. Wheat beer foam showed the best foam stability and the protein in all barley malt beer showed the best migration to the foam. The beer foam properties were influenced by not only protein content but also protein characteristics and/or origin. It is suggested that the barley malt contributed the beer foam ‘skeleton protein’ while protein components from wheat malt kept the foam stable. © 2018 The Institute of Brewing & Distilling     

ISOELECTROPHORETIC SEPARATION OF COMPLEX NITROGENOUS SUBSTANCES OF BEER AND BREWING MATERIALS

Complex nitrogenous materials extracted from barley, malt, wort and beer have been separated by isoelectrophoresis in thin layers of Sephadex gel. Comparison and interpretation of the different isoelectric profiles has been attempted. A preparative isoelectrophoretic procedure has been developed. The isoelectric species of beer have been isolated in quantities sufficient for certain of their physical and chemical properties to be determined. It is concluded that substances with typical protein-like properties account for only about one-third (by weight) of the total complex-nitrogen fraction of beer. An arbitrary but useful classification of the complex-nitrogen fraction of beer is suggested.     

Comparison of beer quality attributes between beers brewed with 100% barley malt and 100% barley raw material

BACKGROUND: Brewing with 100% barley using the Ondea^® Pro exogenous brewing enzyme product was compared to brewing with 100% barley. The use of barley, rather than malt, in the brewing process and the consequences for selected beer quality attributes (foam formation, colloidal stability and filterability, sensory differences, protein content and composition) was considered. RESULTS: The quality attributes of barley, malt, kettle‐full‐wort, cold wort, unfiltered beer and filtered beer were assessed. A particular focus was given to monitoring changes in the barley protein composition during the brewing process and how the exogenous OndeaPro^® enzymes influenced wort protein composition. All analyses were based on standard brewing methods described in ASBC, EBC or MEBAK. To monitor the protein changes two‐dimensional polyacrylamide gel electrophoresis was used. CONCLUSION: It was shown that by brewing beer with 100% barley and an appropriate addition of exogenous Ondea^® Pro enzymes it was possible to efficiently brew beer of a satisfactory quality. The production of beers brewed with 100% barley resulted in good process efficiency (lautering and filtration) and to a final product whose sensory quality was described as light, with little body and mouthfeel, very good foam stability and similar organoleptic qualities compared to conventional malt beer. In spite of the sensory evaluation differences could still be seen in protein content and composition. Copyright © 2011 Society of Chemical Industry     

Processing of bottom‐fermented gluten‐free beer‐like beverages based on buckwheat and quinoa malt with chemical and sensory characterization

Typical beer contains significant amount of gluten, and being the third most popular beverage worldwide, the commercial production of its gluten‐free form is of rising interest. This research aimed to prepare bottom‐fermented beverages from buckwheat and quinoa and to explore their physical, chemical and sensory properties. Compared with barley, the analysis of brewing attributes of buckwheat and quinoa showed a lower malt extracts, longer saccharification times, higher total protein and fermentable amino nitrogen content and higher values of the iodine test and colour. Fermentability values, the wort pH and the soluble protein content were similar for barley and buckwheat, but different for quinoa, whereas only values of viscosity and beverage pH were similar between barley and quinoa. Both beverages, especially the quinoa beverage, contained a superior level of metal cations. The fermentable carbohydrate content in the buckwheat wort was comparable to barley but lower in quinoa; however, worts derived from both pseudocereals contained predominantly glucose. The amino acid content of the buckwheat wort was similar to barley, whereas the content in the quinoa beverage was almost twice as high. The content of volatile compounds commonly associated with beer aroma was comparable between the barley and buckwheat beverage but significantly lower in the quinoa; however, the latter contained some distinctive volatile substances not found in the other beverages. The organoleptic perception of the buckwheat beverage was better than that of the quinoa, although both showed a good general acceptance. In general, buckwheat appears quite similar to barley, whereas quinoa shows many unique properties. Copyright © 2014 The Institute of Brewing & Distilling     

Brewing with 100% Oat Malt

Oats are a cereal with beneficial nutritional properties and also unrealized brewing potential. Furthermore, oats can be tolerated by the majority of people who suffer from celiac disease. Malting of oats produced a malt, which was found suitable for brewing a 100% oat malt beer. The mashing regime, designed by using mathematical modelling, was successfully transferred to a pilot scale plant. The improved lautering performance of oat malt was due to its higher husk content, which also led to a lower extract content in oat wort when compared to barley wort. The protein profile of oat wort, as measured by using Lab‐on‐a‐Chip analysis, revealed that there was no significant difference in the protein profile between oat and barley wort. The fermentation of oat and barley worts followed the same trend; differences could only be seen in the higher pH and lower alcohol content of the oat beer. The flavour analysis of oat beer revealed some special characteristics such as a strong berry flavour and a lower amount of staling compounds when forced aged. This study revealed that it was possible to brew a 100% oat malt beer and that the produced beer was comparable to a barley malt beer.     

Corn grist adjunct – application and influence on the brewing process and beer quality

In the brewing industry, barley malt is often partially replaced with adjuncts (unmalted barley, wheat, rice, sorghum and corn in different forms). It is crucial, however, to preserve constant quality in the beer to meet the expectations of consumers. In this work, how the addition of corn grist (10 and 20%) influences the quality of wort and beer was examined. The following parameters were analysed: wort colour, dimethyl sulphide (DMS) and protein content, non‐fermentable extract, extract drop during fermentation, alcohol content and the attenuation level of the beer, together with filtration performance. The samples (all‐malt, and adjunct at 10 and 20% corn grist) were industrial worts and the beers produced in a commercial brewery (3000 hL fermentation tanks). The application of 10 and 20% corn grist had an effect on the wort colour, making it slightly lighter (11.1 and 10.5°EBC, respectively) than the reference barley malt wort (12.2°EBC). The free amino nitrogen level, DMS and non‐fermentable extract were significantly lower in the worts produced with the adjunct; the alcohol content and attenuation levels were higher in the beers produced with adjunct. The use of corn grist, at the level of up to 20% of total load, appears to affect some of the technological aspects of wort and beer production, but it does not significantly influence the final product characteristics. Copyright © 2014 The Institute of Brewing & Distilling     

Comparison of the impact on the performance of small‐scale mashing with different proportions of unmalted barley,Ondea Pro®, malt and rice

The impact of using different combinations of unmalted barley, Ondea Pro® and barley malt in conjunction with a 35% rice adjunct on mashing performance was examined in a series of small scale mashing trials. The objective was to identify the potential optimal levels and boundaries for the mashing combinations of barley, Ondea Pro®, malt and 35% rice (BOMR) that might apply in commercial brewing. Barley and malt samples used for the trials were selected from a range of Australian commercial barley and malt samples following evaluation by small‐scale mashing. This investigation builds on previous studies in order to adapt the technology to brewing styles common in Asia, where the use of high levels of rice adjunct is common. Mashing with the rice adjunct, combined with differing proportions of barley, Ondea Pro® and malt, resulted in higher extract levels than were observed for reference mashing, using either 100% malt reference or 100% barley reference and Ondea Pro® enzymes. Synergistic mashing effects between barley, Ondea Pro® and malt were observed for mash quality and efficiency parameters, particularly wort fermentability. The optimum levels of barley in the grist (with the relative level of Ondea Pro®) were assessed to be in the range 45–55% when paired with 10–20% malt and 35% rice. When the proportion of malt was reduced below 10% of the grist, substantial reductions in wort quality were observed for wort quality parameters including extract, lautering, fermentability, free amino nitrogen and haze. Extension of this new approach to brewing with rice adjuncts will benefit from further research into barley varietal selection in order to better meet brewer's quality requirements for the finished beer. Copyright © 2016 The Institute of Brewing & Distilling     

The Impact of Malt Derived Proteins on Beer Foam Quality. Part I. The Effect of Germination and Kilning on the Level of Protein Z4, Protein Z7 and LTP1

The effect of barley germination and kilning on three putative beer foam-positive proteins was investigated by immunoblotting and ELISA procedures. These procedures involved the use of specific antibodies raised to purified lipid transfer protein 1 (LTP1)and the two protein Z forms, Z4 (BSZ4) and Z7 (BSZ7). The free fraction of BSZ4 and BSZ7, and all LTP1 were extracted by aqueous salt-solution from barley and malt. The addition of reducing agent allowed the extraction of bound BSZ4 and BSZ7. A previously undescribed fraction of BSZ4 and BSZ7, refered to as latent, was extracted with SDS and reducing agent. The barley combined fraction (free + bound fractions) was surveyed in 93 barley varieties to show that BSZ4 was the dominant isoform, on average constituting ?80 % of all protein Z. Considerable variation was observed between varieties in the level of LTP1 (502–1144 μg/g) and the combined fractions of BSZ4 (18–2136 μg/g) and BSZ7 (38–771 μg/g). The free fraction is expected to be more available for extraction into wort during mashing than the bound or latent fractions. The level of LTP1 did not change substantially during germination, but a significant proportion of the latent and/or bound protein Z fractions was converted into the free fraction. In the seven varieties studied the free fraction of BSZ4 and BSZ7 increased 149–300% and 49–141%, respectively. Proteolytic cleavage in the reactive site loop converts protein Z to heat- and protease-stable forms that survive the brewing process. During germination most of the free BSZ4 and 30–70% BSZ7 was converted to the cleaved form. Kilning was found to reduce the amount of protein Z and LTP1 that could be extracted by 10–30% and 7–37%, respectively, which is likely to be counter productive for foam quality. These results suggest that barley variety selection and optimisation of germination and kilning protocols during malting may be opportunities for improvement of beer foam quality.     

Determination of Prolamins in Beers by ELISA and SDS‐PAGE

Coeliac disease is triggered by exposure to the prolamin protein fraction of wheat, barley, or rye. The prolamin content of five lager beers and one wheat beer were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting and seven lager beers and three wheat beers were analyzed by enzyme‐linked immunosorbent assay (ELISA). Most of the lager beers were made from barley and some had varying amounts of rice or corn as adjuncts. One of the beers was “gluten‐free”, having been produced from corn and buckwheat without barley. The lager beer samples were gel‐filtered before ELISA or SDS‐PAGE analysis. Prolamin proteins were found in all but one beer which was made of corn, rice and barley and which was not the “gluten‐free” beer. ELISA analysis was done using a commercially available gluten assay kit. For lager beers, a barley prolamin standard for ELISA was propanol‐extracted from barley malt instead of using the prolamin standard of the gluten assay kit. As expected, the wheat beers contained much higher amounts of prolamins than the lager beers. The samples were studied by SDS‐PAGE to identify different prolamin fractions. Proteins having a relative molecular mass in the range of 8000–17,000 and 38,000 and above were detected in immunoblotting by the prolamin sensitive antibody in the lager beers.     

Malted and unmalted oats in brewing

Using oats as a raw material in brewing has recently become the focus of increased interest. This is due to research findings that have shown that oats can be consumed safely by coeliac sufferers. It is also a response to consumer demand for products with novel sensory properties. In this study, beer was produced entirely from oat malt, from barley malt and from oat and barley malts mixed with various quantities of unmalted oats. Compared with barley wort, wort made from malted oats provided a lower extract content and had a higher protein content, but a lower free amino nitrogen content (FAN). The oat wort also showed increased viscosity and haze. The addition of unmalted oats during wort production produced significant changes in the physico‐chemical parameters of both oat and barley worts and beers. Unmalted oats caused an increase in wort viscosity and haze, and a reduction in total soluble nitrogen and FAN. Unmalted oats also contributed to lowering the concentration of higher alcohols and esters. Beer made from 100% oat and barley malts exhibited a similar alcohol content. The use of an oat adjunct in both cases resulted in a lower ethanol content. The introduction of enzyme preparations during the production of wort with oat adjunct had many benefits: increased extract content and FAN; a higher volume of wort; and a lower viscosity that led to faster wort filtration. This research suggests that the use of enzymes is necessary to make production using a high proportion of oats in the grist profitable. Copyright © 2014 The Institute of Brewing & Distilling     

Effect of wheat malt on the concentration of metal ions in wort and brewhouse by‐products

The ionic composition of brewer's wort depends on the raw materials and processing employed. The macroelement content is usually sufficient for yeast, but some of the microelements (mainly zinc) often need to be supplemented to the wort. Wheat malt is used as an adjunct in the production of beer, replacing up to 60% of barley malt. In this study, the effect of replacing barley malt with wheat malt on the concentration of magnesium, manganese, iron and zinc ions in brewer's wort was investigated. The ionic content of both the raw materials and by‐products were analysed. Similar amounts of magnesium ions were found in wheat malt and barley malt, whereas, manganese, zinc and iron were more abundant in the wheat malt. Nevertheless, wheat malt did not cause a significant change in ion concentration in the first wort (except for magnesium; its content decreased). The ionic content in the spent grains increased owing to wheat malt addition; the concentration of ions in the wort decreased after wort boiling (Mn²⁺ and Fe) or remained unaffected (Mg²⁺ and Zn²⁺). It was concluded that the ionic composition of the wort depends mainly on the removal rate of ions from the wort during mash filtration and hot trub separation, rather than on the actual amount of ions in the raw materials. Copyright © 2015 The Institute of Brewing & Distilling     

Suitability of unmalted quinoa for beer production

BACKGROUND

This study provides the first detailed investigation into the effect of partially substituting barley malt with quinoa (Chenopodium quinoa Willd.) on the characteristics of wort and beer. Quinoa seeds and flakes were compared in terms of their suitability for brewing. The benefits of applying a commercial enzyme mixture during beer production with quinoa were also investigated.

RESULTS

These findings show that quinoa is a good starchy raw material for brewing. Even without exogenous enzymes, it is possible to substitute barley malt with up to 30% quinoa. The form in which quinoa is used has a negligible influence on the quality of the wort and beer. The foam stability of beer made with quinoa was better than that of all‐malt beer, despite there being a lower level of soluble nitrogen in quinoa beer in comparison with all‐malt beer and more than twice the amount of fat in quinoa in comparison to barley malt.

CONCLUSION

The addition of unmalted quinoa does not give unpleasant characteristics to the beer and was even found to have a positive effect on its overall sensory quality. This offers brewers an opportunity to develop good beers with new sensory characteristics. © 2018 Society of Chemical Industry     

The influence of unmalted barley on the oxidative stability of wort and beer

The aim of this study was to investigate the influences of unmalted barley on the brewing process and the quality of the resulting beer‐like beverages, with the main focus on the oxidative stability, using traditional beer analyses, GC‐MS for the determination of aging compounds and electron paramagnetic resonance spectroscopy to determine free radical activity. For the investigation, brews with different barley proportions and 75% barley brews with a colour malt addition, to compensate for a lower colour using barley, were produced. In general, it can be said that beers with a proportion of up to 50% barley achieved a comparable or higher extract yield and final attenuation owing to the combined effectiveness of the malt and microbial enzymes. Although all analytical values were within the normal range according to Methodensammlung der Mitteleuropäischen Brautechnischen Analysenkommission (MEBAK), a slight decrease in total polyphenols and free amino nitrogen content was observed. Also in response to higher barley portions, an increase of higher molecular weight proteins and β‐glucan was detected. Barley is not exposed to heat and oxidative stress in the malting plant, which explains the lower values of the thiobarbituric acid index and colour as an indicator of Maillard reaction products in the resulting wort and beer. Additionally, the results demonstrate a slower increase of aging compounds during beer storage with increasing barley proportions. Furthermore, it was observed that higher barley proportions led to a better oxidative stability indicated by a lower radical generation (T₄₅₀‐value) in wort and an increasing beverage antioxidant index/endogenous antioxidative potential (BAX/EAP value) in the final beverage. The case of ‘barley beers’ showed that the positive effect of barley on the oxidative beer stability was greater than the negative effect of the addition of colour malt, to adjust the colour of a 100% malt beer. In sensory comparison with beer produced with 100% malt, the beers brewed with a barley proportion up to 50% showed a slight flavour preference and up to a 75% equivalent evaluation. Copyright © 2012 The Institute of Brewing & Distilling     

Fungal Hydrophobins in the Barley‐to‐Beer Chain

Fungal hydrophobins have been shown to induce gushing of beer. In order to study the occurrence and fate of hydrophobins at different stages of the production chain of beer, barley samples artificially infected in the field with Fusarium culmorum, F. graminearum and F. poae were collected during the growing period as well as during various stages of the malting process. In addition, naturally infected malt was brewed in pilot scale and samples were collected throughout the process. The samples were assayed for hydrophobin content using an ELISA method. The results showed that fungi produced hydrophobins that accumulated during barley grain development in the field, but that production was more pronounced during malting. Prolonged storage of barley tended to reduce the ability of fungi to produce hydrophobins in malting. Studies on the fate of hydrophobins during the brewing process revealed that mashing released hydrophobins from the malt into the wort. Some loss of hydrophobins occurred throughout the brewing process with spent grains, cold break (wort boiling) and surplus yeast. In addition, the beer filtration step reduced hydrophobin levels. Despite the substantial loss of hydrophobins during brewing, the level was high enough to induce the gushing detected in the final beer.     

The influence of LOX‐less barley malt on the flavour stability of wort and beer

The aim of this study was to investigate the influence of lipoxygenase‐less (LOX‐less) barley malt on the quality of wort and beer, with the main focus on beer flavour stability. In the current study, pilot‐scale (1000 L) brewing trials were conducted with a control barley malt AC Metcalfe and a LOX‐less barley malt, PolarStar. The results clearly indicated that the LOX‐less barley malt showed less nonenal potential than the control, although LOX activities in both barley malts were relatively low. The beer brewed from the LOX‐less barley malt contained much lower concentrations of trans‐2‐nonenal (T2N) and gamma‐nonalactone, especially after the (forced or natural) aging of the beer, compared with the beer brewed under the same conditions using the control malt. The sensory panel evaluation indicated similar results in the general flavour profile. The freshness scores of beer brewed from the LOX‐less malt were higher than those from the control malt, and this was more pronounced after forced aging. In addition, the beer brewed from LOX‐less malt had a much better foam stability, almost 30 s (NIBEM test). These results confirm that the use of the LOX‐less barley malt was beneficial to beer flavour stability and foam stability. Copyright © 2014 The Institute of Brewing & Distilling     

PROTEIN PROFILES OF BEER

As part of a study of the effect of replacing malt by barley and commercial enzyme preparations, gel chromatography on Sephadex columns and disc electrofocusing in polyacrylamide were used to prepare profiles of beer proteins. Both separation techniques revealed small though consistent differences in the protein profiles of the beers examined. The results of gel chromatography on Sephadex G-100 could be explained on the basis of raw materials composition. With disc electrofocusing correlations were observed between protein profile and composition of the raw materials and between protein profile and mashing procedure. Disc electrofocusing proved to be a reproducible and rapid method for preparing a “fingerprint” of beer proteins.