Beer Foam Stability Research Articles

The Influence of Barley Malt Protein Modification on Beer Foam Stability and Their Relationship to the Barley Dimeric α-Amylase Inhibitor-I (BDAI-I) as a Possible Foam-Promoting Protein

The foam stability of beer is one of the important key factors in evaluating the quality of beer. The purpose of this study was to investigate the relationship between the level of malt modification (degradation of protein, starch, and so on) and the beer foam stability. This was achieved by examining foam-promoting proteins using two-dimensional gel electrophoresis (2DE). We found that the foam stability of beer samples brewed from the barley malts of cultivars B and C decreased as the level of malt modification increased; however, the foam stability of cultivar A did not change. To identify the property providing the increased foam stability of cultivar A, we analyzed beer proteins using 2DE. We analyzed three fractions that could contain beer foam-promoting proteins, namely, beer whole proteins, salt-precipitated proteins, and the proteins concentrated from beer foam. As a result, we found that in cultivar A, some protein spots did not change in any of these three protein fractions even when the level of malt modification increased, although the corresponding protein spots in cultivars B and C decreased. We analyzed these protein spots by peptide mass finger printing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. As a result, all of these spots were identified as barley dimeric alpha-amylase inhibitor-I (BDAI-I). These results suggest that BDAI-I is an important contributor to beer foam stability.

Construction of a SinglePEP4Allele Deletion inSaccharomyces carlsbergensisand a Preliminary Evaluation of Its Brewing Performance

The secretion of proteinase A (encoded by PEP4) from brewer's yeast is detrimental to the foam stability of unpasteurized beer. The aim of this study was to construct mutants of the allopolyploid Saccharomyces carlsbergensis strain TT, which were partially or completely deficient in proteinase A activity. Allelic PEP4 genes were consecutively disrupted by using the Cre-loxP recombination system combined with PCR-mediated gene disruption. A single PEP4 deletion mutant TT-M was successfully constructed. However, no viable mutant could be obtained when the second allelic PEP4 gene was deleted. The brewing performances of the parent strain and the modified strain were compared on a 100 L pilot fermenter scale. Proteinase A activity in fermented wort brewed with mutant strain TT-M was significantly lower (p<0.05) than that of the parent strain TT, whereas no significant difference on either maltose or maltotriose assimilation (p>0.05) was found. The mutant TT-M remained genetically stable, as shown by diagnostic PCR, after re-streaking for 20 generations. The flavor and taste of the final fermented wort, brewed with the mutant strain TT-M, was evaluated by the Tsingtao expert sensory panel, and found to be comparable to that of the parent strain and exhibited no distinct defects. The flavor component profiles of these two finished products were also comparable. The study demonstrated allelic genes in polyploid industrial yeasts could be efficiently and consecutively deleted by the retractive primer disruption strategy, and the mutant of Saccharomyces carlsbergensis partially deficient in proteinase A contributed to an improvement in foam stability.

Characterization of AfpA, an alkaline foam protein from cultures of Fusarium culmorum and its identification in infected malt

The objective of this study was to evaluate the capability of Fusarium culmorum to produce non-hydrophobin surface-active proteins in vitro, to isolate and characterize such proteins from liquid cultures, to analyse their effect on overfoaming (gushing) of beer and to elucidate their prevalence in pure cultures and infected malt. A 20 kDa protein was isolated from liquid cultures of F. culmorum BBA 62182 upon enrichment by foaming. BLAST search with N-terminal and internal sequences of the protein revealed high homology with a hypothetical protein predicted within the F. graminearum PH1 genome sequence. Oligonucleotide primers designed to bind 30 nt upstream and downstream of the predicted gene were used to amplify a 695 nt PCR fragment from genomic DNA of F. culmorum BBA 62182. Cloning and sequencing of the product revealed a 635 nt open reading frame which had 98% homology to the predicted F. graminearium PH1 gene code. Removal of a 59 nt intron and translation resulted in a 191 amino acid protein of 20.754 kDa with a calculated pI of 9.1. Amino acids obtained by Edman sequencing of fragments within the 20 kDa protein were 100% homologous with the sequence deduced from the DNA sequence. According to its properties, the new protein was termed alkaline foam protein A (AfpA). Sequence comparison revealed some homologies with proteins in Emericella nidulans, which are involved in phialide development and response to antifungal agents. Homologies with other hypothetical fungal proteins suggest a new group of proteins, for which we suggest the name fungispumins. Addition of AfpA to beer showed that overfoaming (gushing) is not induced in stable beer but can significantly enhance this effect in beer showing moderate gushing. Use of a polyclonal anti-AfpA antibody in a Western blot revealed that the protein is produced by various F. culmorum strains and also by F. graminearum, but not by other Fusarium spp. tested. PCR testing of 69 species of Fusarium and Trichoderma reesei with a gene specific primer pair revealed that the gene may be present exclusively in F. culmorum, F. graminearum, F. cerealis, F. lunulosporum and F. oxysporum f. sp . dianthi. Immunochemical detection of AfpA in malts artificially inoculated with F. culmorum and F. graminearum showed that the protein was present in gushing inducing malts (gushing test) but absent in malts which were negative in a gushing test. AfpA is a member of a new protein class, fugispumins, and can be isolated from pure liquid cultures of F. culmorum. A homologous protein is synthesised by F. graminearum. The protein is produced in contaminated malt and enhances gushing of beer. The gene coding for AfpA is restricted to Fusarium species presumably involved in the induction of beer gushing. We describe a new class of proteins, fungispumins, the natural function of which remains to be elucidated. Findings add useful information to research on the mechanisms involved in foam stability of beer. AfpA may be useful as a marker for gushing in future quality control applications for the brewing industry.

The Disc Stack Centrifuge and its Impact on Yeast and Beer Quality

The impact of a disc stack centrifuge on brewers' yeast (Saccharomyces cerevisiae) integrity was studied. The physiological state of brewers' yeast is principally dependant on the processing conditions it encounters during wort fermentation and yeast cropping. During cropping by centrifugation, the yeast is subjected to extreme shear stress. The aim of this project was to determine whether the passage of yeast cells through a disc stack centrifuge had a negative effect on cell viability and vitality. Cell viability was determined using flow cytometry and methylene blue staining. Flow cytometeric methods permit quantitative representation of a cell population. The results were compared, and both techniques produced similar results. However, viabilities determined by methylene blue were consistently lower than those determined by flow cytometry. The viability of both the ale and lager yeast strains analyzed decreased as they were cycled through the centrifuge. The ale yeast used was more susceptible to centrifugation damage than the lager yeast strain. In the fermentation trials with the ale yeast strain, the centrifuged cells exhibited slower fermentation rates and increased proteinase A (PrA) activity at the beginning of fermentation. PrA degrades foam-positive polypeptides, resulting in decreased beer foam stability. Beer mannan concentration increased as exposure to shear stress was applied. It was concluded that passage of yeast cells through a disc stack centrifuge resulted in a decrease in cell viability, vitality, physical stability, and beer quality.

Relationship of Proteinase Activity, Foam Proteins, and Head Retention in Unpasteurized Beer

Head retention, proteinase activity, and protein content in unpasteurized beer stored at different temperatures for different storage times were tested. Analysis of variance indicated that treatment (pasteurization), storage time, storage temperature, and associated interactions all had highly significant effects on the level of proteinase activity, head retention, and protein content. The change in beer proteins during storage and the relationship between proteinase A and foam proteins were also studied. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that lipid transfer protein1 was degraded and this foam-active protein was a major substrate of proteinase A. Degradation of lipid transfer protein 1 is a major factor that causes a decrease in beer foam stability.

Effective Prevention of Chill-Haze in Beer Using an Acid Proline-Specific Endoprotease from Aspergillus niger

Chill-haze formation during beer production is known to involve polyphenols that interact with proline-rich proteins. We hypothesized that incubating beer wort with a proline-specific protease would extensively hydrolyze these proline-rich proteins, yielding a peptide fraction that is unable to form a haze. Predigestion of the proline-rich wheat gliadin with different proteases pointed toward a strong haze-suppressing effect by a proline-specific enzyme. This finding was confirmed in small-scale brewing experiments using a recently identified proline-specific protease with an acidic pH optimum. Subsequent pilot plant trials demonstrated that, upon its addition during the fermentation phase of beer brewing, even low levels of this acidic enzyme effectively prevented chill-haze formation in bottled beer. Results of beer foam stability measurements indicated that the enzyme treatment leaves the beer foam almost unaffected. In combination with the enzyme's cost-effectiveness and regulatory status, these preliminary test results seem to favor further industrial development of this enzymatic beer stabilization method.

Effects of the Combination of Hydrophobic Polypeptides, Iso-α Acids, and Malto-oligosaccharides on Beer Foam Stability

The influence of hydrophobic polypeptides concentrated in beer foam, together with the composition of iso-alpha acids and the content of malto-oligosaccharides in beer on foam stability, has been investigated. The objective was to find out whether a shortage of one of these positive contributors to foam stability could be compensated for by an increased presence of another or whether optimum levels of each contributor is necessary. For that purpose, an image analysis method to evaluate beer foam quality was developed. The foam collapse time was the parameter chosen to group beers according to their foam stability. Profiles of hydrophobic polypeptides that concentrate in beer foam, iso-alpha acids, and malto-oligosaccharides of 14 beer brands were acquired by high-performance liquid chromatography. Principal component analysis (PCA) was performed to show the relationship between beer brands and its composition. Beers that contained propylene glycol alginate as a foam enhancer showed high foam stability except for one beer, which had a low content of hydrophobic polypeptides, thereby highlighting the requirement of threshold levels of hydrophobic polypeptides to obtain stable foam. The data of samples that were devoid of a foam additive were subjected to a discriminant statistical analysis. Foam stability declined in proportion to decreases in hydrophobic polypeptides and to a lesser extent to decreases in iso-alpha-acid contents. Apparently, the content of malto-oligosaccharides were found to have no major influence on foam stability. The model of discriminate analysis was found to explain 100% of the variance in data with 85.2% success in classifying all samples according to the model, suggesting that foam stability is mainly governed by the beer constituents evaluated in this study.

Fungal Hydrophobins as Predictors of the Gushing Activity of Malt

Fungal infection of barley and malt, particularly by strains of the genus Fusarium, is known to be a direct cause of beer gushing. We have shown previously that small fungal proteins, hydrophobins, isolated from strains of the genera Fusarium, Nigrospora and Trichoderma act as gushing factors in beer. A hydrophobin concentration as low as 0.003 ppm was sufficient to induce gushing. The gushing-inducing abilities of the isolated hydrophobins varied probably due to their structural differences. The hydrophobins did not affect beer foam stability. A correlation was observed between the hydrophobin level analyzed by the hydrophobin ELISA developed and the gushing potential of malt. The risk of gushing was found to increase with hydrophobin concentrations above 250 μg/g malt. The levels of hydrophobin and the Fusarium mycotoxin deoxynivalenol (DON) in malts were not correlated which indicated that the formation of those two fungal metabolites may not be linked. Furthermore, we did not observe a correlation between the DON content and the gushing potential of the malt studied. Our observations suggest that the accuracy of predicting gushing could be improved by measuring the amount of the actual gushing factors, hydrophobins, in barley or malt.

Impact of different wort boiling temperatures on the beer foam stabilizing properties of lipid transfer protein 1.

Beer consumers demand satisfactory and consistent foam stability; thus, it is a high priority for brewers. Beer foam is stabilized by the interaction between certain beer proteins, including lipid transfer protein 1 (LTP1), and isomerized hop alpha-acids, but destabilized by lipids. In this study it was shown that the wort boiling temperature during the brewing process was critical in determining the final beer LTP1 content and conformation. LTP1 levels during brewing were measured by an LTP1 ELISA, using antinative barley LTP1 polyclonal antibodies. It was observed that the higher wort boiling temperatures ( approximately 102 degrees C), resulting from low altitude at sea level, reduced the final beer LTP1 level to 2-3 microg/mL, whereas the lower wort boiling temperatures ( approximately 96 degrees C), resulting from higher altitudes (1800 m), produced LTP1 levels between 17 and 35 microg/mL. Low levels of LTP1 in combination with elevated levels of free fatty acids (FFA) resulted in poor foam stability, whereas beer produced with low levels of LTP1 and FFA had satisfactory foam stability. Previous studies indicated the need for LTP1 denaturing to improve its foam stabilizing properties. However, the results presented here show that LTP1 denaturation reduces its ability to act as a binding protein for foam-damaging FFA. These investigations suggest that wort boiling temperature is an important factor in determining the level and conformation of LTP1, thereby favoring satisfactory beer foam stability.

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.

Destabilization of Beer Foam by Lipids: Structural and Interfacial Effects

Mechanisms in which fatty acids destabilize beer foam have been studied. Foam stability of a pilot-brewed beer was measured in the presence of a range of concentrations of fatty acids, similar to those found in a range of commercial beers. The foams were sparged with nitrogen and studied using a microconductivity technique. The fatty acid chain length varied from C6 to C18, C18:1, and C18:2. While C6 to C10 fatty acids had no impact on the foam stability over the concentration range studied, the longer-chain fatty acids were more destructive. Thus, C12 to C14, C18:1, and C18:2 reduced foam stability and the surface elasticity of beer. These data suggest that the fatty acids adsorbed into the protein-stabilized surface weakened the adsorbed film, resulting in an increased probability of coalescence. The saturated fatty acids, C16:0 and C18:0, damaged the foam very effectively but did not influence the surface rheology. Light-scattering experiments showed increased numbers of aggregates in these samples, suggesting that these fatty acids destabilized beer foam through a mechanical film-bridging mechanism, similar to that used by particulate antifoam systems.

Application of Immunological Methods to Differentiate between Foam-Positive and Haze-Active Proteins Originating from Malt

Beer foam and storage haze stability are of critical importance to brewers because they are among the first characteristics by which a consumer judges the quality of his or her beer. Key malt-derived, foam-positive proteins, including protein Z4, lipid transfer protein 1 (LTP1), and some members of the hordein storage protein family, interact principally with hop acids to stabilize foam. Silica-mediated beer stabilization procedures remove haze-active protein with high levels of proline (>30%) and glutamine (>30%), suggestive of a hordein origin, without reducing foam stability. Polyclonal antibodies to protein fractions of enriched beer foam proteins and proteins extracted from silica used for beer stabilization were developed. The antibodies were found to measure different malt-derived proteins. The antibodies were subsequently used to select foam-positive and haze-active hordein-recognizing monoclonal antibodies, which enabled the development of enzyme-linked immunosorbent assays (ELISAs). The ELISAs and immunochemical procedures were applied to predict beer foam and haze quality from malt used to brew beer by using pilot- (50 L) and small-scale (0.6–0.8 L) brewing procedures. It is anticipated that the immunochemical methods may be applied to select and manipulate malt for brewing to improve beer foam stability while reducing the likelihood of storage haze formation.

Determination of the Foam Stability of Beer Using the NIBEM-T Meter

Journal of the Institute of BrewingVolume 109, Issue 4 p. 400-402 Free Access Determination of the Foam Stability of Beer Using the NIBEM-T Meter A.A. Ferreira, A.A. FerreiraSearch for more papers by this authorOn behalf of the Analysis Committee of the European Brewery Convention, On behalf of the Analysis Committee of the European Brewery ConventionSearch for more papers by this author A.A. Ferreira, A.A. FerreiraSearch for more papers by this authorOn behalf of the Analysis Committee of the European Brewery Convention, On behalf of the Analysis Committee of the European Brewery ConventionSearch for more papers by this author First published: 09 October 2012 https://doi.org/10.1002/j.2050-0416.2003.tb00614.xCitations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume109, Issue42003Pages 400-402 ReferencesRelatedInformation

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.

The characterisation and mapping of a family of LMW-gliadin genes: effects on dough properties and bread volume.

Analysis of a cDNA library from wheat cv Wyuna endosperm, indicated a significant size and sequence variation among seed-endosperm protein genes. In this study, a family of low-molecular-weight seed protein genes are analysed that are related to the gliadins and the low-molecular-weight glutenin subunits. Sequence analysis and comparison of these proteins showed that they are closely related to a 17-kDa protein from barley, epsilon hordein, which plays a role in beer foam stability in the brewing industry. Mapping of these genes in wheat shows that they are located on group 7 and 4 chromosomes, as opposed to a group 1 and 6 location for the glutenins and gliadins. It is possible that this family of proteins forms a new class of seed-endosperm proteins important in defining the quality characteristics of wheat flour. Therefore, a representative gene from this family was expressed in Escherichia coli and the purified protein was supplemented into a base wheat flour. Rheological analysis showed that the protein effected dough strength and resistance break down during mixing of the dough, and provided a 20% increase in loaf height after baking.

Barley β-Glucan and Beer Foam Stability

Barley β-glucan was examined as a potential foam-enhancing or foam-stabilizing beer component. Neither foam enhancement nor foam stabilization was observed. Treating beer with β-glucanase did not alter the foam properties. β-Glucan did not concentrate in foam collected by the foam tower method from a European Pilsner-style beer with a relatively high known β-glucan content. β-Glucan molecular weight fractionation was not observed. β-Glucan did not have independent foam stability when tested in a model beer system.

Yeast Proteolytic Activity During High and Low Gravity Wort Fermentations and its Effect on Head Retention

The aim was to discover the effect of high gravity brewing on yeast protease activity during fermentation, on the loss of hydrophobic polypeptides from wort during fermentation, and on the foam stability of stored beer. The hydrophobic polypeptide content of low (10° Plato) gravity worts showed a steady decline throughout fermentation, but for the 20° Plato wort there was a rapid decline over the first 8 days of fermentation, followed by little change over the remaining period. The decrease in hydrophobic polypeptides was greater in the high gravity fermentation. Proteinase A increased during fermentations with the highest levels being present at the end of fermentations. High gravity fermentations exhibited levels of yeast protease that from the 3rd to 11th day of fermentation were at least twice the values of the low gravity fermentations. The high gravity brewed beer contained significantly higher levels of proteinase A activity than the low gravity brewed beer. The inclusion of FERMCAP , an antifoam, in high gravity wort did not affect either the hydrophobic polypeptide levels or foam stability of the resultant beer. This suggests that proteinase A, rather than fermenter foaming, must be the major contributor to the lack of foam stability of high gravity brewed beer. Head retention measurements conducted on the high and low gravity brewed bottled beers, over a five month period, demonstrated a steady decline in foam stability for both beers. The declines in head retention did not occur in high and low gravity beers that had been pasteurised.

Molecular Marker for Protein Z Content in Barley (Hordeum vulgae L.).

Protein Z, a major water-soluble seed protein of barley, is thought to be one of the factors influencing foam stability of beer. To find the breeding material for raising the protein Z content in nralting barley, we surveyed the protein Z contents of malting barley varieties. Generally, North American six-row varieties had higher contents than Japanese varieties. Especially, variety Bonanza had the highest protein Z content among the 62 varieties surveyed. Using the lines derived from the crosses between Bonanza and the Japanese malting varieties, we found that the protein Z structural gene region contributed to the protein Z content of seeds. We also estimated the position of protein Z structural gene on chromosome 4H using a set of RFLP markers. Consequently, we concluded that the high protein Z content of Bonanza could be introduced and screened by the RFLP patterns of the protein Z structural gene.

The Impact of Malt Derived Proteins on Beer Foam Quality. Part II: The Influence of Malt Foam-positive Proteins and Non-starch Polysaccharides on Beer Foam Quality

Pilot (50 litre) and small scale (700 mL) brewing trials conducted using, similar brewing protocols with 25 different malts, indicated that differences in malt quality influenced foam stability (Rudin head retention value) by up to 24%. In addition to conventional measures of malt quality, enzyme-linked immunosorbent assays (ELISA) were used to measure the level of the putative foam-positive proteins, BSZ4 (protein Z4), BSZ7 (protein Z7), BSZ7b and lipid transfer protein 1 (LTP1). Regression analysis performed on the combined pilot and small scale data sets identified that malt BSZ4, wort β-glucan and wort viscosity, and beer protein, β-glucan and arabinoxylan were positively correlated with foam stability, while malt Kolbach index (KI), and beer FAN were negatively correlated with foam stability. Potentially foam-positive proteins such as BSZ7 and LTP1 were not correlated with foam stability. The negative correlation of BSZ4 level with KI suggested an additional role for BSZ4 in influencing protein modification. Step-wise multiple regression indicated that up to 82% of the variation in foam stability could be predicted from the malt and beer characteristics measured, demonstrating that there are a number of inter related malt derived factors that influence beer foam stability.

Immunological Study of Hydrophobic Polypeptides in Beer

A panel of 15 monoclonal antibodies (mAbs) has been raised to a fraction of lager beer protein precipitated with ammonium sulfate at 40% saturation. The antibodies have been characterized in terms of their binding to a set of Octyl-Sepharose fractions of lager beer foam with increasing hydrophobicity (groups 1−5) by enzyme-linked immunosorbent assay and immunoblotting methods. The epitopes recognized were present in all of the lager beer foam fractions to some degree, and many of the mAbs bound to both high molecular mass (MM) (>3000 Da) and low MM (<3000 Da) material. Two antibodies, IFRN 1612 and 1613, bound primarily to the high MM material and preferentially recognized the more hydrophobic foam groups important in beer foam formation and stability. All of the mAbs recognized proteins present in malt, with much of the binding directed toward the material soluble in aqueous propanol from the highly modified crystal malt used in the lager beer production. Such a library of mAbs will be of use in investigating the contribution made by proteins and derived polypeptides, such as those from malt, to quality attributes such as foaming. Keywords: Beer; foam; malt; monoclonal antibodies; immunoassay