Induction of Gushing with Recombinant Class II Hydrophobin FcHyd5p from Fusarium culmorum and the Impact of Hop Compounds on its Gushing Potential

Malt‐induced gushing is a problem that has been known for many years. Mechanisms and inducing agents are still not fully understood and identified. Hydrophobins produced by various filamentous fungi are currently under discussion as biological gushing‐inducing compounds. In the current study the class II hydrophobin FcHyd5p, from the cereal pathogen Fusarium culmorum, was employed in beer and other carbonated beverages for gushing experiments and the influence of hop compounds on gushing potential was examined. It was demonstrated that this protein strongly induces gushing in various carbonated beverages, including beer. It was further demonstrated that the resulting gushing volume is susceptible to certain hop compounds and can be decreased significantly by the addition of these substances.     

A Review of Hop Resistance in Beer Spoilage Lactic Acid Bacteria

Hop bitter acids play a major role in enhancing the microbiological stability of beer. However, beer spoilage lactic acid bacteria (LAB) are able to grow in beer by exhibiting strong hop resistance. Recently two hop resistance genes, horA and horC, have been identified in beer spoilage Lactobacillus brevis ABBC45. The horA gene was shown to encode an ATP dependent multidrug transporter that extrudes hop bitter acids out of bacterial cells. In contrast, the product of the horC gene confers hop resistance by presumably acting as a proton motive force (PMF)‐dependent multidrug transporter. Strikingly, the homologs of horA and horC genes were found to be widely and almost exclusively distributed in various species of beer spoilage LAB strains, indicating these two hop resistance genes are excellent species‐independent genetic markers for differentiating the beer spoilage ability of LAB. Furthermore the nucleotide sequence analysis of horA and horC homologs revealed that both genes are essentially identical among distinct beer spoilage species, indicating horA and horC have been acquired by beer spoilage LAB through horizontal gene transfer. Taken collectively, these insights provide a basis for applying horA and horC to the species‐independent determination of beer spoilage LAB, including yet uncharacterized species. In addition to the hop resistance mechanisms mediated by multidrug transporters, proton translocating ATPase and the ATP production system were shown to contribute to the hop resistance mechanisms in beer spoilage LAB by generating PMF and ATP that are necessary for survival in beer.     

Humulus lupulus – a story that begs to be told. A review

The hop cones of the female plant of the common hop species Humulus lupulus L. are grown almost exclusively for the brewing industry. Only the cones of the female plants are able to secrete the fine yellow resinous powder (i.e. lupulin glands). It is in these lupulin glands that the main brewing principles of hops, the resins and essential oils, are synthesized and accumulated. Hops are of interest to the brewer since they impart the typical bitter taste and aroma to beer and are responsible for the perceived hop character. In addition to the comfortable bitterness and the refreshing hoppy aroma delivered by hops, the hop acids also contribute to the overall microbial stability of beer. Another benefit of the hop resins is that they help enhance and stabilize beer foam and promote foam lacing. In an attempt to understand these contributions, the very complex nature of the chemical composition of hops is reviewed. First, a general overview of the hop chemistry and nomenclature is presented. Then, the different hop resins found in the lupulin glands of the hop cones are discussed in detail. The major hop bitter acids (α‐ and β‐acids) and the latest findings on the absolute configuration of the cis and trans iso‐α‐acids are discussed. Special attention is given to the hard resins; the known δ‐resin is reviewed and the ε‐resin is introduced. Recent data on the bittering potential and the antimicrobial properties of both hard resin fractions are disclosed. Attention is also given to the numerous essential oil constituents as well as their contributions to beer aroma. In addition to the aroma contribution of the well‐known essential oil compounds, a number of recently identified sulfur compounds and their impact on beer aroma are reviewed. The hop polyphenols and their potential health benefits are also addressed. Subsequently, the importance of hops in brewing is examined and the contributions of hops to beer quality are explained. Finally, the beer and hop market of the last century, as well as the new trends in brewing, are discussed in detail. Hop research is an ever growing field of central importance to the brewing industry, even in areas that are not traditionally associated with hops and brewing. This article attempts to give a general overview of the different areas of hop research while assessing the latest advances in hop science and their impact on brewing. Copyright © 2014 The Institute of Brewing & Distilling     

Enzymatic release of odourant polyfunctional thiols from cysteine conjugates in hop

Polyfunctional thiols are contributors to the hop varietal aroma of beer. Besides free thiols, a cysteine‐S‐conjugate has recently been shown to be an additional component of the thiol potential of hop. Such cysteine adducts investigated here in four hop cultivars and in different hop forms. Hop hydroalcoholic extracts were purified on a cation exchanger and subjected to apotryptophanase β‐lyase activity. The Cascade hop variety exhibited the highest bound 3‐sulphanylhexan‐1‐ol (grapefruit‐like) potential, while both Tomahawk and Nelson Sauvin cultivars were confirmed to be important sources of bound 3‐methyl‐2‐butene‐1‐thiol (skunky‐like), 3‐sulphanylpentan‐1‐ol and 4‐sulphanyl‐4‐methylpentan‐2‐one (box‐tree‐like). Surprisingly, hop CO₂ extracts proved to contain cysteine conjugates. Although related, the concentrations of cysteine‐bound thiols in hop are not strictly correlated to the amounts of free volatiles found in the derived beers. Copyright © 2013 The Institute of Brewing & Distilling     

FATTY ACIDS IN ISOMERIZED HOP EXTRACTS AND GUSHING

A procedure is described for the analysis of fatty acids (C₁₂-C₁₈ individually and C₁₈₊ as a group) in hops, hop extracts and isomerized hop extracts, and analytical results are quoted for twenty-one different samples of hops or hop products. Isomerized extracts varied widely in their content of fatty acids and isomerization and processing of hops appeared to eliminate some fatty acids selectively so that isomerized extracts were enriched in palmitic acid, linoleic acid and linolenic acid. Some hop extracts had a surprisingly high content of lauric acid. The analytical results are discussed in relation to gushing.     

Possibilities to improve the antioxidative capacity of beer by optimized hopping regimes

Different hopping regimes were evaluated to investigate the effect on the oxidative stability of wort and beer. Compared with a single hop dosage at the beginning of wort boil, it was possible to increase the concentration of α‐acids in pitching wort and beer by applying incremental hop dosage, dry hopping or the use of a pre‐isomerized hop product in combination with an α‐acid extract, which concomitantly resulted in lower iron concentrations and an enhanced flavour stability as indicated by standard wort and beer analyses, atomic absorption spectroscopy, electron spin resonance spectroscopy and sensory analysis of fresh and force‐aged beers. The functional principle of hop dosage variations is explained by saving of α‐acids throughout the wort production process, which yields an increased formation and precipitation of pro‐oxidative acting transition metal ions (e.g. Fe) in α‐acid‐complexes during the whirlpool rest and fermentation. Consequently, fewer reactive oxygen species are generated. Additional laboratory trials simulating wort cooling and beer storage in buffered model solutions proved that un‐isomerized α‐acids are strong iron chelators and confirmed the functional principle of the applied hopping regimes. Negative effects of higher α‐acid contents on fermentation performance and depletion of the zinc concentration, which is an essential nutrient for yeast, could be excluded. Copyright © 2014 The Institute of Brewing & Distilling     

CAMBRIDGE PRIZE LECTURE. STUDIES ON THE SENSITIVITY OF LACTIC ACID BACTERIA TO HOP BITTER ACIDS

Hop bitter acids act as mobile-carrier ionophores. They inhibit the growth of beer-spoilage bacteria by dissipating the transmembrane pH gradient. Their activity is pH dependent. Low pH favours antibacterial activity but high pH reduces it. Resistance to hop bitter acids is a stable character, associated only with beer-spoilage lactic acid bacteria. Hop-resistant organisms can maintain a larger transmembrane pH gradient and ATP pool than can hop-sensitive organisms. Prior exposure of bacteria to trans-isohumulone does not influence the degree of resistance to hop bitter acids. However, in some strains, exposure to trans-isohumulone does induce the ability to spoil beer. The chemistry of these compounds is more complex than previously thought. In aqueous solutions, such as beer, hop acids bind to metal ions and may be covalently hydrated.     

HOP PRODUCTS

Hop products have established an important and permanent position in the brewing raw materials market. Hop pellets and hop extract offer significant advantages to traditional brewers. However, the ultimate hop product is the isomerised hop extract, which can now be produced without the involvement of organic solvents. Still to be resolved and hence controlled is the contribution of the hop oil fraction to beer flavour.     

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.     

The Oxygenated Sesquiterpenoid Fraction of Hops in Relation to the Spicy Hop Character of Beer

Hop‐derived sesquiterpenoid‐type oxidation products have been associated with a spicy or herbal hoppy beer character. However, the flavour threshold values of hitherto identified oxygenated sesquiterpenes are generally much higher than their estimated levels in beer. By applying two‐step supercritical fluid extraction of hop pellets using carbon dioxide, followed by chromatographic purification of the enriched sesquiterpenoid fraction, highly specific varietal hop oil essences containing all main oxygenated sesquiterpenes were obtained. Post‐fermentation addition (at ppb levels) of these purified sesquiterpenoid essences from various European aroma hops led to distinctive spicy or herbal flavour notes, reminiscent of typical ‘noble’ hop aroma. It is concluded that a spicy hop flavour impression in beer depends significantly on minor constituents of the natural sesquiterpenoid hop oil fraction.     

新型酒花制品在啤酒工业的应用

啤酒花是啤酒工业的重要原料之一。阐述了啤酒花中3类物质即酒花树脂、酒花油、多酚物质的主要化学成分，酒花在啤酒酿造中的作用，以及酒花浸膏和酒花油等多种新型的酒花制品在啤酒工业中的应用。     

125th Anniversary Review: The Role of Hops in Brewing

Although hop technology has been a substantial part of brewing science for the last 130 years, we are still far from claiming to know everything about hops. As hops are considered primarily as a flavour ingredient for beer, with the added benefit of having anti‐microbial effects, hop research is focused on hops as a bittering agent, as an aroma contributor and as a preservative. Newer fields in hop research are directed toward the relevance of hops in flavour stability, brewing process utilisation, the technological benefits of hops in brewing as well as hops as a source of various substances with many health benefits. However the more we find out about the so‐called “spirit of beer” the more questions emerge that demand answers. While hop research was only an ancillary research field for decades, during the last ten years more universities and breweries have determined that hops must play a meaningful role in their research efforts. This article gives an overview of the up‐to‐date knowledge on hop aroma, hop derived bitterness, and the role of hops in flavour stability as well as light stability. Hop research is a wide field, therefore in this review only selected topics are reviewed. Other research areas such as hops utilisation, the antifoam potential of hops, or the advances in knowledge pertaining to the physiological valuable substances of hops go beyond the scope of this article.     

Enhanced Quantitative Extraction and HPLC Determination of Hop and Beer Bitter Acids

A pleasant and consistent bitterness is an essential flavour attribute of beer. Hop‐derived iso‐α‐acids are largely responsible for beer bitterness and accurate determination of these primary flavour compounds is very important in relation to quality control. The most widely used way to determine beer bitterness is based on spectrophotometry, measuring the absorbance of an iso‐octane extract of acidified beer. However, this approach is far from specific as it measures all of the extracted compounds, including non‐bittering principles. For that particular reason, High Performance Liquid Chromatography (HPLC) is increasingly applied for the quantitative determination of hop‐derived iso‐α‐acids and, if present, reduced iso‐α‐acids. However, to obtain accurate data on beer bitterness profiles, both quantitative sample preparation and state‐of‐the‐art HPLC are essential. In this paper, several extraction procedures based on solid phase extraction (SPE) and liquid‐liquid extraction (LLE), respectively, were evaluated and an optimised extraction methodology using H₃PO₄ for sample acidification prior to extraction is presented. The proposed extraction/HPLC methodology allows for the quantitative recovery and analysis of hop‐derived beer bitterness.     

Pediococcus damnosus strains isolated from a brewery environment carry the horA gene

Beer is recognized as a safe beverage, owing to its excellent microbiological stability provided by its components, especially iso‐α‐acids from hop and ethanol which have antimicrobial activity. Despite these unfavourable conditions for bacteria, some lactic acid bacteria (LAB) can cause beer spoilage. Resistance to hop compounds is caused, in part, by the product of genes like horA . Understanding how LAB adapts to hop compounds as well as quick detection of these microorganisms is necessary to ensure high‐quality beverages produced by the brewing industry. In this work, we searched for the presence of two main hop‐resistance genes, horA and ORF5, and determined the capacity of four strains of Pediococcus damnosus isolated from a brewery environment to grow in the presence of increasing concentrations of iso‐α‐acids. All strains were able to grow in increasing concentrations of iso‐α‐acids up to 150 μg mL⁻¹. This amount is 10 times greater than the concentration in average beer. Genetic amplification of genes associated with hop‐resistance, demonstrated the presence of horA , but not ORF5 in all tested strains. This communication represents the first report of the presence of horA gene in bacteria isolated from breweries in our country. Copyright © 2017 The Institute of Brewing & Distilling     

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.     

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

All-malt Pilsner beers were brewed with proanthocyanidin-free malt (ant 13 × 13 × Rupal). Hopping was with n-hexane tannin-free hop extract with or without n-hexane extracted hop residue or with whole leaf hops. The different beers were analysed chemically and presented to an expert panel to detect possible preferences and differences in bitterness and astringency. The impact of hop proantho-cyanidins on haze formation is comparable to that of malt proanthocyanidins. No differences between the beers were found in triangular tests. Statistical analysis of the paired comparison tests showed that hop proanthocyanidins do not contribute to the bitterness of beer since beer brewed with tannin-free hop extract is slightly more bitter than beer brewed with whole leaf hops. No differences in astringency were noted between beers brewed with or without hop proanthocyanidins and it was found that the panel expressed a very slight preference for beer brewed with n-hexane hop extract over beer brewed with both n-hexane hop extract and extracted hop residue.     

Assessment of changes in hop resins and polyphenols during long‐term storage

Changes in the content and composition of hop secondary metabolites during storage are reflected in beer quality and in the economics of beer production. A 12‐month storage experiment with T90 pellets of four hop varieties showed different dynamics of hop aging in relation to both storage conditions and hop variety. Negligible effects on the α‐ and β‐acids were detected during storage without air access at +2°C. Storage at +20°C resulted in a final loss of 20–25% α‐acids, but the content of β‐acids did not change significantly. Large decreases in α‐acids (64–88%) and in β‐acids (51–83%) were found in hops stored with access to air at +20°C. The rate of decline accelerated markedly after 6 months of storage. In terms of hop resin changes, Premiant and Sládek were the most and the least stable varieties, respectively. After 12 months, the content of the total polyphenols and flavonoids decreased by 30–40% and by 20–30%, respectively, irrespective of storage conditions. The rate of decline accelerated strongly after 6 months. The DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) antiradical potential decrease was significant only in hops stored under aerobic conditions. The depletion was 9–25% after 1 year; Saaz was the most stable variety. Copyright © 2012 The Institute of Brewing & Distilling     

Heterologous expression of surface-active proteins from barley and filamentous fungi in Pichia pastoris and characterization of their contribution to beer gushing

The spontaneous over-foaming of beer upon opening, i.e. beer gushing, is an unwanted phenomenon for the brewing industry. Currently, surface-active proteins from filamentous fungi and non-specific lipid transfer proteins (nsLTP1) from barley are discussed as gushing inducers. In our study the class I hydrophobin FcHyd3p from Fusarium culmorum, the class II hydrophobin Hfb2 from Trichoderma reesei, the alkaline foam protein A (AfpA) from F. graminearum and nsLTP1 from Hordeum vulgare cv. Marnie (barley) were heterologously expressed in Pichia pastoris and used in gushing tests. The class I hydrophobin FcHyd3p was unable to induce gushing in beer. The class II hydrophobin Hfb2 was able to induce gushing in beer, but proved to be inhibited by heat treatment as well as by the presence of enriched hop compounds. Both resulted in a reduced gushing potential. AfpA and nsLTP1 exhibited no gushing-inducing potential at the amounts added to beer. Addition of these proteins to beer or carbonated water previously treated with class II hydrophobins revealed a gushing reducing character.     

Mechanism of gushing induction by carbon dioxide transfer activator

The article deals with the activation of carbon dioxide transfer from carbonated beverages. As a model compounds agents produced by Trichoderma reesei and harzianum and degraded hop iso‐extract were selected. These substances were added to the carbonated water or beer and their effect was compared. The method of pressure growth in the bottle headspace after rapid piercing and re‐closing the bottle was used. Some carbon dioxide transfer activators accelerated CO₂ escape even at rest; others required shaking for different periods. The pressure growth measurement allowed the prediction of gushing before its occurrence. Copyright © 2017 The Institute of Brewing & Distilling