Beer Haze Research Articles

Identification of the Yeast Mannoprotein Gene HZY1 as a Key Genetic Determinant for Yeast-Derived Haze in Beer

With the sustained popularity of hazy IPAs, brewers have explored multiple approaches to maximizing stable haze that will remain in suspension throughout the shelf life of the beer. Our recent investigations into yeast-dependent haze have uncovered specific brewing yeast strains that promote the formation of haze in heavily dry-hopped beer styles. These brewing strains have been termed “haze-positive” and furthermore, the timing of dry hop additions has been found to be another key factor in producing this stable haze. Classical genetics have identified YIL169C (herein referred to as HZY1) as both necessary and sufficient for the haze-positive phenotype in the yeast strain most widely used for Hazy IPAs. HZY1 encodes a candidate glycoprotein and our recent findings suggest it is localized to the cell wall through a GPI anchor. Surprisingly, using long-read sequencing data we uncovered extensive genetic variation in HZY1 across brewing strains. The haze-positive phenotype correlates with an expansion in the N-terminal serine-rich region. We propose that the Hzy1 glycoprotein is a critical component to yeast-dependent colloidal haze and the genetic variation in this locus contributes the range of haze phenotypes observed across industrial brewing strains.

Activated natural zeolites for beer filtration: A pilot scale approach

A clinoptilolite-rich natural zeolite was tested as a substitute for kieselguhr as a filtering material to eliminate ingredients that cause beer haze formation. Two-grain sizes of micronized natural zeolite were thermally activated to 400 °C, to enhance its adsorption performance and remove the impurities adsorbed in the microporous system of zeolites, followed by their physicochemical characterization. The activated zeolites mixed with four commercial filter aids in different ratios were used for beer filtration at the pilot scale. Most of the physicochemical and sensory characteristics of beers filtered with commercial filter aids and with zeolites were similar. Using zeolite in filtering mixtures significantly reduces the number of microorganisms present in the filtered beer, which can eliminate the necessity of beer sterilization after filtration. The results evidenced that activated natural zeolites, which are cheap materials, are promising candidates as filter aids and can replace kieselguhr without producing any degradation of the beer filtration process.

A novel tannase from Klebsiella pneumoniae KP715242 reduces haze and improves the quality of fruit juice and beverages through detannification

The presence of high tannin content in fruit juices and other beverages generates bitterness and haze formation with a concomitant deterioration of their quality. This problem could be circumvented by treating these food products with the enzyme tannase (tannin acyl hydrolase EC 3.1.1.20), which contains the ability to hydrolyze the ester and depside linkages present in hydrolyzable tannins. In the present study, a low molecular weight (38.20 kDa) tannase purified from Klebsiella pneumoniae KP715242 was used for clarification and detannification of orange juice, beer, and tea cream solubilization. The initial tannin content of orange juice was reduced to 42.45% upon treatment with 4.5 U/mL of purified tannase at 35°C for 3.5 h. Beer haze was clarified by incubating the colloidal suspension with the purified enzyme. Under the optimum condition of temperature (30°C), incubation time (3.5 h), and enzyme concentration (4.5 U/mL), the initial tannin content of beer was reduced to 46.47%. Tea cream solubilization was achieved by treating the tea extract with varying enzyme concentrations at different time intervals and temperatures. The maximum reduction in the tea tannin content (55%) was observed at 35°C when the enzyme was incubated for 3 h at a concentration of 6.0 U/mL. The results of this investigation could be further exploited at an industrial scale in the detannification of fruit juice, beer, and tea.

Preparation, Characterization, and Performance of Natural Zeolites as Alternative Materials for Beer Filtration.

The clarity of the beer is essential to its marketability and good consumer approval. Moreover, the beer filtration aims to remove the unwanted constituents that cause beer haze formation. Natural zeolite, an inexpensive and widespread material, was tested as a substitute filter media for diatomaceous earth in removing the haze constituents in beer. The zeolitic tuff samples were collected from two quarries in Northern Romania: Chilioara, in which the zeolitic tuff has a clinoptilolite content of about 65%, and the Valea Pomilor quarry, containing zeolitic tuff with a clinoptilolite content of about 40%. Two-grain sizes, <40 and <100 µm, from each quarry were prepared and thermally treated at 450 °C in order to improve their adsorption properties and remove organic compounds and for physico-chemical characterization. The prepared zeolites were used for beer filtration in different mixtures with commercial filter aids (DIF BO and CBL3) in laboratory-scale experiments, and the filtered beer was characterized in terms of pH, turbidity, color, taste, flavor, and concentrations of the major and trace elements. The results showed that the taste, flavor, and pH of the filtered beer were generally not affected by filtration, while turbidity and color decreased with an increase in the zeolite content used in the filtration. The concentrations of Na and Mg in the beer were not significantly altered by filtration; Ca and K slowly increased, while Cd and Co were below the limits of quantification. Our results show that natural zeolites are promising aids for beer filtration and can be readily substituted for diatomaceous earth without significant changes in brewery industry process equipment and protocols for preparation.

Phenolic-Degrading Enzymes: Effect on Haze Active Phenols and Chill Haze in India Pale Ale Beer.

The development of green and sustainable biotechnological approaches for preventing chill haze formation is currently under investigation. In this preliminary study, laccase and tannase (pure or combined) were applied as phenolic-degrading enzymes during two crucial brewing steps (i. post-mashing and ii. before the yeast inoculum). In post-mashing and irrespective of the dosage applied (100 μL/L or 1 mL/L), tannase-based treatment ensured the complete removal of haze active (HA) phenols, which was proved by the full prevention of chill haze (about 1 EBC vs. 22 EBC in the control sample). Before yeast inoculum for the alcoholic fermentation, the removal of haze active phenols and the prevention of chill haze were both tannase-dosage-dependent (15 and 2 EBC for the lowest and the highest dosages, respectively) although they failed to completely break down the HA phenols. This biotechnological approach did not significantly affect the chromatic properties of treated beer.

Effects of non-starch polysaccharides from pure wheat malt beer on beer quality, in vitro antioxidant, prebiotics, hypoglycemic and hypolipidemic properties

Pure wheat malt beer (PWMB) with potentially functional properties may attract consumer interest. To explore the functional properties of non-starch polysaccharides (NSPs) in PWMB, NSPs were extracted from PWMB and separated into PWMB non-starch polysaccharides-1 (PNSP-1) and PWMB non-starch polysaccharides-2 (PNSP-2) by Q-Sepharose Fast Flow column. PNSP-1 exhibited more effects on increasing the viscosity of beers, which had higher purity of 92.58% containing 88.53% arabinoxylan with arabinose/xylose (A/X) 0.60 and 2.78% β-glucan but no glucomannan and mannan; while PNSP-2 showed greater effects on promoting beer haze and foam retention, which had more diverse NSPs including 46.03% arabinoxylan with A/X 1.13, 41.47% arabinogalactan, 10.26% glucomannan, and 2.70% mannan but no β-glucan. Diversity of sugar composition resulted in a better antioxidant capacity of PNSP-2. NSPs proliferated Lactobacillus casei LC6117 and LC0331, but did not promote L. plantarum LP-28 and L. fermentium LF-2H. PNSP-2 was also utilized by L. rhamnosus SRG. PNSP-2 had better effects on prebiotics activities. PNSP, PNSP-1 and PNSP-2 all exhibited mild inhibitory effects on α-glucosidase and lipase but not on α-amylase, and showed a binding capacity to fat and cholesterol. This work is of great reference significance for improving the viscosity, haze, foam retention and health value of wheat beer.

Haze in Beer: Its Formation and Alleviating Strategies, from a Protein-Polyphenol Complex Angle.

Beer is one of the oldest and most widely consumed alcoholic beverages. Haze formation in beer is a serious quality problem, as it largely shortens the shelf life and flavor of beer. This paper reviews the factors affecting haze formation and strategies for reducing haze. Haze formation is mainly associated with specific chemical components in malt barley grains, such as proteins. The main factor causing haze formation is a cross-linking of haze active (HA) proteins and HA polyphenols. Many HA proteins and their editing genes or loci have been identified by proteomics and quantitative trait locus (QTL) analysis, respectively. Although some technical approaches have been available for reducing haze formation in beer, including silica and polyvinylpolypyrrolidone (PVPP) adsorbent treatments, the cost of beer production will increase and some flavor will be lost due to reduced relevant polyphenols and proteins. Therefore, breeding the malt barley cultivar with lower HA protein and/or HA polyphenols is the most efficient approach for controlling haze formation. Owing to the completion of barley whole genome sequencing and the rapid development of modern molecular breeding technology, several candidate genes controlling haze formation have been identified, providing a new solution for reducing beer haze.

Non-microbiological turbidity of beer: Part 2 – a case study

Non-microbial beer turbidity of lager beers often indicates a technological problem. Therefore, the occurrence of permanent haze in filtered and stabilized beer should not be underestimated. In this study, practical examples from industrial breweries, where several types of non-microbiological haze of colloidal were identified, are presented. These examples of haze were caused by slightly different factors, and as a result, they had a different microscopic image. It is often accompanied by mechanical impurities and sometimes by microorganisms that function as nucleation centers. Moreover, a very interesting example of almost brilliant permanent beer haze caused by the destruction of yeast cells with the following pouring intracellular contents of cells into beer is introduced. This phenomenon, which could be called “precedent”, was caused by a bad physiological condition of yeasts cells and inappropriately chosen yeast separation technology.

Unfilterable Beer Haze Part I: The Investigation of an India Pale Ale Haze

The nature of undesirable and unfilterable haze particles observed by craft breweries remains nebulous and presents a challenge when the aim is the production of bright beer. A commercial beer was studied in which the brewery had sporadically encountered unfilterable haze. In this study, it was hypothesized that unfilterable haze particles were formed due to increased concentrations of proteins, polyphenols, and/or beta-glucans. Samples of a high haze and low haze India Pale Ale were degassed and digested with enzymes amyloglucosidase, pepsin, and UltraFlo Max (NovozymesTM). Additionally, the protein, polyphenol, and beta-glucan content of each sample was measured. When comparing protein, polyphenol, and beta-glucan concentrations substantial differences between high haze and low haze protein concentrations were observed. Due to the unfilterable nature of these hazes, combined with experimental findings, it was hypothesized that yeast cell-wall proteins were responsible for this haze. Understanding of the source of these hazes offers brewers the opportunity to mitigate against their formation by adjusting brewing practices.

Unfilterable Beer Haze Part II: Identifying Suspect Cell Wall Proteins

The use of various diagnostic techniques has been previously utilized in the assessment of a commercially available India Pale Ale with cases of sporadically occurring unfilterable haze. The results from Part 1 suggested that β-glucans and proteins were the cause of the unfilterable haze and it was postulated that cell wall mannoproteins may also be a culprit of the unfilterable beer haze. In this follow-up study, proteins from high haze and low haze beer samples were precipitated and assessed using SDS-PAGE. Polyphenol interferences observed on the SDS-PAGE indicated that protein purification and targeted analysis was required. Proteins from high haze and low haze samples were fractionated and qualitatively identified via LC-MS. A library was built from FASTA sequences of targeted yeast proteins to qualitatively analyze the high haze and low haze samples. The protein fractionation was successful at purifying and isolating proteins from high and low haze samples. Two protein peaks were observed in the high haze sample, while one protein peak was observed in the low haze sample. The targeted LC/MS analysis discovered the presence of yeast cell wall mannoproteins and flocculation proteins, particularly Flo1 and Flo9. Understanding the source of these hazes can provide an opportunity for brewers to mitigate against their formation by adjusting brewing and yeast management practices.

Influence of gluten-free adjuncts on beer colloidal stability

This work deals with issues related to the colloidal stability of craft beers. It is generally known that haze in beer is directly related to the formation of complexes resulting from the interaction of haze active polyphenols and protein fractions [1–18]. How some variables in the malting process and formulation can contribute to the colloidal stability of the final beer have been considered. Micro malting tests on four glutenfree cereals/pseudocereals (millet, amaranth, buckwheat and quinoa) were carried out in order to identify the optimal conditions for obtaining malts suitable for brewing. Quality of malts was checked analysing diastatic power, beta glucans content and Kolbach Index. The gluten-free malts were under-modified if compared to traditional malts, but potentially suitable as adjuncts (especially buckwheat). Nine beers with different formulations were produced in the laboratory using the malts obtained from the micro malting tests. The evaluation of beer stability was carried out analyzing indices validated by European Brewery Convention (sensitive proteins and cold turbidity) and two unconventional methods (gluten analysis and antioxidant activity). Moreover, three beers (the reference obtained with 100% malted barley, the other two with the buckwheat malts) were subjected to CE analyses (Capillary Electrophoresis) in order to define their protein content. According to the results the use of gluten-free adjuncts, besides relevant process conditions (i.e. alkalinized first steeping water in the malting process), can lead to more stable final products with a gluten content less than 100 mg/L and so potentially suitable by coeliacs according to EU regulation 828/2014.

Development of engineered yeast for biosorption of beer haze-active polyphenols.

Compared to most other alcoholic beverages, the shelf life of beer is much more limited due to its instability in the bottle. That instability is most likely to appear as turbidity (haze), even sedimentation, during storage. The haze in beer is mostly caused by colloidal particles formed by interactions between proteins and polyphenols within the beer. Therefore, beers are usually stabilized by removing at least one of these components. We developed and constructed a Saccharomyces cerevisiae strain with a proline-rich QPF peptide attached to the cell wall, using the C-terminal anchoring domain of α-agglutinin. The QPF peptide served to bind polyphenols during fermentation and, thus, to decrease their concentration. Strains displaying QPF were able to bind about twice as much catechin and epicatechin as a control strain displaying only the anchoring domain. All these experiments were done with model solutions. Depending on the concentration of yeast, uptake of polyphenols was 1.7-2.5 times higher. Similarly, the uptake of proanthocyanidins was increased by about 20%. Since the modification of yeasts with QPF did not affect their fermentation performance under laboratory conditions, the display of QPF appears to be an approach to increase the stability of beer.

Identification of two key genes controlling chill haze stability of beer in barley (Hordeum vulgare L).

BackgroundIn bright beer, haze formation is a serious quality problem, degrading beer quality and reducing its shelf life. The quality of barley (Hordeum vulgare L) malt, as the main raw material for beer brewing, largely affects the colloidal stability of beer.ResultsIn this study, the genetic mechanism of the factors affecting beer haze stability in barley was studied. Quantitative trait loci (QTL) analysis of alcohol chill haze (ACH) in beer was carried out using a Franklin/Yerong double haploid (DH) population. One QTL, named as qACH, was detected for ACH, and it was located on the position of about 108 cM in chromosome 4H and can explain about 20 % of the phenotypic variation. Two key haze active proteins, BATI-CMb and BATI-CMd were identified by proteomics analysis. Bioinformatics analysis showed that BATI-CMb and BATI-CMd had the same position as qACH in the chromosome. It may be deduced that BATI-CMb and BATI-CMd are candidate genes for qACH, controlling colloidal stability of beer. Polymorphism comparison between Yerong and Franklin in the nucleotide and amino acid sequence of BATI-CMb and BATI-CMd detected the corresponding gene specific markers, which could be used in marker-assisted selection for malt barley breeding.ConclusionsWe identified a novel QTL, qACH controlling chill haze of beer, and two key haze active proteins, BATI-CMb and BATI-CMd. And further analysis showed that BATI-CMb and BATI-CMd might be the candidate genes associated with beer chill haze.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1683-1) contains supplementary material, which is available to authorized users.

A codon-optimized endoprotease Endo-Pro-Aspergillus niger: Over expression and high-density fermentation in Pichia pastoris

Abstract The prolyl endopeptidase (endoprotease Endo-Pro- Aspergillus niger , endoprotease EPR) gene from A. niger was optimized according to the codon usage bias in Pichia pastoris . It was successfully expressed using expression vector pPIC9K with high activity (620 ± 25 U/l). The recombinant optimized endoprotease EPR could work in an extremely broad temperature range and over 40% relative activity were remained in the temperature range of 15–70 °C. The optimal pH value and temperature for activity were 4.0–5.0 and 35–40 °C, respectively. The enzyme kinetic parameters K m , K cat and K cat / K m values towards various substrates were also determined. A fed-batch strategy was first developed for high cell-density fermentation and the enzyme activity reached 1890 U/l after cultivation in 7 l fermenter. The broad temperature range and efficient expression made this enzyme possible to apply in the food industry directly. This study also sought to investigate the effect of endoprotease EPR on removing the beer haze protein.

Polysaccharides influence on the interaction between tannic acid and haze active proteins in beer

Colloidal instability in beer is mainly caused by interactions between proteins and polyphenols. These two combine producing a visible haze that reduces the physical shelf life of beer. The haze active proteins (HAPs) react with tannic acid (TA); therefore, this reaction provides a way to determine HAP concentrations in beer. Beers also contain a number of constituents that may influence the protein–polyphenol haze formation. We used a response surface methodology to predict the influence of total polysaccharides (TPS) and proteins on beer haze. Experiments were carried out using the Central Composite Design (CCD) methodology. Samples of beer were prepared with variable concentrations of TPS and proteins. TPS concentrations ranged between 1.34 and 2.23gL−1 and proteins concentrations between 0.11 and 0.18gL−1. Results show that the increase in turbidity in response to TPS addition was similar to that in response to protein addition. Our regression analyses indicate a significant dependency and correlation between the observed values and the predicted response values (R2=97.87% and R2[adj]=95.75%). Furthermore, these values indicate that our experimental model can explain 95.75% of the total variation. Therefore, using TA as an indicator of the interaction of TPS with proteins, as commonly done, can lead to considerable errors, since the polysaccharides also react with TA, and this reaction actually causes a considerable increase in turbidity.

INVESTIGATION OF HORDEINS DURING BREWING AND THEIR INFLUENCE ON BEER HAZE BY PROTEOME ANALYSIS

ABSTRACT Colloidal storage haze is one of the principal indicators of beer quality. In this study, the changes of malt hordeins during the brewing process and the roles of malt hordeins in the formation of beer haze were investigated using two-dimensional electrophoresis (2-DE). It was found that most of malt hordeins disappeared during the brewing process and only the few faint hordein spots (about eight spots) were detected in beer haze. Moreover, these spots were found in wort protein and beer haze protein. The complex 2-DE patterns of malt hordein, wort and beer haze displayed a relationship with some chemical modifications during the brewing. These spots were analyzed and identified as B and γ3 hordein and storage protein (Hordeum vulgare) by mass spectrometry. Our results suggested that hordein might be minor haze-active proteins in beer but was a critical factor in the formation of beer haze. PRACTICAL APPLICATION The formation of haze is a serious quality problem that limits the storage life of beer products, an issue that has become increasingly important to brewers. It is conventionally accepted that proline-rich hordeins play a major role in the formation of haze. In this study, it was found that most of the hordeins disappeared and that small amounts of B hordein and γ3 hordein from the malt in beer haze were detected by proteomics analysis. Moreover, this study demonstrated that hordeins in beer were not a key contributor to beer haze formation. To our knowledge, to date, this result in the existing literature of similar studies has not been reported. This study laid the theoretical foundation for further investigations to determine the mechanism of the formation of beer haze. An improved understanding of the impact of brewing on hordeins could potentially provide further scope for optimizing beer quality.

INVESTIGATION OF THE RELATIONSHIP OF MALT PROTEIN AND BEER HAZE BY PROTEOME ANALYSIS

ABSTRACT The changes in malt proteins during the brewing process and the roles of malt proteins in the formation of beer haze were investigated in this study. Coomassie blue stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) revealed that proteins with molecular weights of ∼40, 25–29 and 6.5–17 kDa were the major components of beer haze protein, originating primarily from malt water-soluble protein, partly from hordein. Two-dimensional electrophoresis and mass spectrometry analysis highlighted malt protein modification occurring during the brewing process. It was found that most of the malt hordeins disappeared during the brewing process, except B and γ3 hordein and storage protein (Hordeum vulgare). The results also suggested that the heat- and proteolysis-stable hydrophobic proteins such as barley trypsin inhibitor CMe protein, germin E (H. vulgare) and protein Z might be the important haze-promoting proteins, and that B and γ3 hordein might be the minor haze-active proteins in beer but were a critical factor in the formation of beer haze. PRACTICAL APPLICATIONS Haze stability is of great importance to brewers as it is the first characteristics by which a consumer judges the quality of their beer. It is conventionally accepted that proline-rich hordeins from malt play a major role in the formation of haze. In this study, it was found that B and γ3 hordein might be the minor haze-active proteins, but the heat- and proteolysis-stable hydrophobic proteins such as barley trypsin inhibitor CMe (BTI-CMe) protein, germin E (Hordeum vulgare) and protein Z might be the important haze-promoting proteins. This study made a further confirmation on this issue that nonhordein proteins are regarded as haze-active and laid the theoretical foundation for further investigations to determine the mechanism of the formation of beer haze. An improved understanding of the impact of brewing on malt proteins could potentially provide further scope for optimizing beer quality.

Allelic Diversity of a Beer Haze Active Protein Gene in Cultivated and Tibetan Wild Barley and Development of Allelic Specific Markers

The formation of haze is a serious quality problem in beer production. It has been shown that the use of silica elute (SE)-ve malt (absence of molecular weight (MW) ∼14000 Da) for brewing can improve haze stability in the resultant beer, and the protein was identified as a barley trypsin inhibitor of the chloroform/methanol type (BTI-CMe). The objectives of this study were to determine (1) the allelic diversity of the gene controlling BTI-CMe in cultivated and Tibetan wild barley and (2) allele-specific (AS) markers for screening SE protein type. A survey of 172 Tibetan annual wild barley accessions and 71 cultivated barley genotypes was conducted, and 104 wild accessions and 35 cultivated genotypes were identified as SE+ve and 68 wild accessions and 36 cultivated genotypes as SE-ve. The allelic diversity of the gene controlling BTI-CMe was investigated by cloning, alignment, and association analysis. It was found that there were significant differences between the SE+ve and SE-ve types in single-nucleotide polymorphisms at 234 (SNP(234)), SNP(313), and SNP(385.) Furthermore, two sets of AS markers were developed to screen SE protein type based on SNP(313). AS-PCR had results very similar to those obtained by immunoblot method. Mapping analysis showed that the gene controlling the MW∼14 kDa band was located on the short arm of chromosome 3H, at the position of marker BPB-0527 (33.302 cM) in the Franklin/Yerong DH population.

Comparison of hazes in freshly bottled and aged beers by multiple angle turbidimetry

The hazes of freshly bottled and aged pale lager beers determined with the 12° and 90° dual angle laboratory hazemeters and 10° to 90° range photogoniometer were compared and evaluated. The instruments were standardised in EBC formazin units. In freshly bottled beer, the forward (12°–25°) haze values were smaller approximately by a factor of three than the nephelometric (90°) values which yielded 0.33 EBC units. In aged beer, the forward haze was greater than the nephelometric one. Whereas the aged beer showed the greatest and the fresh beer the lowest intensity of scattered light, the formazin suspension intensity was in between. Due to the standardisation of the beer scattered intensities by relation to that of formazin, the standardised nephelometric haze in non-aged beer was greater than the forward haze, and vice versa in aged beer. The greater forward than the nephelometric haze in aged beer was caused by the growth of haze particles above the mean size of formazin particles which was larger than 2 &amp;mu;m as confirmed by the particle size distribution measurement.

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.