Haze

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Turbidity gives a first visual impression of the quality of beer to the consumer.

Beer should exhibit a "bright" appearance and haze free character­ istics in order to be fully acceptable by the consumers, qualities ensured by the colloidal stability of beer. The formation of beer haze occurs after the initial steps of fermentation due to the interaction and complexation of polyphenols (e.g. flavonoids and mainly tannins) with proteins, resulting in chill haze and precipitation (permanent haze) (Mosher & Trantham, 2017). The flavan-3-ol monomers (+)-catechin, (-)-epi­catechin and (+)-gallocatechin are able to bind the active haze protein hordein by hydrogen bounds trough polar groups, but also hydrophobic and ionic bounds. Gallic acid, hydroxyphenylactic acid, salicylic acid, chlorogenic acid, vanillic acid, epicatechin, pyrocatechuic acid, ferulic acid, and luteolin may also play an important role in the colloidal changes of beer upon polymerisation into tannins (Jongberg, Andersen, & Lund, 2020).

Especially with craft beers like the New England Indian Pale Ale or traditional German wheat beers, a permanent haze is a desired quality attribute. Apart from protein-polyphenol complexes, haze can be caused by suspended microorganisms or vegetal matter (from late hopping regimens). Here, the desired haze particles are not removed from the beer by filtration. Indeed, among craft beer lovers, there is a growing trend towards cloudy beers. This phenomenon has been termed the "haze craze".

The establishment of "colloidal stability" in beer renders a beer "bright", or haze free. A beer is considered ‘bright’ if no haze forms when chilled to 4°C (40°F) or below.

However, because beer is intrinsically colloidally-unstable, without proper treatment, chill haze (non-permanent) may develop that can lead to sedimentation and precipitation (permanent haze). Beer haze results from the interaction of beer constituents that aggregate to form visible particles in solution that reflect light65. Constituents known to play a role in haze formation include proteins, tannins (polyphenol), carbohydrates, oxygen and metal ions. Several metals have been found in haze: aluminium, barium, calcium, chromium, copper, iron, lead, magnesium, manganese, molybdenum, nickel, phosphorus, silicon, silver, strontium, tin, vanadium and zinc14. Metals such as copper, iron and aluminium can exist in the haze at several thousand fold higher concentrations than in the parent beer. Other metals such as lead, nickel, tin, vanadium and molybdenum are less concentrated in the haze, with manganese, calcium and magnesium even less concentrated. Such a high presence of iron and copper in beer haze is not unexpected as these metals are easily chelated by phenolic, amino and carboxyl groups. It is therefore also not surprising that haze concentrates metals such as iron and copper; protein-polyphenol complexes are the most frequent cause for haze production50,83.

The haze active (HA) protein found in beer derives from barley hordein. Because HA-proteins have on the order of 20 mol% proline46 they display a high affinity for polyphenols (PPs). Siebert et al.104 have described a model (Fig. 2) in which PPs crosslink HA-proteins, in a similar fashion to when beer PPs react with parotid-derived proline-rich lingual proteins to elicit the organoleptic sensation of astringency17,104,107,109. In this model protein-PP complexes form a large network when the number of HA-PP binding sites equal the number of available HA-protein binding sites in the beer matrix. The nature of haze formation in this manner likely involves hydrogen bonding and hydrophobic stacking of proline and PP rings associated with п-bonding (Fig. 3)7 . Formation of protein-PP haze depends on beer pH, alcohol content, ionic strength, as well as phenolic composition7,107–109.

Protein-PP complex formation has received extensive attention and thus precursors to this complex have become the target for beer haze treatments. Several methods have been employed for beer colloidal stabilization including: prolonged cold storage, cold filtration, fining with gelatin, isinglass, or tannic acid, addition of proteolytic enzymes and treatments with adsorbents50,106. One of the more commonly used adsorbent resins, polyvinylpyrrolidine (PVPP) was commercially introduced in 196178 to specifically target and remove beer PPs. While other approaches may be used to target PPs, fining with PVPP is practiced commonly due to its relative ease of use and low cost. PVPP is a neutral polyamide that has an affinity for beerPPs because it is structurally similar to polyproline, a known HA-peptide (both possess a five-membered nitrogen containing ring with hydrogen bonding sites) (Fig. 4)17,50,106. PVPP-PP binding involves hydrogen bonding and hydrophobic stacking, roughly the same mechanism as protein-PP binding7,109.

Although roughly 80 known phenolic compounds have been determined in beer24,45, not all are involved in colloidal instability. HA-PPs must be able to effectively crosslink HA-proline-rich proteins into a stable network to result in precipitation. Flavanoids are known constituents of permanent beer haze. The flavan-3-ol monomers (- )-epicatechin and (+)-catechin and (+)-gallocatechin bind, but do not crosslink HA proteins. However, proanthocyanidin oligomers possess two or more binding sites within the same molecule, allowing them to crosslink HA proteins104. Haze formation varies with proanthocyanidin molecular weight, subunit composition, interflavanoid bond orientation, number and placement of the hydroxyls on the heterocyclic C and aromatic B rings8,75,77,87. Specifically, tri-hydroxy flavanoids bind more readily than dihydroxy flavanoids, vicinal or ortho oriented hydroxyls bind better than meta-oriented hydroxyls, and (-)-epicatechin oligomers bind slightly better than (+)-catechin oligomers. Because trimer, tetramer and higher proanthocyanidin oligomers less readily survive the brewing process, the proanthocyanidin dimers are thought to play the most significant role in beer haze107. However, oxidized flavanols instigate chill haze and once condensed (polymerized) into proanthocyanidins participate in the formation of permanent haze93.

Since the consumer expects a pale lager beer to be clear and bright, the appearance of haze is a limiting factor in shelf life. Therefore, the colloidal or turbidity stability of beer has been a field of active research for the past 100 years. Beer haze first develops as reversible chill haze (noncovalent protein–polyphenol interaction), which re-dissolves when a temperature of 20 °C is reached. Over the course of beer aging, reversible chill haze can develop into permanent haze (covalent protein–polyphenol interactions) (Bamforth, 1999; Steiner, Becker, & Gastl, 2010). According to Steiner et al. (2010), beer haze contains various or- ganic substances, of which proteins form the largest part in com- bination with polyphenols (40% to 75%), polysaccharides (2% to 15%) and inorganic substances. However, protein–polyphenol complexes are seen as the substances that are mainly responsible for the initial haze formation (Bamforth, 1999; Steiner et al., 2010). Protein–polyphenol interactions in food systems were reviewed by Ozdal, Capanoglu and Altay in 2013 (Ozdal, Capanoglu, & Altay, 2013). Polyphenols can bind to proteins both reversibly by hydro- gen bonding, hydrophobic bonding and van der Waals forces and irreversibly through the formation of covalent bonds. Complex formation between proteins and polyphenols leads to a decrease in protein solubility (Ozdal et al., 2013). Since heating induces changes in hydrophobicity by exposing hydrophobic areas of the proteins as potential binding sites, it also changes the nature of protein–polyphenol interactions (Siebert, Troukhanova, & Lynn, 1996). As these interactions and their involvement in beverage haze formation have already been reviewed by (Siebert, 1999), the structural features required for a polyphenol to be haze-active will only be discussed briefly. Even though polyphenols with a higher degree of polymerization are known to be more haze-active than lower molecular weight polyphenols (Gramshaw, 1967), one aro- matic ring bearing two hydroxyl groups is sufficient to interact with proteins (Siebert & Lynn, 1998). In order to cross-link pro- teins and thus form haze, a polyphenol needs to have at least two binding sites (Siebert & Lynn, 1998). Hydroxylation patterns (two vicinal hydroxyls being more active than two separate ones) affect protein–polyphenol interactions, which are weakened by glycosy- lation or methylation (Ozdal et al., 2013). Since higher oligomeric proanthocyanidins are likely to be lost during the brewing process, the main haze-active polyphenols are procyanidin B3 and prodel- phinidin B3 (Siebert & Lynn, 1998). Asano, Ohtsu, Shinagawa, and Hashimoto (1984) found the monomers catechin and epicat- echin and to a greater extent proanthocyanidin dimers to pen- tamers to be haze-active in a buffered model solution of polyphe- nols and proteins. Phenolic acids, on the other hand, had no haze-forming capacity (Asano et al., 1984). Protein–polyphenol interactions in model systems were found to be influenced by pH, ethanol and polysaccharide content (Mercedes Lataza Rovaletti et al., 2014; Siebert & Lynn, 2003). Dimeric proanthocyanidins as well as their monomers can polymerize during storage by oxida- tive and acid-catalyzed mechanisms (Asano et al., 1984; Gramshaw, 1967). Indeed, the haze-forming activity of catechin and dimeric proanthocyanidin solutions was found to increase upon oxidative aging (Asano et al., 1984). Oxidative polymerization involves the formation of an o-quinone group from a catechol group by a free radical mechanism. The o-quinone then polymerizes by addition of other flavonoid or nonflavonoid polyphenols or formation of covalent bonds with proteins or irreversibly linking proteins by oxidation of sulphydryl groups to disulfide bridges (Gramshaw, 1967; Ozdal et al., 2013). Kaneda, Kano, Osawa, Kawakishi, and Kamimura (1990) showed that free radicals formed during ac- celerated storage are involved in chill haze formation and in the decrease of low molecular weight flavanols (Kaneda et al., 1990). These authors later researched the role of transition metal ions (Fe and Cu) in haze formation. They described an accumulation of iron and copper ions in beer haze. As described above, transition metal ions are involved in oxygen activation and form complexes with polyphenols and proteins (Kaneda, Kano, Koshino, & Ohya- Nishiguchi, 1992). Despite the great number of research stud- ies published on the haze-forming activity of beer polyphenols (especially proanthocyanidins) (Asano et al., 1984; Bengough & Harris, 1955; Delcour, Schoeters, Meysman, & Dondeyne, 1984; Gramshaw, 1967; Hall, Harris, & Ricketts, 1959; Harris & Rick- etts, 1959b; McFarlane, Wye, & Grant, 1955; Mikyˇska et al., 2002; Siebert & Lynn, 1998; Steiner & Stocker, 1965), this view was con- tested by Loch-Ahring, Decker, Robbert, and Anderson (2008), who did not detect polyphenolic substances in chill haze. How- ever, they highlighted the importance of hop compounds like α- and β-acids and (iso-) xanthohumol in chill haze formation (Loch-Ahring et al., 2008). Ye, Huang, Li, Li, and Zhang (2016) also described an uncertain relationship between malt polyphenols and haze stability in the alcohol chill test (Ye et al., 2016). In or- der to elucidate the haze-forming potential of polyphenols, more detailed studies of both the protein and the polyphenol part and especially active sites on haze-active protein molecules are needed.

There exist two forms of haze; cold break (chill haze) and age-related haze. Cold break haze forms at 0°C and dissolves at higher temperatures. If cold break haze does not dissolve, age-related haze develops, which is non-reversible. Chill haze is formed when polypeptides and polyphenols are bound non-covalently. Permanent haze forms in the same manner initially, but covalent bonds soon form and insoluble complexes are created which will not dissolve when heated. Condensed tannins (called proanthocyanidins) from the husk of the barley grain are carried from the malt into the wort and are also found after fermentation of the wort in the beer. There they cause precipitation of proteins and haze formation especially after refrigeration of the beer, even if it previously had been filtered to be brilliantly clear.

The mashing stage of brewing affects the amount of haze-active protein in beer. If a beer has been brewed with a protein rest (48–52°C), it may contain less total protein but more haze-active proteins because the extra proteolysis caused release of more haze causing polypeptides.

Polypeptides that are involved in haze formation bind to silica gel so that they are selectively adsorbed, while foam proteins are not affected by silica treatment.

Removal of haze forming tannoids can be effected using PVPP.

Two major proteins in beer are claimed to cause haze formation and influence foam stability; protein Z and LTP 1. Protein Z and LTP1 are heat stable and resistant to proteolytic modification during beer production and appear to be the only proteins of barley origin present in significant amounts in beer.

Hazes are the result of light scattering by colloidal or larger particles suspended in a solution. Particles of greater than colloidal size settle out if there is no agitation to suspend them. True colloids are indefinitely stable suspensions; they arise when both the size of particles is sufficiently small and their density sufficiently similar to the suspending liquid that the particles are kept suspended by Brownian motion.

Many different sources of hazes in beverages have been described. These include both inorganic and organic matter. Oxalate hazes are found in beer and tartrate hazes in grape juice and wine. These are well understood and cause few processing problems. Other inorganic hazes have been associated with adsorbent particles and filter aids; these occur only infrequently and are due to process malfunctions.

Some hazes have been associated with carbohydrate materials or the growth of microorganisms. The latter can result in hazes either because the cells of the organism scatter light directly or through formation of particles caused by metabolic activity. By far the most frequent cause of haze in beverages is protein–polyphenol interaction. Even in products that are initially free of turbidity, proteins and polyphenols can gradually form insoluble complexes that scatter light. The initial combination of protein and polyphenol may be soluble. If the complex grows to sufficient size to become insoluble it results in turbidity. The particles may grow further still and become so large that they sediment. Analysis of haze material isolated from beverages often shows a large proportion of carbohydrate, but since stabilization can be achieved by reducing only protein or polyphenol, the carbohydrate is not involved in the haze formation mechanism, but otherwise incorporated into haze particles.

A number of approaches for stabilization of beverages against haze formation have been employed. A very traditional approach is storage of a beverage at low temperature; this leads to the settling out of material that would otherwise lead to haze in the package. The product is then typically decanted from the storage tank and filtered.

Fining (typically adding a substance to a beverage when entering a storage tank) can be carried out with the addition of either a protein (frequently gelatin or isinglass) or a polyphenol (most often TA). Both gelatin and isinglass are rich in proline and are thus HA proteins. TA is a gallotannin that can attach to proteins in two or more places and which forms haze readily. Fining thus serves to shift the protein–polyphenol ratio and facilitates precipitation and removal of material that would otherwise persist into the package and lead to haze formation.

Treatment with adsorbents can be used to reduce the amount of either HA protein or HA polyphenol. Adsorbents that remove proteins include bentonite and silica. Bentonite removes proteins indiscriminately. That makes it unsuitable for treating beer, where it is undesirable to remove the protein involved in foam formation. Silica, on the other hand, has been shown to be highly specific in removing haze protein and sparing foam protein. It was demonstrated that this occurs because the silica binds to prolines in the protein and is thus specific for the proteins capable of binding polyphenols. Silica has only limited effectiveness in removing HA protein from apple juice; this appears to be because most of the sites to which silica can attach are blocked by polyphenols. This presumably would also be the case with other fruit juices and with wines.

The most commonly used polyphenol adsorbent is polyvinylpolypyrrolidone (PVPP), which bears a remarkable resemblance to polyproline; both have five-membered nitrogen containing rings and amide bonds. The pattern of PVPP effectiveness is opposite to that seen with silica. In beer, PVPP removes only a small proportion of the HA polyphenol; that appears to be because most of this polyphenol is sandwiched between beer HA protein molecules and thus inaccessible. In apple juice, however, PVPP is quite effective in removing HA polyphenol, often along with some of the attached protein.

For fruit juices, ultrafiltration through a membrane that retains proteins can be carried out. This would be unsuitable for beer or sparkling wines, as it would remove protein that is needed for foam.

The β-glucans and arabinoxylans, the main non-starch polysaccharides in malt (beer), are responsible for problems during wort filtration. They form the dietary fiber fraction, present at considerable concentrations in final beers, which, among others, can lead to the formation of beer hazes.

Reactions between haze active proteins and polyphenols are at their best at high and balanced concentration and when pH is near 5.

During the mash, starch and large dextrins can cause a beta glucan haze in the resulting beer.

Several examples can be given regarding the effects of inorganic ions on the stability of colloidal systems.

1. Yeast flocculation is improved by Ca2+ 23,25,26; most yeast strains require at least 50 mg/L Ca2+ ions for good flocculation. Calcium ions almost certainly act by binding to mannoproteins on yeast cell walls and so cross-link cells in a lectin-like manner.
2. The interactions among proteins, polyphenols, and hop iso-α-acids are influenced by several ions, including Ca2+, Mg2+, Fe3+, and PO4 3−. Formation of complexes such as these can lead to improved wort clarification during boiling and improved beer clarification during maturation, leading to enhanced haze stability.
3. Protein precipitation during wort boiling (trub formation) occurs not only because of thermal denaturation but also because of the neutralizing effect of cations (especially Ca2+) on the negatively charged polypeptides. It has been estimated that a minimum level of 100 mg/L Ca2+ ions is required for good-quality protein break formation.
4. Oxalate derived from malt is precipitated as calcium oxalate.27 Ideally, this should occur during wort production because subsequent formation of calcium oxalate crystals in beer can lead to gushing and haze formation.17,28,29 It is recommended that 70 to 80 mg/L Ca2+ ions should be present during mashing to eliminate excess oxalate during beer storage.

Some proteins or protein fragments are involved in binding to polyphenols giving adducts which can form hazes in beers. These "haze-forming proteins" can be selectively removed from beer by adsorbtion onto silica hydrogels, or can be selectively degraded by proteolytic enzymes such as papain. These proteins and polypeptides appear to be distinct from those which add to the `body' of the beer and those which help form and stabilize foam.

Most haze formation is considered to be the result of oxidation of beer phenols (presumably free), followed by polymerisation and association with hydrophobic proteins. However, some results results suggest that the phenol association with protein precede further oxidation of either the sulfhydryl groups or phenol-protein or peptide derivatives that lead to polymerization and beer haze formation. The further oxidation in beer, during storage, might lead to formation of more hydrophobic proteins of even higher molecular weights and hence haze formation. The involvement of smaller peptides in the oxidation and further polymerization of proteins is a possibility.

Oxidation in the brewhouse promotes polymerization, precipitation of proanthocyanidins. In other words, wort produced under aerobic conditions is substantially more turbid than wort made under rigorous anaerobic conditions. This mechanism is promoted by the action of peroxidases.

Many proteins and other macro-molecules derived from barley and cereals can directly affect wort filterability and haze, including b-glucans, arabinoxylan, and prolamin (hordeins) to name a few.

Increased abun- dance of peroxidases in Dan’er malts, which facilitate oxidative polymerization between phenolic substances and proteins, leads to increased wort turbidity (Jin et al., 2013). Along with peroxidases, chitinases, which breakdown cell walls of fungi, are also known to cause haze and are higher in abundance in Dan’er malts.

Undoubtedly, haze depends on interactions between specific proteins and polyphenols (tannins), but the specific factors and mechanisms ruling haze formation are still unclear.

CMb and CMe trypsin inhibitors appears to be haze-active proteins as they have been isolated from silica adsorbates after beer clarification (Leiper et al., 2003b; Robinson et al., 2007; Iimure et al., 2009). However, these proteins can be nucleation and growth factors of colloidal haze rather than actual haze-active proteins. Hordein peptides can also be involved in haze formation, but the coagulation of proteins and the colloidal aggregation with polyphenols strongly depends on thermal processing of brewing steps. Opportune wort boiling and cooling steps can remove great part of colloidal haze, while the acceptance of unclarified and unfiltered beer is increasing.

Haze is generally formed on cold conditioning of freshly fermented beer and can be due to multiple materials as yeast cells and colloidal particles of organic or mineral origin. Much research has been conducted to characterize the haze-forming materials in beer as well as in other beverages by analyzing the chemical composition of the sediment. Significantly, haze-forming molecules are mainly composed of polypeptides derived from the storage proline-rich proteins of barley endosperm (i.e. hordeins) and polyphenols from malted barley and hops. Another protein, the barley trypsin inhibitor, that is not a proline-rich proteins in regard to hordeins, also seems to play a role in the formation of beer haze. Although carbohydrates are present in high amount in the sediment, it was shown that they are not involved in haze formation but co-aggregate with haze particles. Haze is mainly due to the formation of protein and polyphenol aggregates on cold conditioning. The polyphenol–protein complex grows to suffi cient size to result in turbidity and finally can form large particles that can sediment.

The appearance of haze is a visual clue to the reduced flavor stability of beer since the haze and flavor stability are both directly influenced by oxidation processes during storage (6,7). Critical to the rate of haze formation is the content of oxygen both during brewing and especially once the beer is packaged. cited:

• Bamforth, C. W. (1999). Beer haze. J. Am. Soc. Brew. Chem. 57:81-90.
• Bamforth, C. W. (1999). The science and understanding of the flavour stability of beer: A critical assessment. Brauwelt Int. 17:98-110.
• Bamforth, C. W. (1988). Processing and packaging and their effects on beer stability. Ferment 1:49-53.
• Back, W., Forster, C., Krottenthaler, M., Lehmann, J., Sacher, B., and Thum, B. (1999). New research findings on improving taste stability. Brauwelt Int. 17:394-405.

Proteins with high levels of proline and polyphenols with higher degrees of polymerization are most likely to form haze. Haze-active (HA) proteins isolated from beer have been found to be derived primarily from fragments of the barley storage protein group, the hordeins. These protein fragments consist of several different molecular weights (MWs) and are relatively rich in proline.

To improve the colloidal stability of beer, the residual HA protein, HA polyphenol, or a portion of both needs to be removed. This is typically achieved by using stabilization treatments, such as silica hydrogel (HA proteins) or polyvinylpolypyrrolidone (PVPP) (HA polyphenols). Siebert, K. J., and Lynn, P. Y. (1997). Mechanisms of beer colloidal stabilization. J. Am. Soc. Brew. Chem. 55:73-78.

Some barley varieties are more prone to haze based on their genetics.

The application of a nitrogen-rich atmosphere produced beer with relatively poor colloidal stability compared with that of beer produced under a normal atmosphere (Fig. 1). The initial chill hazes for both beers were less than 0.3 EBC FU (Fig. 1). It was expected that brewing under nitrogen would improve beer colloidal stability. A possible explanation may be that some oxidation is needed during brewing to ensure that the load of beer HA proteins/polyphenols are precipitated during boiling and maturation so that they do not carry through into the finished beer to be present to allow for more rapid haze formation. The oxidation of polyphenols during wort production is known to lead to the polymerization of these compounds and to binding with protein, forming large insoluble complexes that precipitate during boiling, benefiting overall colloidal stability (6).

Using proteome analysis, Iimure et al. (2008, 2009) identified additional beer foam proteins (BDAI-1 and yeast thioredoxin) and haze active proteins (BDAI-1, CMb and CMe).

In beer, hordein peptides positively influence foam formation but are also suspected to form a complex with polyphenolic compounds, causing precipitation and haze formation (Evans and Sheehan, 2002).

Turbid lautering is assumed to cause a lower non-biological stability80,90 and the reason for this might be that turbid worts sweep along more anthocyanogens resulting in a higher affinity for the formation of haze in bottled beer.

β-glucans, in addition to proteins, polyphenols, and arabinoxylans, participate in haze formation in wort and beer.

Beer stabilization can be ensured in different ways. Cold storage (the colder the better) for a short period reduces the potential haze-active material (protein-polyphenol complexes) in beer [96]. Utilization of adsorbents specific to proteins or polyphenols, proteolytic enzymes, and the addition of isinglass or tannic acid are common methods to achieve colloidal stability [83,97].

When haze-active proteins and haze-active polyphenols are combined in a buffered model system,(207,214) haze rises, peaks, and then declines as the concentration of hazeactive agents increases. The pH also has a huge impact. Much more haze is produced near pH 4.0 than at pH 3.0 or above pH 4.2. At the beer pH, ethanol at low concentration causes a modest decline of haze, whilst strong haze is observed at higher concentrations. To preserve beer colloidal stability, brewers usually remove haze-active materials.(215) To get rid of haze-active proteins, precipitation with tannic acid, hydrolysis with papain, and adsorption to bentonite(216) or silica gel(217,218) are very effective, but unfortunately in some cases, such procedures also remove foam proteins. To remove haze-active polyphenols, the most usual way is adsorption to polyvinylpolypyrrolidone-PVPP. Because of the structural analogy between these compounds and proline(219) (Fig. 11), pyrrolidone rings bind polymerized flavanoids through hydrogen and ionic bonds. New combined absorbents are now proposed to brewers, such as PVPP mixed with silica xerogel, PVP bound onto silica, and tannin linked to silica.(209,220) Another innovative way is the use of flavan-3-ol and proanthocyanidin-free malt which allows affording an excellent colloidal stability.(221)

A lag phase is usually observed in lager beers before chill-haze development.(210,222,223) As depicted in Fig. 12 for different batches(1,2,3), the longer the lag phase, the better the colloidal stability. Chill haze (or reversible haze), defined by non-covalent bonds between polyphenols and active proteins, can eventually turn into permanent haze that no longer dissolves as the beer warms.

As explained above, catechin does not rapidly induce strong haze. Upon storage, however, it does. Likewise, colloidal instability caused by dimers and trimers is enhanced after oxidation (not true for tetramers and pentamers).(208,213,224) Free radicals are known to enhance haze.(225) Tannoids have been defined by Chapon (103) as intermediates in the oxidation of simple flavanoids to tannins, forming complexes with proteins. On the other hand, according to O’Rourke et al., (224) oxidized flavanols cause chill haze but only subsequent polymerization leads to tannoids and permanent haze (Fig. 13a). Leemans et al. (223) have recently proposed a model in which aldehydes and oxygen play key roles in tanning polyphenol formation (Fig. 13b). The time needed to form critical amounts of tanning polyphenols leading to visible chill-haze particles corresponds to the lag phase. Not only dissolved oxygen but also shaking, higher temperature, polyphenol-rich raw materials, light, and heavy metals will significantly increase colloidal instability.

The interactions during brewing between phenolics and proteins are certainly important. Malts made from many sorghums are so rich in phenolic tannins that during mashing many of the enzymes are inhibited and conversion is insufficient. In mashes, and indeed in wort, there is a partition of proanthocyanidins between being free in solution, being bound to soluble proteins and being bound to insoluble proteins. The associations may be reversible, as in chill hazes, or may be irreversible, as in permanent hazes.

Polyphenols containing 4-25 aromatic rings can bond with high-molecular-weight proteins to form haze.

During cold storage, potential haze-active material (for example, protein-polyphenol complexes) is precipitated and thus eliminated from the beer. Miedl and Bamforth (2004) suggested storing beer at the lowest temperature possible for shorter periods of time to obtain high colloidal stability (Miedl & Bamforth, 2004). Apart from this traditional approach to colloidal stabilization, techniques involving the use of adsorbents specific to proteins or polyphenols, proteolytic enzymes, the addition of isinglass or tannic acid are applied (Siebert & Lynn, 1998; Siebert et al., 1996). PVPP is commonly used in brewing as a means to enhance the colloidal stability of beer by removal of haze-active polyphenols after fermentation. It is chosen as a stabilizing agent due to the structural similarity to polyproline. Proline-rich proteins are mainly involved in the formation of turbidity during storage. However, when applied to beer, part of the haze-active polyphenols are protected from removal by binding to proteins (Siebert & Lynn, 1998). Complex formation between polyphenols and PVPP and proline-rich proteins is determined by the same forces: hydrogen bonding, π-bond overlap, hydrophobic and polar interactions (Magalhaes et al., 2010). In a study by McMurrough, Madigan, and Kelly (1996a), 48% of total polyphenols (78% of total flavanols, 90% of prodelphinidin B3, 96% of procyanidin B3, 79% of (+)-catechin and 88% of (-)-epicatechin) were removed from beer treated with 100 g/hl PVPP (McMurrough et al., 1996a). At lower levels of PVPP, phenolic compounds with higher degrees of hydroxylation and oligomerization are preferably adsorbed, whereas high levels of PVPP indiscriminately remove polyphenols (Gramshaw, 1967; McMurrough, Madigan, & Smyth, 1995). Magalhaes et al. (2010) found adsorption equilibrium constants of phenolic compounds on PVPP to increase with the number of hydroxyl groups (Magalhaes et al., 2010). This was especially evident with benzoic and cinnamic acid derivatives, whereas flavonoids had higher adsorption constants than expected from their number of OH groups alone. After removal from beer, PVPP with adsorbed polyphenols can be recovered by treatment with a sodium hydroxide solution. (Magalhaes et al., 2010).

Numerous studies have mentioned a significant decrease in phenolics content during storage due to the fact that the phenolic compounds participate in the formation of colloidal hazes, and are oxidatively polymerized to higher-molecular-weight species.

Polyphenolic molecules can be found in different stages during the brewing process and react with proteins: during wort boiling, they form the hot break; during cooling, they form the cold break; and during post-fermentation, they are involved in the formation of chill haze and permanent hazes and facilitate the removal of undesirable compounds with filtration. However, they tend to react with proteins in packaged beer and form undesirable haze after the expiration date [66].

Polymerization reactions, protein–polyphenol interactions, and precipitation have a significant impact as well. It is predicted that during wort boiling, about 70% of polyphenols are extracted from hops [17,79], but according to their polarity and their tendency to form complexes with wort proteins, this number is variable [79]. Narziss and Bellmer [80] reported that protein precipitation greatly depends on the polyphenol polymerization index and that hop anthocyanogens exhibit higher reactivity than malt anthocyanogens. Besides, lower polymerization index or less oxidized polyphenols were more active in protein precipitation [80]. Hot trub contains 40–70% of proteins, 7–32% of bitter substances, 20–30% of organic substances, and 5% of ash [81]. Higher oligomeric phenolic compounds are prone to form complexes with proteins, and small phenolic molecules like phenolic acids easily get adsorbed to hot trub [82]. This leads to the conclusion that a significant share of higher oligomeric proanthocyanidins can be removed from wort with the settling of hot trub or get adsorbed to yeast cells during fermentation [82]. Proanthocyanidin tetramers and pentamers can be completely removed from worts after boiling and settling, and catechin and procyanidin B3 were found in the finished beer as reported by [83]. It has been reported that during whirlpool rest, phenolic compounds show a significant decrease, mostly because of their adsorption to hot trub. Catechin or dimeric proanthocyanidins are showing a significantly higher decrease than monophenolic compounds [76,77], with a noted decrease in antioxidant activity [84]. Leitao, however, declared no significant changes [85]. Following the process stages (fermentation, warm rest, and chill-lagering) contributes to the additional decreases in phenolic substances [75,84]. However, several phenolic acids and catechin, except for ferulic acid (35% decrease during warm rest), were not affected [76]. In another study, ferulic acid showed an increase during fermentation. This was attributed to yeast metabolic activity [72].

Beer contains less haze-active polyphenols than haze-active proteins (207). Derived from barley hordeins, haze-active proteins (10 kD-30 kD) are acidic hydrophilic polypeptides, rich in both proline and glutamic acid (208) and glycosylated.(209) Flavan-3-ol monomers do not induce haze as strongly as higher polymers (Table 32). Among dimers, procyanidin B3 and prodelphinidin B3 are very strong haze inducers, especially the latter.(58,176,210,211) Procyanidin trimer C2 is even more haze-active. The polymerization degree appears to be more determinant than the number of hydroxyl groups (211). Phenolic acids and flavonols do not participate in beer haze formation (Table 32).(212,213) No similar data are available in the literature for stilbenes, prenylchalcones, or derived flavanones.

Polyphenols specifically interact with binding sites of haze-active proteins. Haze-active proteins have a fixed number of haze active polyphenol binding sites specified by the content of the amino acid proline. Haze-active polyphenols have fixed numbers (two or more) of binding ends. Haze-active polyphenols are, above all, proanthocyanidins (dimers, trimers of catechin, epicatechin and gallocatechin)1,24,36. Polyphenols form bridges between protein molecules. Protein–polyphenol complexes are held together by weak non-covalent interactions, mainly hydrophobic interactions. The amount of haze formed depends both on the concentration of proteins and polyphenols and on their ratio. Two basic approaches to stabilize beer have been developed: reducing the concentration of the haze active proteins, or reducing the concentration of the haze active polyphenols. Beer, in contrast to other beverages, has a significant excess of haze-active proteins over haze active polyphenols42. Therefore, reducing the polyphenol content is an efficient way to haze stabilize beer. Removal of haze active polyphenols can be accomplished by adsorption using PVPP or by fining. However, non-haze active polyphenols are removed by these treatments and polyphenol substances are considered to be important antioxidants.

the amount of (+)–catechin in beer has been linked to haze formation by oxidative polymerization (1).

Higher concentrations of polyphenols can increase haze formation.

simple polyphenols have little or no chill-haze forming capabilities until they have been oxidatively polymerized. Haze formation occurs readily with high temperature even with fairly low oxygen levels.

Some brewers consider polyphenols, including hop polyphenols, a harmful factor since polyphenols are well known to promote colloidal instability through the formation of complexes with proteins, thus leading to reversible and ultimately irreversible turbidity or haziness in beer (21,31,38). In fact, during brewing, efforts are sometimes made to reduce the dosage of polyphenols, e.g., by using specific barley varieties free of proanthocyanidins (e.g., barley cultivars Caminant and Galant) or by using polyphenol-free hop extracts. Furthermore, in view of improved colloidal stability, polyphenols are often partly removed from beer by adsorption on polyvinylpolypyrrolidon (PVPP) during filtration. Such efforts to minimize the polyphenol content in the final product are in line with the general trend towards crystal clear beers.

Although the formation of chill haze increased (data not shown) when total hop polyphenol extract was used, the for- mation of permanent haze was reduced in the beers with addition of hop polyphenols to the brewing and sparging liquor whether prepared with isomerized hop extract or with non-isomerized hop extract (see Fig. 6). The increase in chill haze was expected, as polyphenols are known to interact reversibly with proteins to form temperature-dependent precipitates (7). However, such chill haze formation can be avoided, for instance, by passage of fermented beer over a silica gel filter to remove haze-sensitive hydrophilic proteins, a standard procedure that was not applied to any of the experimental brews of this study. In contrast, the reduction in the formation of permanent haze in beers made with added hop poly- phenols during brewing is somewhat unexpected and suggests that hop polyphenols slow down the oxidative transformations that take place upon beer storage.

Cold or chill haze, which is formed initially, is visible when the beer is cold (at temperatures below 0°C) but disappears when the beer is warmed to room temperature. This is caused by proteins and polyphenols binding using weak non-covalent hydrogen bonds. With the temperature increase, hydrogen bonds break, leading to the reversible reaction. However, this soluble complex is soon converted to an insoluble one, where covalent bonds are not disrupted with heating. This is called permanent haze (31), which represents an irreversible process and can be caused by many factors such as temperature, oxygen, heavy metals, agitation and light.

A higher proteolytic temperature (52°C) and a prolonged time interval (20 min) had as a consequence an increase in the haze in all of the beers, while the haze in the dark beers was created after 10 min at the same temperature.

(+)-catechin caused the formation of hazes in much lower concentration than ferulic acid. Ferulic acid took part in the formation of colloidal hazes at higher concentrations, and this was dependent on different factors such as haze protein concentrations, pH, and the presence of other phenolic compounds.

Low molecular polyphenols increase the reduction power of beer, while when simple phenols or polyphenols monomers react with proteins, no haze is formed (Siebert 1999). Haze is composed of complexes between condensed polyphenols and water-soluble proteins.

Colloidal instability due to interactions between polyphenols and proteins limits the shelf life of beer. A lag phase is usually observed in lager beers before chill-haze development [23–25]. The time needed to form critical amounts of tanning polyphenols leading to visible chill-haze particles corresponds to the lag phase. As described by Leemans et al. [25] for different batches, the longer the lag phase, the better the colloidal stability. Chill haze (or reversible haze), defined by non-covalent bonds between polyphenols and active proteins, can eventually turn into permanent haze that no longer dissolves as the beer warms.

Catechin does not rapidly induce strong haze. Upon storage, however, it does. Likewise, colloidal instability caused by dimers and trimers is enhanced after oxidation (not true for tetramers and pentamers) [26–28]. Free radicals are known to enhance haze [29]. Tannoids have been defined by Chapon [30] as intermediates in the oxidation of simple flavanoids to tannins, forming complexes with proteins. On the other hand, according to O’Rourke et al. [28], oxidized flavanols cause chill haze, but only subsequent polymerization leads to tannoids and permanent haze [28, 31]. Leemans et al. [25] have proposed a model in which aldehydes and oxygen play key roles in tanning polyphenol formation [25, 31]. Not only dissolved oxygen but also shaking, higher temperature, polyphenol-rich raw materials, light, and heavy metals will significantly increase colloidal instability [25, 31].

Beer contains less haze-active polyphenols than haze-active proteins. Derived from barley hordeins, haze-active proteins (10–30 kDa) are acidic hydrophilic polypeptides, rich in both proline and glutamic acid [26] and glycosylated [32]. Much more haze is produced near pH 4.0 than at pH 3.0 or above pH 4.2. At the beer pH, ethanol at low concentration causes a modest decline of haze, while strong haze is observed at higher concentrations [33]. To preserve beer colloidal stability, brewers usually remove haze-active materials [34]. To get rid of haze-active proteins, precipitation with tannic acid, hydrolysis with papain and adsorption to bentonite [35] or silica gel [36, 37] are very effective, but unfortunately in some cases, such procedures also remove foam proteins. To remove haze-active polyphenols, the most usual way is adsorption to polyvinylpolypyrrolidone-PVPP. Because of the structural analogy between these compounds and proline [38], pyrrolidone rings bind polymerized flavanoids through hydrogen and ionic bonds. New combined absorbents are now proposed to brewers, such as PVPP mixed with silica xerogel, PVP bound onto silica, and tannin linked to silica [23, 39]. Another innovative way is the use of flavan-3-ol and proanthocyanidin-free malt which allows affording an excellent colloidal stability [40].

Preserving colloidal stability in lager beers is a difficult issue for the brewing industry. Interactions between haze active polyphenols (proanthocyanidins) and proteins can result in irreversible bounding which has a negative impact on the "shelf life" of beer. Also polysaccharides, metal ions and minerals can be responsible for the forming of haze. Colloidal instability in beer is caused mainly by interactions between polypeptides and polyphenols; this has already been reported by several authors [11, 12, 14, 16, 22, 25, 27]. The natural haze active polyphenols in beer are mainly proanthocyanidins because of their relatively large and complex structure. A haze active polyphenol binds at least with two proteins. The haze forming capacity of those proteins is dependent on their proline content [28].. The nature of protein-polyphenol complexes is reversible in the early stages of their formation. This expresses itself as chill haze which can be dissipated by warming the beer to room temperature [23, 28]. Permanent haze originates from protein-polyphenol interactions that are irreversible.

In general, the use of stabilisation products during wort boiling, more specifically at the end of the boiling process, has a positive impact on the shelf life of the final beers. The suffi cient removal of the haze-active polyphenols by a PVPP treatment (10 g/hL and 3 minutes contact time) has a positive impact on the colloidal sta-bility, and thus on the shelf life, of the finished beer. The sensory evaluation of the stabilised beers has shown that PVPP treatment does not deteriorate the flavor stability, which is in contrast with the results on the analytical evaluation of the reducing capacity. pH adjustment (5.2) at mashing-in, combined with application of both PVPP and gallotannins in the upstream brewing process, seems promising because of the explicitly prolonged shelf life. Addition of gallotannins in the boiling kettle at the end of the boiling process results in an improved physical stability. They also contribute to a better antioxidative capacity and a prolonged flavour stability. The beers brewed with addition of gallotannins at the end of wort boiling were preferred by the taste panel because of their higher sensorial freshness and fullness. In view of the obtained results discussed above, it is possible to produce a beer with long shelf life and improved fl avour stability by addition of gallotannins in the upstream brewing process. Application of gallotannins at boiling is a very convenient way of physico-chemical stabilisation without the need for extra filtration, which reduced beer losses and results in beers with an improved flavour stability.

Transition metal ions play an important role in polyphenol-protein association and precipitation (61, 62). Other factors that will influence the size and grade of polyphenol-protein complexation are the presence of oxidising agents (e.g. hydrogen peroxide), heightened temperatures and oxygen exposure, as these factors facilitate the formation of covalent bonds that irreversibly link polyphenols to proteins (63, 64).

See also

• Siebert, K. "Haze formation in beverages." 2006.

Potential sources

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