Haze

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

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.

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].

See also

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

Potential sources

• https://www.mdpi.com/2311-5637/4/4/91/pdf
• Conventional and alternative principles for stabilization of protein and polyphenol fractions in beer
• "Adsorption by Polyvinylpolypyrrolidone of Catechins and Proanthocyanidins from Beer." PVPP removes phenolic compounds?
• "Contribution of Wheat and Wheat Protein Fractions to the Colloidal Haze of Wheat Beers"
• Siebert, K. et al. "Formation of Protein−Polyphenol Haze in Beverages."
• "Nature of Polyphenol−Protein Interactions."
• http://www.craftbrewersconference.com/wp-content/uploads/2015_presentations/R1320_Roland_Pahl.pdf
• https://www.sciencedirect.com/science/article/pii/B9780127999548000125
• see Handbook of Brewing 10.7
• Nature of Beer Hazes
• http://www.lowoxygenbrewing.com/wp-content/uploads/2017/04/kusche_0207.pdf Kinetics of haze formation in beer – turbidity and flavan-3-ols
• http://www.lowoxygenbrewing.com/wp-content/uploads/2017/04/loch_ahring_0408.pdf Chill-haze – Identification and Determination of Haze-active Constituents by HPLC and Mass Spectrometry
• https://bsgcraftbrewing.com/kerry-biohaze-1-kg
• https://www.tandfonline.com/doi/full/10.1080/03610470.2020.1803468
• https://www.tandfonline.com/doi/full/10.1080/03610470.2019.1705045
• Leiper, K. A., Stewart, G. G., and McKeown, I. P. Beer polypeptides and silica gel. Part I. Polypeptides involved in haze formation. J. Inst. Brew. 109:57-72, 2003.
• Leisegang, R., and Stahl, U. Degradation of a foam-promoting barley protein by a proteinase from brewing yeast. J. Inst. Brew. 111:112-117, 2005.
• https://www.mdpi.com/2304-8158/10/12/3114/pdf
• The Interaction Between Malt Protein Quality and Brewing Conditions and Their Impact on Beer Colloidal Stability
• Beer Haze Bamforth
• Mechanisms of Beer Colloidal Stabilization