Beer haze may also be addressed during maturation (pre-filtration) using precipitants. Proteolytic enzymes such as papain can be used to remove (hydrolyse) haze proteins, although the enzyme can also have a negative impact upon finished product, as it is relatively non-specific and often hydrolyses foam active proteins to destroying a beers head. However, research conducted by Eden et al (2005), using a proline specific protease enzyme (from the microorganism Aspergillus niger), resulted in the prevention of chill haze in beers without having a negative impact on the foam active proteins, as they are very low in proline. This enzyme may prove to be a viable option for brewers in the future.

Tannic acid (a polyphenol) interacts with HA proteins to form insoluble precipitates in the beer. These precipitates are frequently voluminous, thus beer losses via this mechanism can be large.

Fining with agents such as gelatine can also remove HA protein.

Microbrewers often rely on the more traditional method of extended cold storage and filtration to precipitate and remove HA material, which can result in a less stable product.

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continuing our article on Beer Clarity and hazes

Chill hazes consist of small (0.6µm) particles and are a result of HA protein-polyphenol (catechin) cross linking, via hydrogen or hydrophobic bonding, at temperatures of 0 - 4°C. Such hazes re-dissolve upon warming to 20°C as these bonds are weak; however, polyphenols such as catechin are subject to oxidation during beer storage, producing polymers, which form large particles (1 - 10µm) and permanent hazes when covalently cross-linked with HA proteins.

Beer haze is typically measured via nephelometric methods, which are based upon the principle of light scattering when a beam of white light passes through a solution carrying a dispersion of particles (Fischer, 1966). The size, concentration, shape and differing refractive index (compared to the media) of the particles dictate the amount and intensity of the light scattered over a range of angles. Increasing amounts of particles (haze) in the medium corresponds with increased capacity to scatter light. Nephelometric instruments primarily measure the intensity of light scattered at a 90° angle from the incident beam. Measurements at narrow angles, i.e. 13°, are sensitive to large particles (1 - 10µm) that often form visible hazes, whereas 90° angles are sensitive to pseudo or invisible hazes that form from smaller particles < 0.1µm. Larger particles are (usually) removed during the filtration of beer, but smaller particles may remain in the final product to promote colloidal instability. Thus, beer haze measurements are usually performed at 90° angles.

Large, modern commercial breweries address the problem of protein-polyphenol colloidal instability via the use of adsorbents during beer filtration, such as silica gels, which bind to HA proteins, or Polyvinylpolypyrrolidone (PVPP), which binds to HA polyphenols. Both compounds are highly selective for their respective HA material.

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Beer is subject to colloidal instability during storage. Chill filtered beer initially remains haze free, however, as the product ages it passes through a series of metastable colloidal states, whereby chill hazes and eventually insoluble (permanent) hazes form, primarily as a result of protein-polyphenol interactions within the product. Haze reactions are significantly accelerated with increasing temperature, due to an increase in the rate of reactions (Kunze, 1999).

Proteins and polyphenols are natural components of beer which are extracted from malt and hops. Haze active (HA) proteins contain high percentages of the amino acid proline and are thought to be the degradative products (as a result of the malting and brewing process) of barley hordein. Globulin and albumin proteins are also regarded as haze active, as they associate with polyphenols in the beer to form hazes upon removal of the proline containing proteins.

The polyphenols primarily involved in haze formation are barley flavanoids such as catechin, or polymers of the latter, proanthocyanidins.

The mechanism of haze formation is the following: Haze active proteins contain a number of active sites, thought to be the proline residues, which have a specific affinity for polyphenols. If polyphenols have two binding ends, they are capable of linking protein molecules together, forming colloidal particles, which create haze. Smaller colloidal particles initially form in beer, as there are insufficient amounts of polyphenols to link any more than two protein molecules together, hence light hazes form. Larger particles and increased haze can form when polyphenol levels approach those of the protein (due to oxidative reactions during storage ), as polyphenol linking of the proteins can form large networks.

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However, as packaged beer may contain amino acids and reducing sugars in solution Maillard reactions may still occur in the product, particularly if the beer is subjected to storage under conditions that favour this reaction, namely elevated temperatures, long time periods or a combination of both. Studies have attributed colour increases during ageing to such Maillard reactions.

The polyphenols in beer are also subject to degradation via oxidation reactions catalysed by light, oxygen, oxidising agents, and heavy metals, to form darker coloured quinonoid bases that result in an increase in beer colour.

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Key to the diagram: 1 : Ethanol, 2: Lactic acid, 3: Ethyl Acetate, 4: pH, 5: Extract content, 6: Acetic acid

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The finished product is characterised by high levels of lactic acid, acetic acid, ethyl acetate and ethyl acetate. Final gravities are in the region of 1.008 (2.2 oP) 1.012, 3 oP, DMS levels are reduced to approx 100pb and diacetyl concentrations between 45-80ppb. Final 2,3 butanediol levels are not available.

Beers of the lambic style are described as having vinous aromas and tastes accompanies by ‘horse blanket’ or ‘goaty’ characterisitics, which appears to be attributable to the contained acids and products from Brettanomyces activity. Thin mouth feel is also noted, due to the low levels of residual dextrins.

The spontaneous nature of lambic fermentation and the varied spectra of bacteria and yeasts encountered in differing breweries that are responsible for such fermentation, may produce beers with differing characterisitics.

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Tetrahydropyridines are also produced from ethanol and the amino acid lysine, which impart a ‘mousy’ or ‘horsy’ aroma to the beer. Volatile phenolics, with medicinal, ‘barnyard’ or ‘animal’ type odours are also from from 4-ethyl phenol and 4-ethyl guaiacol compounds.

Brettanomyces activity also leads to a significant increase in caprylic (C8) and caprix (C10) acids (small amounts are formed during earlier Saccharomyces acitivity) along with their associated esters, ethyl caprylate and ethyl caprate. These compounds produce a characteristic ‘goaty’ aroma/flavour to the final product. Capric and caprylic acids are short chain fatty acids and are thought to be by-products of yeast metabolism, produced during lipid synthesis by a yeast cell and released into the medium via leakage through membranes damaged by ethanol, or as a consequence of an autolytic mechanism. Higher temperatures, aeration and agitation during fermentation reduce the amounts of fatty acids in beer. Reduced aeration/agitation produced higher concentrations. The combined threshold for C6-C12 acids is 10ppm, beyond which ‘goaty’ aromas arise. C8 acid levels in gueuze as 12.4 - 21.85 ppm and C10 acids as 2.3 - 3.9p ppm.

Brettanomyces is active until the end of the fermnetaion period. Acetic acid bacteria of the genus Acetomonas and a number of oxidative yeasts (Pichia, ) are also found during lambic fermentations, however their influence is often minimal.

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Fermentation at temperatures above 20oC are required for P. damnosus growth, therefore increasing temperatures beyond this level will stimulate lactic acid production as cell numbers increase. P. damnosus is a also a facultative anaerobe, thus the exclusion of air during P. damnosus activity will presumably facilitate the further production of lactic acid.

P. damnosus is also a producer of diacetyl, prodcuing up to 200ppb during this period. Acetoin is also produced as a consequence . The production of large amounts of lactic acid result in a drop in beer pH from 4 to 3, with a gradual lowering of residual extract.

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And when they begin to bloom:

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