European Food Research and Technology

, Volume 232, Issue 2, pp 191–204

Protein changes during malting and brewing with focus on haze and foam formation: a review


    • Lehrstuhl für Brau- und GetränketechnologieTechnische Universität München
  • Martina Gastl
    • Lehrstuhl für Brau- und GetränketechnologieTechnische Universität München
  • Thomas Becker
    • Lehrstuhl für Brau- und GetränketechnologieTechnische Universität München
Review Paper

DOI: 10.1007/s00217-010-1412-6

Cite this article as:
Steiner, E., Gastl, M. & Becker, T. Eur Food Res Technol (2011) 232: 191. doi:10.1007/s00217-010-1412-6


Beer is a complex mixture of over 450 constituents and, in addition, it contains macromolecules such as proteins, nucleic acids, polysaccharides, and lipids. In beer, several different protein groups, originating from barley, barley malt, and yeast, are known to influence beer quality. Some of them play a role in foam formation and mouthfeel, and others are known to form haze and have to be precipitated to guarantee haze stability, since turbidity gives a first visual impression of the quality of beer to the consumer. These proteins are derived from the malt used and are influenced, modified, and aggregated throughout the whole malting and brewing process. During malting, barley storage proteins are partially degraded by proteinases into amino acids and peptides that are critical for obtaining high-quality malt and therefore high-quality wort and beer. During mashing, proteins are solubilized and transferred into the produced wort. Throughout wort boiling proteins are glycated and coagulated being possible to separate those coagulated proteins from the wort as hot trub. In fermentation and maturation process, proteins aggregate as well, because of low pH, and can be separated. The understanding of beer protein also requires knowledge about the barley cultivar characteristics on barley/malt proteins, hordeins, protein Z, and LTP1. This review summarizes the protein composition and functions and the changes of malt proteins in beer during the malting and brewing process. Also methods for protein identification are described.


ProteinsBarleyMaltBeerHaze formationFoam formation

Proteins in barley and malt

Barley (Hordeum vulgare L.) is a major food and animal feed crop. It ranks fourth in area of cultivation of cereal crops in the world. Barley is commonly used as raw material for malting and subsequently production of beer, where certain specifications have to be fulfilled. These specifications are among others: germinative capacity, protein content, sorting (kernel size), water content, kernel abnormalities, and infestation. Malting includes the controlled germination of barley in which hydrolytic enzymes are synthesized, and the cell walls, proteins, and starch of the endosperm are largely digested, making the grain more friable [13]. Proteins in beer are mainly derived from the barley used. The mature barley grain contains a spectrum of proteins that differ in function, location, structure, and other physical and chemical characteristics. Barley seed tissues have different soluble protein contents and distinct proteomes.

The three main tissues of the barley seed are the aleurone layer, embryo, and starchy endosperm that account for about 9, 4, and 87%, respectively, of the seed dry weight [4, 5]. The level of protein in barley is an important determinant in considering the final product quality of beer, for example for cultivar identification or as an indication of malting quality parameters [4], and it is influenced by soil conditions, crop rotation, fertilization, and weather conditions. For malting barley, the balance between carbohydrates and proteins is important, since high protein content reduces primarily the amount of available carbohydrates. Proteins present in barley seeds are important quality determinants. During malting, barley storage proteins are partially degraded by proteinases into amino acids and peptides which are critical for obtaining high-quality malt and therefore high-quality wort and beer [1, 6, 7].

Germination provides the necessary hydrolytic enzymes to modify the grain, which are, in the case of proteins, endoproteases, and carboxypeptidases. These enzymes degrade storage proteins, especially prolamins (hordeins) and glutelins [8] and produce free amino acids during germination by cleavage of reserve proteins in the endosperm [9]. According to Mikola [10], there exist five seine carboxypeptidases in germinating barley, which have complementary specificities and mostly an acidic pH optimum. All of these carboxypeptidases consist of 2 identical subunits, each compose of two polypeptide chains, cross-linked by disulphide bridges [9, 11, 12]. Barley malt endoproteases (EC.3.4.21) develop multiple isoforms mainly during grain germination and pass through kilning almost intact [8, 13]. Jones [1317] surveyed those enzymes and their behavior during malting and mashing. Cysteine proteases (EC 3.4.22) are clearly important players in the hydrolysis of barley proteins during malting and mashing. However, it seems likely that they do not play as predominant a role as was attributed to them in the past [15, 16, 1822]. It has been found out that metalloproteases (EC 3.4.24) play a very significant role in solubilizing proteins, especially during mashing at pH 5.8–6.0 [23]. All current evidence suggests that the serine proteases (EC 3.4.21) play little or no direct role in the solubilization of barley storage proteins [23, 24], even though they comprise one of the most active enzyme forms present in malt [22]. While none of the barley aspartic proteases (EC 3.4.23), that have been purified and characterized, seem to be involved in hydrolyzing the seed storage proteins, it is likely that other members of this group do participate. Jones [17] investigated endoproteases in malt and wort and discovered that they were inactivated at temperatures above 60 °C. Jones et al. [6] examined the influence of the kilning process toward the endoproteolytic activity. These enzymes were affected by heating at 68 and 85 °C, during the final stages of kilning, but these changes did not influence the overall proteolytic activity.

Other proteins are involved in protein folding, such as protein disulfide isomerase (EC, which catalyzes the formation of protein disulfide bridges. Due to their heat-sensitivity, proteinases are inactivated when the temperature rises above 72 °C [2530]. They are almost totally inactive within 16 min [1, 7, 13].

Summarizing the most important factors for the protein composition, as origin in finished beer are barley cultivar and the level of protein modification during malting, which is judged by malt modification which is conventionally measured in the brewing industry as the Kolbach index (soluble nitrogen/total nitrogen*100) [31, 32].

To get an overview of the main proteins in malt and beer, the most studied proteins are described in the next paragraphs. Proteins can be classified pursuant to their solubility. Osborne [3337] took advantage of this fact and developed a procedure to separate the proteins. Proteins are divided into water-soluble (albumins), salt-soluble (globulins), alcohol-soluble (prolamins), and alkali-soluble (glutelins) fractions [3436, 38, 39]. Osborne fractionation is a relatively simple, fast, and sensitive extraction–analysis procedure for the routine quantitation of all protein types in cereals in relative and absolute quantities, including the optimization of protein extraction and of quantitative analysis by RP-HPLC. High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify, and purify the individual components of the mixture.

Not only Osborne fractionation and HPLC but also several other methods exist to separate and identify proteins in barley, malt, wort, and beer. To get an overview over the applications of the described methods in the review, a description follows in the next paragraphs.

Several authors [5, 3960] characterized barley and barley malt proteins with help of 2D-PAGE. Other authors [25, 26, 29, 30, 32, 41, 6165] used 2D-PAGE and mass spectrometry to fingerprint the protein composition in beer and to evaluate protein composition with regard to foam stability and haze formation. Klose [39] followed protein changes during malting with the help of a Lab-on-a-Chip technique and validated the results with 2D-PAGE. Iimure et al. [64] invented a protein map for the use in beer quality control. This beer proteome map provides a strong detection platform for the behaviors of beer quality–related proteins, like foam stability and haze formation. The nucleotide and amino acid sequences defined by the protein identification in the beer proteome map may have advantages for barley breeding and process control for beer brewing. The nucleotide sequences also give access to DNA markers in barley breeding by detecting sequence polymorphisms.

Hejgaard et al. [6673] worked with immunoelectrophoresis and could identify several malt and beer proteins. Shewry et al. [54, 7478] determined several methods for investigation of proteins in barley, malt, and beer mainly with different electrophoresis methods. Asano et al. [62, 63] worked with size-exclusion chromatography, immunoelectrophoresis and SDS–PAGE. Mills et al. [79] made immunological studies of hydrophobic proteins in beer with main focus and foam proteins. He discovered that the most hydrophilic protein group contained the majority of the proteinaceous material but it also comprised polypeptides with the least amount of tertiary structure.

Vaag et al. [28] established a quantitative ELISA method to identify a 17 kDa Protein and Ishibashi et al. [80] used an ELISA technique to quantify the range of foam-active protein found in malts produced in different geographic regions, and using different barley cultivars. Van Nierop et al. [30] used an ELISA technique to follow LTP1 content during the brewing process.

Osman et al. [1820] investigated the activity of endoproteases in barley, malt, and mash. Hence, protein degradation during malting and brewing is very important for the later beer quality (mouthfeel, foam, and haze stability). It was suggested that estimation of the levels of degraded hordein (the estimation of the levels of hordein degraded during malting truly reflects the changes in proteins during malting and can measure the difference in barley varieties related to proteins and their degrading enzymes) during malting is a sensitive indicator of the total proteolytic action of proteinases as well as the degradability of the reserve proteins. And therefore, it is possible to predict several beer quality parameters according the total activity of all proteinases and the protein modification during malting.

To obtain good results, those separation and identification methods can be combined. Van Nierop et al. [30], for example, used ELISA, 2D-PAGE, RP-HPLC, electrospray mass spectrometry (ESMS), and circular dichroism (CD) spectrophotometry to follow the changes of LTP1 before and after boiling.

Since there exist various methods to separate and identify proteins in this review, an overview over existent proteins in barley, malt, wort, and beer is provided according to only one method, which is Osborne fractionation. These fractions are described more closely in the next sections.

Barley glutelin

About 30% of barley protein is glutelin that dissolves only in diluted alkali [54]. Glutelin is localized almost entirely in the starchy endosperm (Fig. 1), is not broken down later on, and passes unchanged into the spent grains [81, 82].
Fig. 1

Shematic longitudinal section of a barley grain [81]

Glutelin is the least well-understood grain protein fraction. This is partly because the poor solubility of the components has necessitated the use of extreme extraction conditions and powerful solvents which often cause denaturation and even degradation (e.g., by the use of alkali) of the proteins, rendering electrophoretic analysis difficult. Also, because glutelin is the last fraction to be extracted, it is frequently affected by previous treatments and contaminated with residual proteins from other fractions, notably prolamins, which are incompletely extracted by classical Osborne procedures [83]. It has not been possible to prepare an undenatured glutelin fraction totally free of contaminating hordein [3].

Barley prolamin

The prolamin in barley is called hordein and it constitutes about 37% of the barley protein. It dissolves in 80% alcohol and part of it passes into spent grains. Hordein is a low-lysine, high-proline, and high-glutamine alcohol-soluble protein family found in barley endosperm (Fig. 1). It is the major nitrogenous fraction of barley endosperm composing 35–55% of the total nitrogen in the mature grain [1, 8486]. Hordeins are accumulated relatively late in grain development, first being observed about 22 days after anthesis (when the grain weighs about 33% of its final dry weight) and increasing in amount until maximum dry weight is reached [87]. The major storage proteins in most cereal grains are alcohol-soluble prolamins. These are not single components, but form a polymorphic series of polypeptides of considerable complexity [88]. Hordein is synthesized on the rough endoplasmic reticulum during later stages of grain filling and deposited within vacuoles in protein bodies [89, 90]. Silva et al. [91] ascertained that the exposure of hordeins to a proteolytic process during germination reduces its content and originates in less hydrophobic peptides. Some malt water–soluble proteins result from the hordein proteolysis. Hordeins are the most abundant proteins in barley endosperm characterized by their solubility in alcohol. These storage proteins form a matrix around the starch granules, and it is suggested that their degradation during malting directly affects the availability of starch to amylolytic attack during mashing [92].

Shewry [75, 77] divided the hordeins according to their size and amino acid composition in four different fractions (A-D), dependent on their size and amino acid composition. A-hordeins (15–25 kDa) seem to be no genuine storage proteins as they contain protease inhibitors and α-amylases. B-hordeins (32–45 kDa) are rich in sulfur content and are, with 80%, the biggest hordein fraction. B-hordeins have a general structure, with an assumed signal peptide of 19 aminoacid residues, a central repetitive domain rich in proline and glutamine residues, and a C-terminal domain containing most of the cysteine residues are encoded by a single structural locus, Hor2, located on the short arm 1 of chromosome 1H(5), 7–8 cM distal to the Hor1 locus which codes for the C-hordeins. C-hordeins (49–72 kDa) are low in sulfur content, and D-hordeins (>100 kDa) are the largest storage proteins and are encoded by the Hor3 locus located on the long arm of chromosome 1H(5) [85, 87, 93, 94].

Cereal prolamins are not single proteins but complex polymorphic mixtures of polypeptides [54]. During malting, disulfide bonds are reduced and B- and D-hordeins are broken down by proteolysis. Well-modified malt contains less than half the amount of hordeins present in the original barley. D-hordeins are degraded more rapidly than their B-type counterparts, and the latter are more rapidly degraded than C-hordeins [3, 95].

Barley albumins and globulins

Many researchers extract a combined salt-soluble protein fraction, because water extracts contain globulins as well as albumins. The two classes of proteins may be separated by dialysis, but there is considerable overlap between the two [83]. Albumins and globulins consist mainly of metabolic proteins, at least in the cereal grains [96] and are found in the embryo and the aleurone layer, respectively [81, 82]. Whereas prolamins are degraded during germination, albumins and other soluble proteins increased during the germination process [92].


The globulin fraction of barley is called edestin. It dissolves in dilute salt solutions and hence also in the mash. It forms about 15% of the barley protein. Edestin forms 4 components (α, β, γ, and δ) of which the sulfur-containing β-globulin does not completely precipitate even on prolonged boiling and can give rise to haze in beer. Enzymes and enzyme-related proteins are mainly albumins and globulins [42].


The albumin of barley is called leucosin. It dissolves in pure water and constitutes about 11% of the protein in barley. During boiling, it is completely precipitated. α-Amylase, protein Z, and lipid transfer proteins are barley albumins and are important for the beer quality attributes: foam stability and haze formation [97]. Albumins can be further divided into protein Z and lipid transfer proteins as functional proteins

Protein Z

Protein Z belongs to a family of barley serpins and consists of at least four antigenically identical molecular forms with isoelectric points in the range 5.55—5.80 (in beer: 5.1–5.4), but same molecular mass near 40 kDa [1, 55, 67, 68, 98]. Protein Z is hydrophobe and exists in free and bound forms in barley, like α-amylase, and there also exist heterodimers. Protein Z contains 2 cysteine and 20 lysine residues per monomer molecule and is relatively rich in leucine and other hydrophobic residues. Protein Z accounts for 5% of the albumin fraction and more than 7% in some high-lysine barleys [67, 99]. The content of protein Z in barley grains depends on the level of nitrogen fertilization [67, 100]. Protein Z makes up to 20–170 mg/L of beer protein [79]. In mature seeds, protein Z is present in thiol bound forms, which are released during germination [101]. The function of the protein is at present unknown but it is known that it is deposited specifically in the endosperm responding to nitrogen fertilizer, similar to the hordein storage proteins. The synthesis is regulated during grain development at the transcriptional level in dependence of the supply of nitrogen [98, 100, 102, 103]. It is stated that upregulation of transcript levels could be effectuated within hours, if ammonium nitrate was supplied through the peduncle, and equally rapid reduced when the supply was stopped [103]. Finnie et al. [49] investigated the proteome of grain filling and seed maturation in barley. They identified a group of proteins that increased gradually both in intensity and abundance, during the entire examination period of development and were identified as serpins. Also Sorensen [55] and Giese [98] could detect the expression of protein Z4 (a subform of protein Z) only during germination. Protein Z4 has an expression profile similar to β-amylase and seed storage proteins (hordeins).

Three distinct serpin sequences from barley could be found in the databases SWISSPROT and TREMBL: protein Z4, protein Z7, and protein Zx. These different protein Z forms are thought to have a role as storage proteins in plants, due to their high “Lys” content and the fact that serpin gene expression is regulated by the “high-Lys” alleles lys1 and lys3a [49, 104].

Hejgaard et al. [68] suggest that the precursors of protein Z originate from chromosomes 4 and 7, and thus, they are named protein Z4 and protein Z7. Rasmussen and co-workers [105] were able to estimate the size of protein Z mRNA at 1.800 b. This is sufficient to code for the 46.000 or 44.000 MW precursor peptides found in vitro translations plus leave 400–500 b for the 5′ and 3′ non-coding regions. Doll [106] and Rasmussen [107] suggest that protein Z could be a candidate for modulation of the barley seed protein composition to balance the nutritional quality of the grain. Giese and Hejgaard [98] found out that during germination, protein Z becomes the dominant protein in the salt-soluble fraction in developing barley. The proteins in barley malt are known to be glycated by D-glucose, which is a product of starch degradation during malting [108]. Bobalova et al. [109] investigated in their research the glycation of protein Z and found out that protein Z glycation is detectable from the second day of malting. The role of protein Z in beer is described more detailed in the sections foam and haze formation.

Lipid transfer protein

Lipid transfer proteins (LTPs) are ubiquitous plant lipid-binding proteins that were originally identified by their ability to catalyze the transfer of lipids between membranes. LTPs are abundant soluble proteins of the aleurone layers from barley endosperm. The compact structure of the barley LTP1 comprises four helices stabilized by four disulfide bonds and a well-defined C-terminal arm with no regular secondary structure [110] which is shown in Fig. 2, where a 3D and surface protein of barley LTP native protein (here called 1LIP, red) is shown [111]. In comparison with other plant lipid transfer proteins, the barley protein has a small hydrophobic cavity but is capable of binding different lipids such as fatty acids and acyl-CoA [25, 112, 113]. According to molecular mass, this multigene family is subdivided into two subfamilies, ns-LTP1 (9 kDa) and ns-LTP2 (7 kDa); both located in the aleurone layer of the cereal grain endosperm [56, 114]. LTP1 and LTP2 are expressed in barley grain but only LTP1 has been able to be detected in beer. LTP1 is claimed to be an inhibitor of malt cysteine endoproteases [14, 115]. The role of LTP1 in beer is described more detailed in the sections foam and haze formation.
Fig. 2

3D and surface protein of barley LTP native protein (1LIP, red) is shown [111]

Protein Z and LTP1

Evans [116, 117] investigated the influence of the malting process on the different protein Z types and LTP1. He discovered that the amount of LTP1 did not change during germination but a significant proportion of the bound/latent protein Z was converted into the free fraction. He claims that during germination, proteolytic cleavage in the reactive site loop converts protein Z to a heat and protease stable forms, and hence, they can survive the brewing process. He ascertained also that kilning reduced the amount of protein Z and LTP1 [66, 118].

Evans [116] analyzed feed and malting barley varieties and could not find any differences in the level of protein Z and LTP1. He also ascertained malt-derived factors that influence beer foam stability, such as protein Z4, β-glucan, viscosity, and Kohlbach index. Beer components (protein Z4, free amino nitrogen, β-glucan, arabinoxylan, and viscosity) were correlated with foam stability [117]. Protein Z4, protein Z7, and LTP1 have been shown to act as protease inhibitors [116, 119, 120].

Proteins in wort and beer

Proteins influence the whole brewing process not only in the form of enzymes but also in combination with other substances such as polyphenols. As enzymes, they degrade starch, β-glucans, and proteins. In protein–protein linkages, they stabilize foams and are responsible for mouthfeel and flavor stability, and in combination with polyphenols, they are thought to form haze. As amino acids, peptides, and sal ammoniac, they are important nitrogen sources for yeast [121]. Only about 20% of the total grain proteins are water soluble. Barley water-soluble proteins are believed to be resistant to proteolysis and heat coagulation and hence pass through the processing steps, intact or somewhat modified, to beer [116, 122, 123]. Several aspects of the brewing process are affected by soluble proteins, peptides, and/or amino acids that are released. No more than one-third of the total protein content passes into the finished beer which is obtained throughout mainly two processes; mashing and the wort boiling. Mashing is the first biochemical process step of brewing and completes the enzymatic degradation started during malting. Enzymes synthesized during malting are absolutely essential for the degradation of large molecules during mashing. These enzymes are displayed in Table 1 [1, 7]. The three biochemical basic processes taking place during malting are cytolysis, proteolysis, and amylolysis, which are indicated by β-glucan, FAN, and extract concentration, respectively. In order to get good brews, part of the insoluble native protein must be converted into “soluble protein” during malting and mashing. This fraction comprises a mixture of amino acids, peptides, and dissolved proteins, and a major portion of it arises by proteolysis of barley proteins [23]. During the brewing process, there are three possibilities to discard the (unwanted) proteinic particles. The first opportunity is given during wort boiling, where proteins coagulate and can be removed in the “whirlpool”. The second, during fermentation, where the pH decreases and proteinic particles can be separated by sedimentation. The third step is during maturation of beer. During maturation, proteins adhere on the yeast and can be discarded [124].
Table 1

Enzymes in barley and barley malt [1, 7, 166, 167]







Matrix linked β-glucan

Soluble, high molecular weight β-glucan

Endo-β-(1-3) glucanase

Soluble, high molecular weight β-glucan

Low molecular weight β-glucan, cellobiose, laminaribiose

Endo-β-(1-4) glucanase

Soluble, high molecular weight β-glucan

Low molecular weight β-glucan, cellobiose, laminaribiose


Cellobiose, laminaribiose








Peptides, free amino acids


Proteins, peptides

Free amino acids


Proteins, peptides

Free amino acids



Free amino acids



High and low molecular weight α-glucans

Melagosaccharides, oligosaccharides







Limit dextrinase

Limit dextrins



α-1,6-d-glucans in amylopectin, glykogen, pullulan

Linear amylose fractions



Lipids, lipidhydroperoxide

Glycerine, free fatty acids, fatty acid hydroperoxide


Free fatty acids

Fatty acid hydroperoxide

It has also been demonstrated that yeast proteins are present in beer, but only as minor constituents [73]. Beer contains ~500 mg/L of proteinaceous material including a variety of polypeptides with molecular masses ranging from <5 to >100 kDa. These polypeptides, which mainly originate from barley proteins, are the product of the enzymatic (proteolytic) and chemical modifications (hydrogen bonds, Maillard reaction) that occur during brewing, especially during mashing, where proteolytic enzymes are liable for those modifications [125]. A beer protein may be defined as a more or less heterogeneous mixture of molecules containing the same core of peptide structure, originating from only one distinct protein present in the brewing materials [126]. Jones [1317] surveyed proteinases and their behavior during malting and mashing. Proteinases are not active in beer anymore; hence, they are inactivated when the temperature rises above 72 °C, which happens already during mashing [1, 7, 13, 2530].

Proteins influence two main quality aspects in the final beer: 1st haze formation and 2nd foam stability. In the following lines, these quality attributes are described in a more detailed way.

Haze formation

Proteins play a major role in beer stability; hence, they are, beside polyphenols, part of colloidal haze. There exist two forms of haze; cold break (chill haze) and age-related haze [127]. 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 [128]. Proanthocyanidins (condensed tannins) from the testa tissue (seed coat) 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 [129]. Proteins, as the main cause of haze formation in beer, can be divided into two main groups: 1st proteins and 2nd their breakdown products. Protein breakdown products are characterized by always being soluble in water and do not precipitate during boiling. Finished beer contains almost only protein breakdown products [126]. The content of only 2 mg/L protein is enough to form haze [118]. Beer contains a number of barley proteins that are modified chemically (hydrogen bond formation, Maillard reaction) and enzymatically (proteolysis) during the malting and brewing processes, which can influence final beer haze stability. Leiper et al. [130, 131] found out that 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. Asano et al. [62] investigated different protein fractions and split them in 3 categories: 1st high, 2nd middle and 3rd low molecular weight fractions being high molecular weight fractions: >40 kDa, middle molecular weight fraction: 15–40 kDa and low molecular weight fraction: <15 kDa. Nummi et al. [132] even suggested that acidic proteins derived from albumins and globulins of barley are responsible for chill haze formation (Table 2).
Table 2

Distribution of hordeins in barley according to their size [75]


MW (kDa)

% of total hordeins













Researchers proofed that proline-rich proteins are involved in haze formation [63, 65, 124, 127, 128, 130, 131, 133137]. Outtrup et al. [138] say that haze-active proteins are known to be dependent on the distribution of proline within the protein. Nadzeyka et al. [127] suggested that proteins in the size range between 15–35 kDa comprised the highest amount of proline. It was also investigated that proline and glutamic acid-rich hordeins, in the size range between 10–30 kDa, are the main initiators causing haze development [63, 74]. β-Amylase, protein Z, and two chymotrypsin inhibitors have relatively high-lysine contents [100]. Barley storage proteins that are available for hydrolysis are all proline-rich proteins [15]. Dadic and Belleau [139, 140] on contrary say that there is no specific amino acid composition for haze-active proteins. Leiper [130, 131] even says that not only the mainly consistence of proline and glutamic acid of the glycoproteins is responsible for causing haze but also that the carbohydrate component consists largely of hexose. It was found out that the most important glycoproteins for haze formation are 16.5 and 30.7 kDa in size. Glycation is a common form of non-enzymatic modification that influences the properties of proteins [109]. Non-enzymatic glycation of lysine or arginine residues is due to the chemical reactions in proteins, which happen during the Maillard reaction [109]. It is one of the most widely spread side-chain-specific modifications formed by the reaction of α-oxoaldehydes, reducing carbohydrates or their derivatives with free amine groups in peptides and proteins, such as e-amino groups in lysine and guanidine groups in arginine [141, 142]. The proteins in barley malt are known to be glycated by D-glucose, which is a product of starch degradation during malting [108]. D-glucose reacts with a free amine group yielding a Schiff base, which undergoes a rapid rearrangement forming more stable Amadori compounds.

Haze-sensitive proteins

Polypeptides that are involved in haze formation are also known as sensitive proteins. They will precipitate with tannic acid, which provides a mean to determine their levels in beer. Proline sites of these polypeptides bind to silica gel hydroxyl groups so that haze-forming proteins are selectively adsorbed, since foam proteins contain little proline and are thus not affected by silica treatment [143]. Removal of haze forming tannoids can be effected using PVPP [143]. To assure colloidal stability, it is not necessary to remove all of the sensitive proteins or tannoids. Identification of a tolerable level of these proteins can be used to define a beer composition at bottling that delivers satisfactory haze stability [94, 99]. To prolong stability of beer, stabilization aids are used. Haze-forming particles are removed with: (a) silica, which is used to remove proline-rich proteins that have the ability to interact with polyphenols to form haze in bright beer, or (b) PVPP, which is used to remove haze-active polyphenols.

Evans et al. [144] investigated the composition of the fractions which were absorbed by silica. This analysis revealed that the mole percentage of proline ranged between 33.2 and 38.0%, and of glutamate/glutamine between 32.7 and 33.0%, consistent with the proline/glutamine–rich composition of the hordeins [144]. Iimure et al. [65] stated in their studies that proteins adsorbed onto silica gel (PAS) are protein Z4, protein Z7, and trypsin/amylase inhibitor pUP13 (TAI), rather than BDAI-1 (α-amylase inhibitor), CMb, and CMe. Lázaro et al. [145] investigated the CM proteins CMa, CMb, and CMe. The CM proteins are a group of major salt-soluble endosperm proteins encoded by a disperse multigene family and act as serine proteinase inhibitors. Genes CMa, CMb, and CMe are located in chromosomes 1, 4, and 3, respectively. Protein CMe has been found to be identical with a previously described trypsin inhibitor. Furthermore, Iimure et al. [64] analyzed proline compositions in beer proteins, PAS, and haze proteins. It was proofed that the proline compositions of PAS were higher (ca. 20 mol%) than those in the beer proteins (ca. 10 mol%), although those of the haze-active proteins such as BDAI-1, CMb, and CMe were 6.6–8.7 mol%. These results suggest that BDAI-1, CMb, and CMe are not predominant haze-active proteins, but growth factors of beer colloidal haze. Serine proteinase inhibitors have also been called trypsin/α-amylase inhibitors, and it has been proposed that some of them might inhibit the activities of barley serine proteinases. However, none have been shown to affect barley enzymes [16]. Robinson et al. [146] identified a polymorphism for beer haze-active proteins and surveyed by immunoblot analysis throughout the brewing process. In this polymorphism, some barley varieties contained a molecular weight band at 12 kDa, while in other varieties, this band was absent. Pilot brewing trials have shown that the absence of this 12 kDa protein conferred improved beer haze stability on the resulting beer. This band was detected by a polyclonal antibody raised against a haze-active, proline/glutamine–rich protein fraction; it was initially assumed that the band was a member of the hordein protein family [144, 147].

Foam formation

Beer foam is an important quality parameter for customers. Good foam formation and stability gives an impression of a freshly brewed and well-tasting beer. Therefore, it is necessary to investigate mechanisms that are behind foam formation. Beer foam is characterized by its stability, adherence to glass, and texture [148]. Foam occurs on dispensing the beer as a result of the formation of CO2 bubbles released by the reduction in pressure. The CO2 bubbles collect surface-active materials as they rise. These surface-active substances have a low surface tension, this means that within limits they can increase their surface area and also, after the bubbles have risen, they form an elastic skin around the gas bubble. The greater the amount of dissolved CO2 the more foam is formed. But foam formation is not the same as foam stability. Foam is only stable in the presence of these surface-active substances [1]. Beer foam is stabilized by the interaction between certain beer proteins, for example LTP1, and isomerized hop α-acids, but destabilized by lipids [30, 148]. The intention is to find a good compromise of balancing foam-positive and foam-negative components. Foam-positive components such as hop acids, proteins, metal ions, gas composition (ratio of nitrogen to carbon dioxide), and gas level, generally improve foam, when increased. Whereas foam negatives, such as lipids, basic amino acids, ethanol, yeast protease activity, and excessive malt modification, decrease foam formation and stability. Free fatty acids, which are extracted during mashing, have a negative effect on foam stability [64, 65, 80, 85, 88, 128131, 166].

Foam-positive proteins can be divided into high molecular weight proteins (35–50 kDa) and low molecular weight proteins (5–15 kDa) which primary originate from malt but in small amount can also originate from yeast [62, 73, 148]. It is thought that during foam formation, amphiphile proteins surround foam cells and stabilize them by forming a layer. They arrange themselves into bilayers, by positioning their polar groups toward the surrounding aqueous medium and their lipophilic chains toward the inside of the bilayer, defining a non-polar region between two polar ones [149]. There are two main opinions concerning the nature of foaming polypeptides in beer. The first position claims the existence of specific proteins which basically influence foam stability. Those proteins are known as protein Z and LTP1 [150, 151]. The second argument claims the existence of a diversity of polypeptides which stabilize foam; the more hydrophobic their nature, the more foam active they are [122, 152], like hordeins that are rich in proline and glutamine content and exhibit a hydrophobic β-turn-rich structure [74]. Kapp [153] investigated the influence of albumin and hordein fractions from barley on foam stability, because both are able to increase the foam stability. The ability to form more stabile foams seems to be higher by albumins than by hordeins. Denaturation of these proteins causes an increase in their hydrophobic character and also in their foam stability. This confirms the already known opinion that the more hydrophobic the protein, the better is the foam stability [122, 152]. The foams from albumins are more stable than those from hordeins. This may also be the reason for the increased ability of albumin fractions to withstand the presence of ethanol. The foam stability of both albumins and hordeins is increased by bitter acids derived from hops.

Whereas the barley LTP1 does not display any foaming properties, the corresponding beer protein is surface active. Such an improvement is related to glycation by Maillard reactions on malting, acylation on mashing, and structural unfolding on brewing which was ascertained by Perrocheau et al. [25]. During the malting and brewing processes, LTP1 becomes a surface-active protein that concentrates in beer foam [55]. LTP1 is modified during boiling and this modified form influences foam stability [28, 150]. The two forms have been recovered in beer with marked chemical modifications including disulfide bond reduction and rearrangement and especially glycation by Maillard reaction. The glycation is heterogeneous with variable amounts of hexose units bound to LTPs [112]. The four lysine residues of LTP1 are the potential sites of glycation [112]. Altogether, glycation, lipid adduction, and unfolding should increase the amphiphilic character of LTP1 polypeptides and contribute to a better adsorption at air–water interfaces and thus promote foam stability.

Van Nierop et al. [30] established that LTP1 denaturation reduces its ability to act as a binding protein for foam damaging free fatty acids and therefore boiling and boiling temperature are important factors in determining the level and conformation of LTP1 and so enhance foam stability. Perrocheau et al. [25] showed that unfolding of LTP1 occurred on wort boiling before fermentation and that the reducing conditions are provided by malt extract. Van Nierop et al. [30] showed that the wort boiling temperature during the brewing process was critical in determining the final beer LTP1 content and conformation. It was discovered that higher wort boiling temperatures (102 °C) resulted in lower LTP1 levels than lower wort boiling temperatures (96 °C). Combination of low levels of LTP1 and increased levels of free fatty acids resulted in low foam stability, whereas beer produced with low levels of LTP1 and free fatty acids had satisfactory foam stability. LTP1 has been demonstrated to be foam promoting only in its heat denatured form [55, 150, 154].

Perrocheau et al. [26] investigated heat-stable, water-soluble proteins that influence foam stability. Most of the heat stable proteins were disulfide-rich proteins, implicated in the defense of plants against their bio-aggressors, e.g., serpin-like chymotrypsin inhibitors (protein Z), amylase and amylase-protease inhibitors, and lipid transfer proteins (LTP1 and LTP2). Leisegang et al. [9597] identified LTP1 as a substrate for proteinase A, which degrades LTP1, but does not influence protein Z and may have a negative influence on beer foam stability. Iimure et al. [32] invented a prediction method of beer foam stability using protein Z, barley dimeric α-amylase inhibitor-1 (BDAI-1) and yeast thioredoxin and confirmed BDAI-1 and protein Z as foam-positive factors and identified yeast thioredoxin as a possible novel foam-negative factor. Jin et al. [155, 156] found out in their research that structural changes of proteins during the wort boiling process are independent of the malt variety. It was discovered that barley trypsin inhibitor CMe and protein Z were resistant to proteolysis and heat denaturation during the brewing process and might be important contributors to beer haze formation. Vaag et al. [28] found a new protein of 17 kDa which seemed to influence foam stability even more than protein Z and barley like LTP1. She could support this theory by the correlation of the content of this so called 17 kDa protein and the foam half-life of lager beers. LTP1 and the 17 kDa protein exhibit some similarities; their tertiary structures are characterized by disulfide bridges, both are rich in cysteine and are modified during heating to a more foam promoting form. Ishibashi et al. [80] agrees that both malting and mashing conditions influence the foam-active protein levels in experimental mashes. Proteinaceous materials in beer have as well been implicated in the stabilization of beer foam. Molecular weight has been reported to be important for foaming potential, while the hydrophobicity of polypeptides has been cited as a controlling factor [62]. Kordialik-Bogacka et al. [157, 158] investigated also foam-active polypeptides in beer. In contrary to Osman et al. [123] in this investigation, it was confirmed that fermentation influences the protein composition of beer and particularly in beer foam.

Yeast polypeptides were also found in beer foam. It was noted that, especially during the fermentation of high gravity wort, excessive foaming may occur, and this may be one of the reasons why beer brewed at higher gravities has a poor head. It was detected that polypeptides of molecular weight about 40 kDa present in fermented wort and foam originated not only from malt but also from yeast cells. Okada et al. [159] studied on the influence of protein modification on foam stability during malting. They found that the foam stability of beer samples brewed from barley malts of 2 cultivars decreased as the level of malt modification increased, but the foam stability of another cultivar did not change. In this research, they defined BDAI-I as an important contributor to beer foam stability.


Proteins do not only influence haze formation; furthermore, they play an important role for mouthfeel and foam stability. These aspects are important for brewers, since consumer judge beer also according to these quality attributes. As it is known, most foam-positive proteins are also haze active, Evans et al. [144] made an investigation to immunologically differentiate between those two protein forms (foam and haze-active proteins) and concluded that no barley variety or growing condition have any significant influence on beer stability. It was also demonstrated in a regression analysis that a prediction of foam stability is not possible, which underlines the complexity of these problems. It is suggested that both foam-active and foam-negative components should be measured and that the amount of hordeins and protein Z4 are somehow related. It was also ascertained that foam and haze-active proteins share some epitopes and that oxygen during the brewing process influence haze stability of beer [147].

Leiper et al. [130, 131] studied beer proteins that are involved in haze and foam formation. All proteins were found to be glycosylated to varying degrees. The size range of the polypeptides which make up the glycoprotein fraction of beer is relatively narrow and the range was found to be from 10 to 46 kDa. The glycoproteins were found to consist of proteins, six carbon sugars (hexoses), and five carbon sugars (pentoses). Beer glycoproteins were found to exist in three forms; those responsible for causing haze, those responsible for providing foam stability, and a third group that appeared to have no role in physical or foam stability. Approximately 25% of beer glycoproteins are involved in foam and foam stability. As 3–7% of beer glycoproteins have been identified as being involved in haze formation, this leaves around 70% of beer glycoproteins that appear to have no role in either physical and/or foam stability. This fraction contains the most abundant beer polypeptide, protein Z, which is glycosylated with both hexoses and pentoses. It has been estimated that about 16 % of the lysine content of protein Z are glycated during the brewing process through Maillard reaction [61, 126].

There are three major groups of proteins in beer. The first consists of a group of proline-rich fragments originating from hordein ranging in size from 15–32 kDa which are involved with haze formation. The second is LTP1 (9.7 kDa in pure form) that is involved in foam stability and the third is protein Z (40 kDa) that appears to have no direct function, but may play a role in stabilizing foam once it has been formed [130, 131]. Several authors [25, 30, 49, 66, 70, 73, 125, 126, 160, 161] investigated haze-active proteins in beer. 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. It is presumed that protein Z causes haze and is all the same positive for foam stability [70, 73]. LTP1 is claimed not to influence foam stability but the quantity of foam generated [55, 117]. Protein Z is homologous to serine protease inhibitors and these inhibitory properties might be the reason that protein Z is not degraded by proteolytic enzymes during malting and mashing [104, 126, 162, 163]. Curioni et al. [125] showed that glycation of protein Z improved foam stability and might prevent precipitation of protein during the wort boiling step. Both glycation and denaturation increase the amphiphilicity of LTP1 polypeptides and contribute to a better adsorption at air–water interfaces of beer foam [55, 164]. Jin et al. [155, 156] found out in their research that structural changes of proteins during the wort boiling process are independent of the malt variety. It was discovered that barley trypsin inhibitor CMe and protein Z were resistant to proteolysis and heat denaturation during the brewing process and might be important contributors to beer haze formation. It is known that foam-active hydrophobic protein fractions in beer can be hydrolyzed by proteinases leading to a decrease in foam stability.

Besides proteins, other beer constituents such as iso-alpha acids, peptides, amino acids, proteinase, fatty acids, and melanoidins were suggested to influence haze formation and foam properties [154, 165]. The contents of these constituents in beer were influenced by brewing material variables such as barley varieties, malt types, hop usage, yeast strains, and malting and brewing processes.

Copyright information

© Springer-Verlag 2010