Interactions between polypeptides derived from barley and other beer components in model foam systems

The impact of a range of variables on the foaming properties of hordein‐ and albumin‐derived fractions from barley has been investigated. When foamed using nitrogen, the hordein‐derived polypeptides produce the most stable foams. However, when carbon dioxide was the foaming agent, the previously reported observations were repeated, namely that proteolysis weakens the foaming capabilities of albumin, whereas partial hydrolysis enhances the stability of foams derived from hordein. At lower levels of bitterness (iso‐α‐acid) addition there is a reduction in foam stability of albumin‐derived polypeptides, although hordein‐derived polypeptides can withstand this destabilising effect. Higher bitterness levels in both instances lead to a stabilisation of foam. It is likely that this involves ionic interactions between the acid anion (pK of iso‐α‐acids is ca 3.1) and divalent cations, because higher pHs (in the range 3.8–4.6) and the addition of zinc lead to an enhancement of foam stability, particularly of the albumin‐derived fractions. The foaming of the latter, however, is significantly impaired by the presence of ethanol, whereas hordein‐based foams are enhanced by increasing concentrations of ethanol. Copyright © 2003 Society of Chemical Industry     

The foaming properties of proteins isolated from barley

Albumin and hordein protein fractions have been isolated from barley by extraction with salt and aqueous ethanol respectively. Both produce stable foams as assessed by a shaking procedure, and in each case foaming was enhanced by denaturation of the proteins. Foam power was increased rather more for albumin than for hordein, especially by heat, and this was accompanied by an increase in hydrophobic character as assessed by the fluorochrome 1‐anilino‐8‐napthalenesulphonic acid. Iso‐α‐acids and reduced iso‐α‐acids increased the foam stability of both albumin and hordein, with there being a proportionately greater increase for the undenatured proteins. Albumin is resistant to hydrolysis by the cysteine proteinases ficin and papain but is hydrolysed by the serine proteinase trypsin. In the latter instance, hydrolysis is accompanied by a loss of foam stability. By contrast, hordein is resistant to digestion by trypsin but is susceptible to the action of ficin and papain. In these instances, limited proteolysis leads to a substantial enhancement of foam stability from hordein, though prolonged proteolysis is detrimental. Proteinase A from yeast hydrolyses both protein classes and lowers their ability to give stable foams. It is concluded that polypeptides derived from both albumin and hordein could make a contribution to the foam potential of beer. © 2002 Society of Chemical Industry     

The foaming of mixtures of albumin and hordein protein hydrolysates in model systems

Although hydrolysed albumin and hydrolysed hordein derived from barley are independently capable of stabilising foams in model beer solutions, when both are present together, the net foam stability is less than anticipated. In particular, it seems that hordein, even in an unhydrolysed state, interferes with the ability of albumin‐derived polypeptides to stabilise foam. It appears, therefore, that the observed foam stability of a product such as beer is not only dependent on the absolute level of individual foam‐stabilising polypeptides but also on the relative proportions of polypeptides derived from the albumin and hordein protein subsets. Copyright © 2004 Society of Chemical Industry     

Characterisation of polyclonal and monoclonal antibodies against glycinin (11S storage protein) from soya (Glycine max)

Polyclonal and monoclonal antibodies have been raised against the soya (Glycine max L) 11S storage protein, glycinin. The characteristics of the antibodies have been studied using enzyme-linked immunosorbent assay (ELISA) and immunoblotting techniques. The polyclonal antibodies showed strong recognition of the storage proteins from pea, and smaller but significant interactions with storage proteins from other seeds. Two monoclonal antibodies were virtually completely soya specific, recognising different continuous epitopes from the acidic polypeptides believed to be present on the surface of the native protein. A third monoclonal showed a much wider specificity in the ELISA, including the recognition of certain storage proteins from other seed types to a greater extent than soya. The epitope for this antibody may have been present on the surface of the native protein and was discontinuous, dependent on spatial organisation for recognition.     

The effect of nitrogen nutrition on the lysine content and protein composition of barley seeds

Increases in the total grain nitrogen of barley (cv. Julia and Bomi), resulting from increased N fertilisation, are accompanied by decreases in the relative lysine content of the grain proteins. Extraction of the grain proteins shows that the high-N grain have increased relative and total amounts of the lysine-poor storage protein (hordein) fraction. There are only small increases in the total amounts of the other more lysine-rich protein fractions (salt-soluble proteins and glutelin+residue proteins). There is also little or no effect on the amino acid compositions of the fractions. Consequently, it is concluded that the decreased relative lysine content of the whole grain is due to the increased synthesis of hordein. Further evidence for this hypothesis comes from similar studies with the high-lysine mutant, Risø 1508, in which the high-lysine character results from a decrease in the relative and total amount of hordein. The hordein fraction does not increase disproportionately with increasing grain N, nor does the relative lysine content of the grain decrease. There is no effect of grain N on the polypeptide compositions of the salt-soluble proteins of either line. However, in Bomi and Julia increased grain N does result in an increase in the relative amount of sulphur-poor ‘C’ hordein polypeptides in the hordein fraction. It is speculated that this results from limitation of S relative to N.     

The Relative Significance of Physics and Chemistry for Beer Foam Excellence: Theory and Practice

Bubble nucleation sites in beer glasses should be uniformly small to ensure that the foam produced comprises homogenously small bubbles with attendant reduced degrees of disproportionation. The latter phenomenon is also hindered by increased film thickness in foam, which in turn will be enhanced by increased localised viscosity, encouraged perhaps by polysaccharide‐polypeptide complexes. Erring on the “high end” of specification for carbon dioxide will promote replenishment of foam through beading. Nitrogen at a relatively low level (15–20 ppm) will hinder disproportionation with limited suppression of hop aroma and introduction of undesirable textural features. Hydrolysed hordein appears selectively to enter beer foams at the expense of the more foam‐stabilising albuminous polypeptides, suggesting that any future procedures that might selectively eliminate hydrolysed hordein components from beer would be expected not only to enhance haze stability but also foam, by eliminating species that preferentially enter into the bubble but have less foam‐stabilizing capability once they are there.     

Beer Polypeptides and Silica Gel Part II. Polypeptides Involved in Foam Formation

Beer contains approximately 500 mg/L protein depending on the brewing procedures employed. This protein is in the form of polypeptides, the majority of which lie within the 10–40 kD size range. Some of these polypeptides are responsible for causing colloidal haze, some enhance foam stability and the remainder appear to have no function in beer except to contribute to mouth‐feel. Those polypeptides involved in haze formation were described in a previous paper. To continue these studies, data is presented to show that foam polypeptides are highly glycosylated and that purified foam glycoprotein contains low levels of the amino acid proline. As silica preferentially adsorbs polypeptides rich in proline, it is unlikely to adsorb this material and damage foam stability. The molecular sizes and composition of glycoproteins recovered from untreated beer, purified foam and beer from which the foam component has been removed are presented. These fractions include the polypeptides responsible for foam stability and those that appear to have no role in physical stability.     

POLYPEPTIDES WITH ENHANCED FOAM POTENTIAL

The ability of beer to produce a good foam is strongly influenced by the level of foam active polypeptides. It has previously been proposed that a means of ensuring an adequate level of such species is to add an exogenous preparation of foam active protein³. One such preparation, hydrolysed liquid egg white (HLEW), has been shown to impart a good foam to beer with substandard foam performance, without detriment to product quality. The foam active properties of this material are the subject of the work described here . HLEW was characterised by a combination of reverse phase chromatography and polyacrylamide gel electrophoresis to reveal a heterogeneous mixture of low molecular weight (2100–6000 daltons) relatively hydrophilic polypeptides. When this material was subjected to foaming and the foam positive and foam negative fractions similarly characterised, it was apparent that the foam positive fraction consisted almost exclusively of hydrophobic polypeptides. Accordingly, preparative reverse phase chromatography was used to isolate foam active fractions from the hydrophobic species present. These fractions were subsequently subjected to both functional and physical characterisation. It was apparent from a small-scale Rudin test that the foam activity per unit dry weight of protein was enhanced in the foam active factions; in one case an enhancement of 2 fold greater than the HLEW itself was observed. Moreover, upon addition to beer a positive effect on HRV was achieved with as little as 0.0025 mg protein/ml beer. Physical characterisation of the foam active material revealed the presence of tightly bound polypeptide aggregates which could only be separated by the use of protein denaturing agents.     

Use of acid gel electrophoresis in the characterisation of ‘B’ hordein protein in relation to malting quality and mildew resistance of barley

The ‘B’ polypeptides of the storage protein (hordein) of barley were studied in 84 varieties. On the basis of acid gel electrophoresis the varieties were classified into 13 distinct groups. Contrary to the suggestion that varieties preferred for malting tend to show less intensity of staining of the faster moving bands of the ‘B’ fraction of hordein, no consistent association was found in this study. Thus the malting potential of some varieties may be less affected by protein composition than by other factors. The genetic linkage between the Hor-2 locus, specifying the ‘B’ group of polypeptides, and the Mlalocus, determining resistance to powdery mildew (Erysiphe graminis), was illustrated by the similar groupings of varieties carrying the same resistance alleles, but exceptions were observed. Examples of the inheritance of hordein patterns are illustrated and discussed.     

IDENTIFICATION AND CHARACTERISATION OF BEER POLYPEPTIDES DERIVED FROM BARLEY HORDEINS

Two groups off hordein-reactive antibodies (cross-reactive in barley with B/C monomers or subunits from the B/D-hordein aggregates) were used for the analysis of malt, wort and beer. A number of additional lower molecular weight hordein-derived polypeptides were present in water extracts of malts and worts, and protease inhibitors or higher mash-in temperature had a stabilising effect on certain hordein-derived polypeptides . The solubilisation profiles of hordein polypeptides monitored by specific immunological methods were clearly different from total wort polypeptides monitored by the Bradford method. There were also differences between the profiles for the two groups of hordein-derived polypeptides with the solubilisation of polypeptides recognised by antibody with specificity for the subunits from the B/D aggregates being especially temperature dependent . Beer samples mainly contained hordein-derived polypeptides of lower molecular weight than barley hordeins with polypeptides in the B and C molecular weight regions being lost during the brewing process. Beer polypeptides (Mr > 50,000) were recognised by antibodies cross-reactive with the subunits from the B/D aggregates in barley. A number of lower molecular weight polypeptides (Mr < 30,000) were recognised by antibodies with specificity for B and C hordeins; a subset of the latter antibody group bound to a specific Mr 23,000 beer polypeptide .     

The Gel Filtration Chromatographic‐Profiles of Proteins and Peptides of Wort and Beer: Effects of Processing — Malting,Mashing, Kettle Boiling,Fermentation and Filtering

Barley and malt proteins, of infusion (IoB) and decoction (EBC) mashing worts as well as commercial wort and beer, obtained from the Castlemaine Perkins brewery, Brisbane, were gel filtered, with or without further treatments. A general, similar pattern of protein and peptide profiles emerged from barley malt and beer. This confirmed the widely assumed fact that beer proteins descend from barley, some transformed and others perhaps mostly unchanged by processing. In the gel‐filtrate profiles, a maximum of 8 or 9 fractions were discerned. These fractions were collected and quantified for protein contents and amino acid compositions. The first four fractions contained the proteins and polypeptides of molecular weight higher than 14,000. Consequently, the remaining fractions contain the smaller peptides (<14,000), that were completely removed by dialysis. The effects of processing on proteins and peptides varied contingent upon the type of processing step considered and the pre‐chromatographic treatment. Malting was the most effective process remarkably increasing the soluble protein contents, especially the smaller peptide fractions and the colour development. This is the first report, as far as we are aware of, on the gel filtration profiles of wort and beer low molecular weight peptides including those of barley wort. The importance of the smaller peptides in foam formation and retention cannot be overemphasised. The amino acid composition of the fractions revealed much more diversity than was observed in the comparison of the profiles. Proline content of fraction 1 resembled that of barley soluble proteins while fractions F2, F3 and F4 that of glutelin and only fraction 8 that of hordein. The latter, suggests that hordeins or, at least the peptide products rich in proline, are likely to be completely digested to amino acids, during malting.     

AN IMMUNOCHEMICAL ANALYSIS OF BEER FOAM

Beer foam produced in a continuous foaming tower in volumes representative of commercial dispense, was analysed by immunoelectrophoretic and immunoblotting techniques to identify antigens involved in foam structural stability. In crossed immunoelectrophoresis (CIE), only one antigen precipitated from foam in the homologous foam antiserum. This antigen was shown to be of malt origin by rocket-line immunoelectrophoresis and was also present in 11 commercial beers (5 bitters, 4 lagers and 2 stouts). However, the foam preparation separated into more than 20 polypeptides by SDS polyacrylamide gel electrophoresis. Immunoblotting showed that at least 12 of these reacted with foam antiserum and that they originated from either malt or yeast. Similar polypeptides were also identified in the antigen precipitated in CIE, suggesting that these polypeptides were probably present in the foam as a complex. It is concluded that the stability of foam reflected molecular interactions between these polypeptides (and possibly other components such as carbohydrates) in the liquid film of the bubble structure.     

The ratio of B to C hordeins in barley as estimated by high performance liquid chromatography

The ratio of B to C hordeins was determined using high performance liquid chromatography (h.p.l.c.) for a range of spring barley cultivars and the values obtained varied from 1.79 for Kneifel to 4.15 for Goldmarker. Fine grinding of seed samples resulted in a significant increase in the quantity of protein solubilised but relatively little difference was observed in the B and C hordein ratio, which appeared to be independent of protein extraction efficiency. Increased nitrogen content significantly changed the ratio by increasing the proportion of C hordein. A highly significant correlation was found between the B and C hordein ratio and sample nitrogen content. Analysis of single seeds selected at random from within a single sample of Goldmarker revealed considerable variability in the B and C hordein ratios with values differing by over 2 units. Due to the sensitivity of the B to C hordein ratio to various environmental effects, it is unlikely that the ratio will be of great value as an unambiguous aid in the identification of barley cultivars.     

Variation in in vitro digestibility of barley protein.

In vitro digestibility of protein was measured with pepsin/pancreatin in 321 spring barley lines grown in the field. The variation in digestibility was far less than the variation in the protein content. A small environmental influence on the digestibility was found. Two entries had slightly impaired digestibilities; these findings were partially verified in a repeated field trial, but were not confirmed in vivo. In vitro digestibilities of barleys grown in pots at various N-levels were positively correlated with protein or hordein content. In vitro digestibility was negatively correlated with the fibre content in decortification fractions of Bomi and the high-lysine mutant Risø 1508. The digestibility was positively correlated with the hordein concentration for the Bomi fractions, but not for the 1508 fractions.     

A comparison of methods for the extraction and separation of hordein fractions from 29 barley varieties

Methods have been developed for extracting and comparing the hordein fractions of barley seed as a means of identifying different varieties. A rapid method involving a single extraction with 55% (v/v) propan-2-ol+2% (v/v) 2-mercaptoethanol at 60°C removed slightly less nitrogen from milled grain than a more detailed procedure in which three sequential extractions were made with the same solvent. The hordein fractions extracted by these two procedures were alkylated and their component poly-peptides separated by four one-dimensional and two two-dimensional systems. The results showed that the hordein extracted by both techniques had the same polypeptide composition and thus the rapid method was satisfactory for use in comparing varieties. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focusing were used to compare the polypeptide composition of the hordein fractions from 29 varieties. Using the former system a total of 11 different patterns were recognised while further small differences between several varieties were revealed by isoelectric focusing or two-dimensional analysis. If the polypeptide patterns obtained by these techniques are considered in conjunction with other grain characters it should be possible to identify almost all of the 29 varieties.     

Preparation of polyclonal antibodies specific for wine proteins

Immunology is an expanding area of research with potentially important applications in the analysis of many biological molecules. Polyclonal antibodies were raised in rabbits against specific proteins as well as against the total protein from a Portuguese wine. FPLC cation exchange chromatography was used to isolate the total protein fraction and, when in combination with denaturing electrophoresis, to purify individual wine polypeptides. To obtain a high titre, an injection of each antigen followed by three boosters were given in the immunisation of each rabbit. The titre of the antisera was measured by the ELISA technique and the specificity of the antibodies detected by immunoblotting. The antibodies produced were shown to be highly specific for the corresponding antigens. However, antibodies obtained specifically against a highly purified wine polypeptide seem to recognize the other major wine polypeptides, raising the possibility of structure similarity between different wine proteins. Neither the anti‐total wine protein antibodies not the anti‐specific wine protein antibodies originated a signal when used to probe thaumatin or chitinase. © 1999 Society of Chemical Industry     

Characterisation of monoclonal antibodies against β-conglycinin from soya bean (Glycine max) and their use as probes for thermal denaturation

A panel of four monoclonal antibodies (Mabs) was raised against β-conglycinin, the 7S globulin from soya bean. The antibodies were characterised by using direct and competitive enzyme-linked immunosorbent assays (ELISAs), immunoblotting procedures and by analysis of subunit fractions obtained after anion exchange chromatography. All the Mabs were specific for β-conglycinin, recognising the acidic α-and α-subunits. One of the Mabs (IRFN 0089) was used to probe the structural changes that take place during thermal denaturation of β-conglycinin. A two-site ELISA was developed to observe structural changes in β-conglycinin with heating. Antibody recognition of heated conglycinin increased with temperature reaching a maximum at 65°C; β-conglycinin heated to this temperature was recognised three-fold better than unheated β-conglycinin. At a higher temperature (65–95°C) β-conglycinin immunoreactivity remained at least two-fold higher than that of the unheated protein. Differential scanning calorim-etry data showed the maximum binding of Mab 0089 to correspond with the thermal transition of the β-conglycinin molecule. The use of the panel of antibodies as structural probes is discussed together with further possible applications of this technology in food chemistry.     

新型冠状病毒核衣壳蛋白抗原的重组表达及其单克隆抗体的制备

目的 制备新型冠状病毒SARS-CoV-2核衣壳蛋白重组抗原及其单克隆抗体。方法 根据公布的新冠病毒全基因组序列,构建其核衣壳蛋白的表达载体pET-28a-N, 并转化至E. coil BL21(DE3)进行原核表达, ELISA及Western blot鉴定纯化后的核衣壳蛋白重组抗原的特异性并免疫Balb/c系小鼠, 基于杂交瘤融合技术筛选获得可持续分泌新冠病毒核衣壳蛋白单克隆抗体的细胞株, 并基于ELISA及生物膜层干涉技术鉴定单克隆抗体的特异性和亲和力。结果 基于大肠杆菌原核表达获得新冠病毒核衣壳蛋白的重组抗原, 其纯度大于90%, 筛选获得7株可分泌特异性结合新冠病毒核衣壳蛋白的杂交瘤细胞株, 经Western blot、ELISA及分子相互作用分析等方法证实所获得的单克隆抗体与核衣壳蛋白具有优良的结合特异性和结合活性, 其中的7E11、7E8单克隆抗体与核衣壳蛋白抗原的亲和力常数分别为1.69×10-9、2.031×10-9M, 可作为抗体元件应用于新冠病毒的快速免疫分析领域。结论 获得高特异性的新型冠状病毒SARS-CoV-2核衣壳蛋白重组抗原及其单克隆抗体。     

Isolation of beer foam polypeptides by hydrophobic interaction chromatography and their partial characterisation

Beer foam polypeptides have been separated into five groups based on their relative hydrophobicity. Foam stability increases with increasing hydrophobicity of the polypeptide groups. The most hydrophobic polypeptide group contains a large proportion of Coomassie blue-binding polypeptides. Analysis by SDS-PAGE reveals that each polypeptide group is composed of several differently-sized polypeptides. Further purification by anion-exchange chromatography results in five fractions, each of which has a different polypeptide profile on SDS-polyacrylamide gels.     

The Impact of Malt Derived Proteins on Beer Foam Quality. Part I. The Effect of Germination and Kilning on the Level of Protein Z4, Protein Z7 and LTP1

The effect of barley germination and kilning on three putative beer foam-positive proteins was investigated by immunoblotting and ELISA procedures. These procedures involved the use of specific antibodies raised to purified lipid transfer protein 1 (LTP1)and the two protein Z forms, Z4 (BSZ4) and Z7 (BSZ7). The free fraction of BSZ4 and BSZ7, and all LTP1 were extracted by aqueous salt-solution from barley and malt. The addition of reducing agent allowed the extraction of bound BSZ4 and BSZ7. A previously undescribed fraction of BSZ4 and BSZ7, refered to as latent, was extracted with SDS and reducing agent. The barley combined fraction (free + bound fractions) was surveyed in 93 barley varieties to show that BSZ4 was the dominant isoform, on average constituting ?80 % of all protein Z. Considerable variation was observed between varieties in the level of LTP1 (502–1144 μg/g) and the combined fractions of BSZ4 (18–2136 μg/g) and BSZ7 (38–771 μg/g). The free fraction is expected to be more available for extraction into wort during mashing than the bound or latent fractions. The level of LTP1 did not change substantially during germination, but a significant proportion of the latent and/or bound protein Z fractions was converted into the free fraction. In the seven varieties studied the free fraction of BSZ4 and BSZ7 increased 149–300% and 49–141%, respectively. Proteolytic cleavage in the reactive site loop converts protein Z to heat- and protease-stable forms that survive the brewing process. During germination most of the free BSZ4 and 30–70% BSZ7 was converted to the cleaved form. Kilning was found to reduce the amount of protein Z and LTP1 that could be extracted by 10–30% and 7–37%, respectively, which is likely to be counter productive for foam quality. These results suggest that barley variety selection and optimisation of germination and kilning protocols during malting may be opportunities for improvement of beer foam quality.