Immunological characterisation of barley polypeptides in lager foam

Eight monoclonal antibodies (mAb) recognising barley polypeptides have been identified from a library developed to wheat prolamins. The specificity or the mAb has been determined using enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Six were of broad specificity, recognising D, B, C and γ-hordeins to varying degrees by both techniques. IFRN 0610 preferentially recognised γ-hordeins by ELISA but was highly specific for this hordein group by immunoblotting. Another mAb, IFRN 0624, bound to a M_r ∽ 18000 polypeptide belonging to the CM protein (trypsin/α-amylase inhibitor) family by immunoblotting. This, or a related protein, was detected by 0624 in all hordein fractions using ELISA. These mAb, together with two others described previously and found to recognise the repeat motif of C hordein, were used in ELISA and immunoblot analysis of Octyl-Sepharose fractions of lager foam. Hordein polypeptides were found in all foam fractions, indicating that much foam protein originates from the malt. The CM-like protein was found present in a virtually unmodified form. In contrast, the repeat motif of C hordein was not detected, indicating that it had either been destroyed or masked by other beer constituents. The foam stabilising agent, propylene glycol alginate (PGA), increased the apparent hydrophobicity of hordein fragments suggesting that at least part of the activity of PGA is mediated by interactions with the hordein components of foam.     

Differences in protein content and foaming properties of cloudy beers based on wheat malt content

To investigate differences in protein content, all barley malt beer, wheat/barley malt beer and all wheat malt beer were brewed, and the protein during mashing, wort, fermentation and beer determined. It was shown that protein was mainly extracted during mashing and the protein rest phase, decreased in the early stages of fermentation and remained almost steady during wort boiling and cooling, in the middle and late stages of fermentation. By separating beer foam from beer, similar protein bands of 51.7, 40.0, 27.3, 14.8, 6.5 and < 6.5 kDa appeared in the three beers, defoamed beers and beer foams using the sodium dodecyl sulphate polyacrylamide gel electrophoresis. Quantitatively, protein bands of 6.5–14.8 and <6.5 kDa had the highest contents in the three beers. Unique bands at 34, 29.2, 23.0, 19.7 and 17.7 kDa were found in beer, defoamed beer and beer foam from wheat beer and all‐wheat malt beer, respectively. Wheat beer foam showed the best foam stability and the protein in all barley malt beer showed the best migration to the foam. The beer foam properties were influenced by not only protein content but also protein characteristics and/or origin. It is suggested that the barley malt contributed the beer foam ‘skeleton protein’ while protein components from wheat malt kept the foam stable. © 2018 The Institute of Brewing & Distilling     

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.     

Investigation of foam‐active polypeptides during beer fermentation

During ale fermentation there was an accumulation of total and hydrophobic polypeptides in the foam relative to the wort. Comparisons were made not only of the total and hydrophobic polypeptide contents but also of the molecular weights of these polypeptides present in wort, partially fermented wort and its concomitant foam. Wort, fermented wort and foam fractions had very similar polypeptide compositions with a major group having molecular weights of 40–43 kDa. Material of molecular weight in the range of 5–17 kDa and at 66 kDa was also detected. The polypeptides accumulated in foam displayed both hydrophobic and non‐hydrophobic character. The presence of yeast polypeptides in foam was confirmed. Comparison was also made between the fermentations of 10°Plato and 15°Plato wort. The results of the work may contribute to a better understanding of the mechanism of foam formation during beer fermentation, leading to reduced foaming and enabling an increase in the working capacities of fermenters. Copyright © 2004 Society of Chemical Industry     

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.     

FRACTIONATION OF HIGH MOLECULAR WEIGHT POLYPEPTIDES FROM BEER USING TWO DIMENSIONAL GEL ELECTROPHORESIS

High molecular weight polypeptides from beer were fractionated by a two dimensional gel electrophoretic method comprising isoelectric focusing in the first dimension followed by gradient gel electrophoresis in the second dimension. Silver stained gels revealed a complex pattern of spots which is consistent with individual polypeptide species having both discrete isoelectric points and molecular weights. The pattern of silver stained spots was reproducible both within and between beers and was independent of the sample preparation method or the composition of the sample solubilisation mix. Beers produced from grists containing malt plus 20% wheat flour were shown to contain four polypeptides of approximate molecular weight 30 000, 30 000, 16 000 and 15 000 which were absent from all malt control beers.     

大豆蛋白用作啤酒泡沫稳定剂的研究

本研究以脱脂大豆蛋白粉为原料,碱溶酸沉法制备大豆分离蛋白(SPI)和大豆乳清蛋白(WSP),根据大豆分离蛋白酶解过程中溶解性及泡沫稳定性的变化情况,确定大豆分离蛋白的酶解程度。再通过对酶解大豆分离蛋白(ESPI)及大豆乳清蛋白(WSP)的性能实验及应用实验进一步考察大豆蛋白用作啤酒泡沫稳定剂的可行性。结果表明,酶解30min后,ESPI溶解性和泡沫稳定性均有所增强;ESPI和WSP不仅能改善低度熟啤酒的泡持性,而且可以明显改善纯生啤酒货架期内的泡持性;大豆蛋白的添加不会影响啤酒的非生物稳定性。     

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     

The Impact of Malt Derived Proteins on Beer Foam Quality. Part II: The Influence of Malt Foam-positive Proteins and Non-starch Polysaccharides on Beer Foam Quality

Pilot (50 litre) and small scale (700 mL) brewing trials conducted using, similar brewing protocols with 25 different malts, indicated that differences in malt quality influenced foam stability (Rudin head retention value) by up to 24%. In addition to conventional measures of malt quality, enzyme-linked immunosorbent assays (ELISA) were used to measure the level of the putative foam-positive proteins, BSZ4 (protein Z4), BSZ7 (protein Z7), BSZ7b and lipid transfer protein 1 (LTP1). Regression analysis performed on the combined pilot and small scale data sets identified that malt BSZ4, wort β-glucan and wort viscosity, and beer protein, β-glucan and arabinoxylan were positively correlated with foam stability, while malt Kolbach index (KI), and beer FAN were negatively correlated with foam stability. Potentially foam-positive proteins such as BSZ7 and LTP1 were not correlated with foam stability. The negative correlation of BSZ4 level with KI suggested an additional role for BSZ4 in influencing protein modification. Step-wise multiple regression indicated that up to 82% of the variation in foam stability could be predicted from the malt and beer characteristics measured, demonstrating that there are a number of inter related malt derived factors that influence beer foam stability.     

ASSESSMENT OF BEER FOAM STABILITY BY HIGH PERFORMANCE LIQUID IMMUNOAFFINITY CHROMATOGRAPHY (HPLIAC)

An assay based on high performance liquid immunoaffinity chromatography (HPLIAC) was developed for measuring foam stability using an antiserum raised against foam produced from a commercially available lager (lager C). Foam stability of ten commercial beers (4 bitters, 4 lagers including lager C and 2 stouts) tested by HPLIAC showed a correlation of 0.733 with head retention value (HRV). This correlation increased to 0.998 when the data obtained for the lagers were considered on their own. Fast protein liquid chromatography (FPLC) of three fractions (fractions 1–3), depicting foam drainage over 30 min, identified differences between the lagers and the rest of the beers tested in fraction 3. Two peaks (I & II) which adsorbed on Superose 12 were present in similar concentrations in all the beers tested but a third peak (III), which eluted in the void volume, was present in low concentrations in the lagers as compared to the rest. It is suggested that components of I and II are essential for foaming but that HRV depends upon the concentration of III. Hence an antiserum raised against a beer of high HRV might provide a generic immunoaffinity adsorbent for assessing foam stability.     

The Influence of Ethanol on the Foaming Properties of Beer Protein Fractions: A Comparison of Rudin and Microconductivity Methods of Foam Assessment

The effect of ethanol on the foaming properties of beer protein fractions was studied using a microconductivity method and nitrogen gas to generate the foam. Increasing the ethanol concentration resulted in a decrease in foam stability. Interfacial studies including thin film drainage and dilational elasticity measurements indicated that ethanol reduced the rigidity of the adsorbed protein layer resulting in accelerated drainage from the foam lamellae and increased probability of film rupture. These results conflict with data from the Rudin method (using nitrogen gas to generate the foam) which indicate that, at low concentration, ethanol improves foam stability. These apparently conflicting results may be explained by the foam positive effects of a decline in bubble size and increase in bulk viscosity observed for the Rudin method, contrasted with the negative influence of a reduction in surface viscosity observed for the microconductivity foam assessment method.     

Low‐molecular‐weight materials from heavily roasted barley and malt with strong foam‐stabilising potential

Extracts of roasted barley and black malt made both at room temperature and using a ramped temperature mashing regime contained relatively low concentrations of protein. However cold water extracts displayed strong foaming power even at very low measurable protein concentrations. The observation that very little material appeared to be precipitated out by ammonium sulphate based salt fractionation is consistent with the very low protein levels and it is inferred that the powerful foaming potential is not due to polypeptides. The foaming material in black malt was investigated in some detail. It was purified under conditions that indicated that it is of molecular weight <4000 and is relatively anionic. A solitary foam‐active fraction was recovered using preparative HPLC and time of flight mass spectrometry revealed one large peak and two small peaks. The larger peak is tentatively identified as pyridyl pyrazine, while we believe that the smaller peaks are due to peptides, which we tentatively identify as a hexa‐ and a tetrapeptide. The Fourier transform infrared spectrum is consistent with pyridine, pyrazine and peptide being components of this foaming entity. © 2018 The Institute of Brewing & Distilling     

PGA对纯生啤酒泡沫稳定性的影响研究

在纯生啤酒中添加微量的PGA,可有效地提高纯生啤酒的泡沫稳定性。实验发现,在初过滤之后,终过滤之前,向纯生啤酒中添加35mgPGA/L啤酒,静置5min后,泡持值可从294s平均增加到402s,即泡持值提高了36.7%。经过60d贮藏后,测得其泡持值为390s。     

啤酒高分子蛋白检测方法的评估及应用

通过对考马斯亮蓝法检测啤酒高分子蛋白含量的操作方法进行优化,提升其方法的重复性和稳定性,优化后该方法的重复性和稳定性相对标准偏差(RSD)分别为3.68%和3.92%。同时,对该方法检测的高分子蛋白含量与啤酒泡持性进行了相关性分析。结果表明,巴氏杀菌啤酒泡持值与高分子蛋白含量的Pearson相关系数为0.923(P＜0.01);纯生啤酒在贮存3 d、1个月、2个月和4个月的泡持值和其高分子蛋白含量的Pearson相关系数分别为0.700(P＜0.01)、0.739(P＜0.01)、0.899(P＜0.01)和0.883(P＜0.01)。表明该方法不仅能用于啤酒高分子蛋白含量的检测,还能用于巴氏杀菌啤酒和纯生啤酒泡持性的监控。     

纯生啤酒泡沫稳定性的研究

纯生啤酒的泡持随着货架时间的延长会逐渐衰减 ,严重影响啤酒的外观质量。大量的文献资料证实 ,纯生啤酒泡持性的下降是由酒液中存在的蛋白酶A造成的。通过对成品酒泡持性的跟踪测定 ,重点讨论了发酵及啤酒过滤过程控制对泡持衰减趋势的影响。     

THE NITROGENOUS COMPLEXES OF HAZE AND FOAM AND THEIR MEASUREMENT

Earlier work and a review of recent studies have led to the view that the complex nitrogenous substances of beers are all, to some extent, irreversibly co-complexed either with glucose polymers or with polyphenols, or with both. Where amino-groups are still free, there can, in addition, be reversible combination with complex acids in worts and beers. The proportions of these different types of co-complex are considered to have important effects on beer properties, so that methods for the measurement of the amounts present are regarded as essential. With this as objective, it has been found that the relatively unmodified “free-amino” nitrogenous complexes, as well as those co-complexed with polyphenols, can be precipitated by suitable acidic dyes. The resulting precipitates can then be dissolved to give a simple and rapid colorimetric measurement of the amounts of these complexes present. By using the specific reagent polyvinyl pyrrolidone (AT) to absorb and remove the polyphenolic co-complexes, the remaining “free-amino” nitrogenous complexes can be precipitated alone, so that separate measures of both types can be obtained. Using bromophenol blue as the dye, some precipitation, mainly of the higher molecular weight fractions, occurs around pH 5 and by progressively lowering the pH to 3, or by progressively increasing the dye concentration, larger and larger amounts are precipitated to give a spectrum of the nitrogenous complexes present. Using a method based on these findings, wide variations have been found in the amount of different complexes in different beers. Studies have been made of the selective migration of complexes into foam and of the changes occuring during increase of haze. The methods have also been used to study the changes produced by proteolytic enzymes and by silica gel absorption, as well as those produced by changes in the nitrogen content of malts used. In one stout, dye-precipitable complexes are present in very small amounts. It appears that only non-reactive “crypto-nitrogenous” co-complexes persist after the prolonged mashing period used: a rapid method of measurement has been found for these also.     

Suitability of unmalted quinoa for beer production

BACKGROUND

This study provides the first detailed investigation into the effect of partially substituting barley malt with quinoa (Chenopodium quinoa Willd.) on the characteristics of wort and beer. Quinoa seeds and flakes were compared in terms of their suitability for brewing. The benefits of applying a commercial enzyme mixture during beer production with quinoa were also investigated.

RESULTS

These findings show that quinoa is a good starchy raw material for brewing. Even without exogenous enzymes, it is possible to substitute barley malt with up to 30% quinoa. The form in which quinoa is used has a negligible influence on the quality of the wort and beer. The foam stability of beer made with quinoa was better than that of all‐malt beer, despite there being a lower level of soluble nitrogen in quinoa beer in comparison with all‐malt beer and more than twice the amount of fat in quinoa in comparison to barley malt.

CONCLUSION

The addition of unmalted quinoa does not give unpleasant characteristics to the beer and was even found to have a positive effect on its overall sensory quality. This offers brewers an opportunity to develop good beers with new sensory characteristics. © 2018 Society of Chemical Industry     

黄酒糟蛋白作为啤酒泡沫强化剂的研究

利用黄酒糟蛋白作为啤酒泡沫强化剂,改善啤酒的起泡性和泡持值,添加后的啤酒起泡性提高到3左右,泡持值增加到410s,泡沫品质高。     

基于不同消泡模型的泡沫评价方法的对比

Sigma法与Constant法是国际上常用的两种啤酒泡沫评价方法,其特点是通过建立消泡模型,以模型的特征参数表征泡沫质量。不同的是,两种方法倚赖的消泡模型不同。通过考察21种啤酒的泡沫稳定性发现,Sigma法与Constant法倚赖的两种消泡模型在测量区间(1～5 min)内都高度显著,但其预测能力存在较大差别,且泡沫稳定性的评价结果也不一致,Sigma法对整体消泡时间的预测较准确。相关性分析结果表明,三种啤酒泡沫稳定性评价方法中,Sigma法与国标秒表法线性显著相关。理化指标分析进一步揭示了高分子蛋白含量对啤酒的泡沫稳定性的决定性作用。     

啤酒泡沫泡持性测定的研究进展

通过对国内外现行泡持测定法的介绍，优缺点分析和比较；说明了泡沫测定对泡沫的质量评判，工艺改进等的重要意义，并为泡持的精确测定提供极具价值的参考。