Proteome analysis of the wort boiling process

A comprehensive proteome map was constructed of brewers wort. The map consisted of protein identification on two-dimensional gel electrophoresis (2DE) images and identified 63 out of 202 protein spots, which were categorized into 20 protein species. To analyze the modification of protein Z during wort boiling, protein Z spots on 2DE gel of the sweet wort, the boiled wort and the trub were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF-MS) and then the spectra were compared. The analysis identified several specific signals detected only in the trub, suggesting that specific modification occurred in the precipitated protein Z during wort boiling. The protein Z spot on the 2DE gel of the precipitate was further analyzed by liquid chromatography mass spectrometry/mass spectrometry. The analysis identified low molecular weight fragment (1.3 kDa) derived from wound induced protein (barwin) in the protein Z spot of the trub. These results suggested that protein Z was precipitated by binding with comparatively small size specific fragment(s) derived from sweet wort protein, i.e., barwin during wort boiling. Our results and understandings have application for quality assurance and control in commercial brewing practice, and development of DNA markers for malting barley breeding.     

Proteome analysis of Pithecellobium dulce seeds using two‐dimensional gel electrophoresis and tandem mass spectrometry

BACKGROUND: Pithecellobium dulce Benth. belongs to the Leguminosae family, which contains several members that are important components of human diets owing to their high protein content and quality. In this study the seed proteins from P. dulce were separated and identified using two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry respectively. RESULTS: The 2‐DE protein map revealed a total of 317 distinct protein spots, including a cluster of about 12 proteins located in the region of pI 5–6 with molecular masses of 55–97 kDa that accounted for more than 50% of the total proteins. Ninety‐six of the most abundant protein spots were analysed using nano liquid chromatography/tandem mass spectrometry (LC/MS/MS), from which 27 were successfully identified through the query of acquired tandem mass spectral data used in MASCOT searching against a custom legume protein database. A further four proteins from the highly abundant protein cluster were putatively identified using mass spectrometry‐driven BLAST (MS‐BLAST) homology searches. CONCLUSION: This research has generated a 2‐DE proteome reference map for P. dulce seeds and used LC/MS/MS to characterise the proteins. The identification of proteins from P. dulce was carried out using the sequence database successively for MASCOT and MS‐BLAST homology‐based searches. Copyright © 2009 Society of Chemical Industry     

The chemical components in malt associated with haze formation in beer

In bright beer, haze formation is a serious quality problem, which reduces beer storage and shelf life. In this study, haze‐active proteins, alcohol chill haze formation ability, α‐amylase activity, the contents of total polyphenol, protein and its fractions and amino acids were analysed using 23 barley accessions to investigate the relationship between the quality components in the malt and the haze character in beer. The results showed that there were largely genotypic variations for all examined traits among the 23 barley accessions. However, there was no significant correlation between the haze character and α‐amylase activity. All haze characteristics were significantly and positively correlated with total protein content, albumin, globulin and the hordein content, as well as the glutamic acid (glutamine), proline and phenylalanine content, and were not correlated with total polyphenols. A model describing the relationship between the chill haze in the beer and the protein content in the malt was developed. Copyright © 2016 The Institute of Brewing & Distilling     

奶啤的研制

以牛奶、麦芽和酒花为主要原料,通过二次生物发酵研制出具有奶香和啤酒风味的奶啤饮料。通过实验确定奶啤的最佳配方及生产工艺流程并对奶啤的稳定性进行探讨,从而研制出即有啤酒的低酒精度、泡沫丰富和适度的二氧化碳,又具有酸乳饮料的酸甜可口、营养全面的奶啤饮料。     

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

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.     

不同种类麦芽对下面发酵啤酒酿造风味的影响

为了从源头控制下面发酵啤酒的发酵生产并提高其品质,通过啤酒发酵模拟体系,系统评价英国麦芽（Eng）、加拿大麦芽（Can）和德国麦芽（Ger）对下面发酵啤酒酿造风味的影响。分别测定了3种麦芽的品质指标及所酿啤酒的理化指标;并采用顶空固相微萃取（HS-SPME）和气相色谱质谱联用（GC-MS）法检测了所酿啤酒中风味物质组成和含量上的差异。结果表明,加拿大麦芽的库尔巴哈值（45%）、浸出率（≥77%）、糖化力（414 WK）和α-氨基酸态氮（118 mg/L）等品质指标和酒精度（2.97%vol）、原麦汁浓度（8.63 °P）、实际浓度（2.76%）、外观浓度（1.17%）、实际发酵度（67.97%）和外观发酵度（86.41%）等所酿啤酒的理化指标适中,啤酒中风味物质种类多样（75种）、相对含量丰富（271.82%）、比例协调,是最适合酿造Lager啤酒的麦芽种类。该结果可为高品质啤酒的工业化生产提供理论依据。     

Influence of the malting parameters on the haze formation of beer after filtration

Malting changes the chemical and enzymatical composition of barley. During malting, enzymes are synthesized, cell walls (pentosans, proteins, etc.) degraded and starch becomes available for enzymatic attack. The progress of germination defines the final beer quality and processability in several aspects: mouthfeel, foam and haze formation (different proteins), processability (viscosity caused by certain substances, like β-glucan), fermentation progress (FAN, sugar content), etc. The objective of this research was to study the influence of different modified malt on turbidity in beer after filtration. This was achieved by analyzing selected malts at different germination stages and afterward studying their influence on the final beer composition, focusing on protein content and composition. Protein fractions were analyzed using a Lab-on-a-Chip technique, which separates the proteins—based on their molecular weight—by capillary electrophoresis. This analysis was supported by the use of two-dimensional gel electrophoresis (2D-PAGE). Additionally, common malt and beer analyses and turbidity and filterability measurements were performed. The protein composition could be followed from malt to beer with both the Lab-on-a-Chip technique and 2D-PAGE. No differences in protein composition could be seen in the final protein composition of the beer. However, it could be observed, with Lab-on-a-Chip technique, that high amounts of a protein fraction with a size of 25–28 kDa caused increased turbidity in the beer.     

南瓜保健啤酒的研制

研究了在啤酒酿造过程中添加南瓜汁的工艺流程。通过正交试验和感官评定的方法得出最佳工艺为：在主发酵前添加2％的南瓜汁，发酵15d。实验结果表明，酿出的成品啤酒具有突出的南瓜香、麦芽香及酒花香，三香协调，口味纯正柔和，适宜饮用。     

Two-dimensional gel analysis of the proteome of lager brewing yeasts

Modern lager brewing yeasts used in beer production are hybrid strains consisting of at least two different genomes. To obtain information on the identity of the parental strains that gave rise to industrial lager yeasts, we used two-dimensional (2-D) gel electrophoresis and analysed the proteomes of different Saccharomyces species isolated from breweries. We found that the proteome of lager brewing yeasts and of the type strains of S. carlsbergensis, S. monacensis and S. pastorianus can be interpreted as the superimposition of two elementary patterns. One originates from proteins encoded by a S. cerevisiae-like genome. The other corresponds to a divergent Saccharomyces species whose best representative is a particular S. pastorianus strain, NRRL Y-1551. A map of industrial lager brewing yeasts has been established, with the individual origin of proteins and with identification of protein spots by comparison to known S. cerevisiae proteins. This 2-D map can be accessed on the Lager Brewing Yeast Protein Map server through the World Wide Web. This study provides the first example of the use of proteome analysis for investigating taxonomic relationships between divergent yeast species.     

Characterisation of proteins from date palm sap (Phoenix dactylifera L.) by a proteomic approach

The proteins contained in juice tapped from date palm (Phoenix dactylifera L.), from Deglet Nour variety, were analysed by the application of two-dimensional electrophoresis (2DE). Identification was carried out by mass spectrometry analyses. The SDS–PAGE patterns showed more than 100 spots of which 52 spots were identified. A proportion of the identified proteins were related to Saccharomyces cerevisiae that may belong to the natural microflora of date palm sap. These proteins are principally involved in glycolysis. While other proteins were assigned to be vegetable proteins, probably a mixture of proteins from the vascular system, which have several biological functions within the palm tree. Thus, we found enzymes involved in stress and defence reactions, in glycolysis, and photosynthesis reactions. Other enzymes are associated with carbohydrates and proteins metabolisms.     

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.     

混合微生物发酵生产啤酒醋及饮料开发

以大麦芽为主要原料,进行啤酒醋发酵,调配制备麦香啤酒醋饮料。优化后的啤酒醋发酵工艺条件为：麦芽糖化后接种啤酒酵母进行酒精发酵,24 h后接种生香酵母,继续发酵4 d,啤酒酵母和生香酵母的添加比例为4∶1;醋酸发酵阶段醋酸菌接种量为10%,30 ℃条件下发酵,发酵结束时啤酒醋酸度为（3.21±0.09） g/100 mL,乙醇含量＜0.05%vol。开发麦香啤酒醋饮料,最佳配方为啤酒醋12%,麦芽汁70%,白砂糖3%,橙色素0.04‰,最终产品的酸度为（0.38±0.07） g/100 mL,糖度为（84.32±0.05） g/L,酸糖比为1∶22。感官评定表明,该方法制备的麦香啤酒醋饮料色泽金黄、澄清透明、酸甜爽口,醋香和麦芽香气浓郁。     

A study on malt modification,used as a tool to reduce levels of beer hordeins

Storage proteins from barley, wheat and rye are toxic to gluten sensitive consumers. These consumers include those suffering from coeliac disease, which account for up to 1% of the global population, and non‐coeliac gluten sensitivity that may affect even greater numbers of the population. Codex Alimentarius has published guidelines and limits of gluten in gluten‐free foods, which are applied in Europe, and similar guidelines apply in the rest of the world. The storage proteins present in barley are hordeins. These proteins are broken down and used by the plant as a source of amino acids during germination and growth of the barley embryo. The objective of this study was to extend the germination stage of the malting process and look at the effect on beer hordeins. Standard MEBAK methods were used to develop an extended malting process and produce three different malts, germinated for 3, 5 or 7 days. The quality of malt was assessed and model beers were produced from each malt to test the effect of modification on levels of beer hordeins. Malt germinated for 7 days produced beer 18 mg/kg hordeins corresponding to a reduction of 44% compared with the beer made from malt germinated for 3 days characterized by a hordein content equal to 32 mg/kg. The malting loss was increased during the 7 days of germination but otherwise all malts were of high quality. The results showed that malting conditions have a significant impact on beer hordeins. Copyright © 2018 The Institute of Brewing & Distilling     

Impact of the brewing process on the concentration of silicon in lager beer

Silicon is an important trace element that is believed to be essential, especially for the proper functioning of connective tissues. Beer is claimed to be an excellent source of bioavailable silicon. However, little is known about the impact of the brewing process on the concentration of silicon in beer. To clarify this relationship, the concentration of silicon in various lager beer samples was determined by instrumental neutron activation analysis, and the mass balance of silicon during the brewing process was calculated. The concentration of silicon ranged from 13.7 to 44.2 mg/L and was highly dependent on the type and quantity of the raw materials used, as well as on the brewing technology. The concentration of silicon in beers of the same brands brewed by distinct breweries did not differ significantly. The silicon mass balance showed that the main silicon source in beer is the barley malt and that the concentration of silicon in solution increases significantly after mashing, whereas it decreases after fermentation. The majority of silicon remains in the spent grains and the resulting beer contains only about 10% of the silicon present in the raw materials. The results confirmed that beer is a rich source of silicon in the human diet. In addition, the important factors that determine the silicon concentration in beer were identified. Copyright © 2014 The Institute of Brewing & Distilling     

蜂蜜艾尔精酿啤酒酿造工艺研究

以澳洲淡色艾尔大麦芽、澳洲水晶焦香麦芽、酒花和蜂蜜为原料,选用上面发酵的WB06型艾尔酵母进行发酵,酿制蜂蜜艾尔 精酿啤酒。以感官评分为评价指标,通过单因素试验和响应面试验研究了原麦汁浓度、发酵温度、葡萄糖添加量及蜂蜜添加量对蜂蜜 艾尔精酿啤酒感官品质的影响。结果显示,蜂蜜艾尔精酿啤酒的最佳酿造工艺条件为：原麦汁浓度12.56 °P,发酵温度18 ℃,葡萄糖添 加量7 g/L,蜂蜜添加量3.05%。 采用该工艺条件酿造的蜂蜜艾尔精酿啤酒产品,各项指标符合GB/T 4927—2008《啤酒》中特种啤酒质 量要求,啤酒的感官评分为96.64分,酒体呈琥珀色,泡沫丰富细腻,挂杯持久,麦香、酒花、蜂蜜香气协调,口感细腻,酒体饱满。     

Influence of high molecular weight polypeptides on the mouthfeel of commercial beer

Low malt beers have high sales volumes in Japan, but improving their mouthfeel, including softness, smoothness and decreasing astringency, is challenging because the compounds responsible remain unclear. In this study, beer was fractionated by preparative size‐exclusion chromatography, with the polypeptide and maltodextrin fractions purified using solid‐phase extraction and ion‐exchange resin. Sensory data from a spike test showed that the mouthfeel (softness, smoothness, and reduced astringency) of low malt beer was improved both by the degree of polymerisation (DP) of maltodextrins (DP of 2‐10; at increased concentration of 40 to 60%; P < 0.01) and by 10 ‐ 20 kilodalton (kDa) high molecular weight (HMW) polypeptide and 2‐3 kDa low molecular weight polypeptide fractions (at a 50% increase in concentration; P < 0.01). Furthermore, highly purified 10 to 20 kDa HMW polypeptides improved the softness and smoothness and decreased the astringency (at a 25% increase in concentration). This report is the first to provide experimental sensory data indicating that HMW polypeptides improve the mouthfeel of beer. Based on these findings, a new low malt beer was developed that showed significantly higher levels of the 10‐20 kDa HMW polypeptides with an overall improved mouthfeel. Mass spectrometric analysis of the 10 to 20 kDa proteins identified several unique foam positive proteins, including barley dimeric alpha‐amylase inhibitor‐1 and non‐specific lipid‐transfer protein 1. These 10‐20 kDa HMW proteins are likely to be responsible for the improved mouthfeel of beer. © 2020 Kirin Holdings Kabushik Kaisha Co. Ltd. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling     

国产麦芽酿造的应用分析

目前许多啤酒生产企业为了降低生产成本,采用国产麦芽替代进口麦芽来酿造啤酒,但国产麦芽的品质会给啤酒产品质量带来一些影响。文中指出了国产麦芽的质量缺陷和酿造性能,并提出了应用国产麦芽生产啤酒的工艺改进办法和优化措施。     

COLD STERILE FILTRATION: A SMALL SCALE FILTRATION TEST AND INVESTIGATION OF MEMBRANE PLUGGING

Beer brewed from 24 commercially and bag malted samples by a small scale brewing method was assessed by a micro-filtration efficiency (MFE) test designed to emulate the cold-sterile (membrane or micro-) filtration process. The level of malt derived beer components with the potential to reduce MFE, such as β-glucan, arabinoxylan, protein and polyphenol, were consistent over duplicate beer batches suggesting that beer quality was reproducible using the small scale method. The small scale MFE test was able to differentiate (P<0.001) between beer brewed from distinct malt samples in a reproducible fashion, suggesting that the test is effective in assessing beer MFE in the laboratory. Subsequently, the effects of various malt derived beer components on micro-filtration were investigated. MFE (measured as <i>V_max) was negatively correlated with beer arabinoxylan content (r=–0.62, P<0.01), suggesting that the arabinoxylan content of malt, and subsequently beer, may influence MFE. Total beer β-glucan was not significantly related to beer MFE (r=-0.36). However, it was likely that β-glucan molecules of high molecular weight influenced MFE more so than the total β-glucan content. Beer viscosity, which was correlated to both beer β-glucan and arabinoxylan content (r=0.86, P<0.001 and r=0.68, P<0.05, respectively), correlated with V_max (r=-0.81, P<0.001) .     

In vitro studies on the main beer protein Z4 of Hordeum vulgare concerning heat stability,protease inhibition and gushing

Protein Z4, the most abundant form of serpin protein Z of Hordeum vulgare, is one of the major constituents of beer foam and inhibits the activity of serine proteases. The possible influence of protein Z4 on malt proteases, which impact malt quality, is of interest for brewers. In addition, the persistence of Z4 in the brewing process and the ensuing presence of Z4 in beer matters to brewers because of its impact on foam. In order to analyse the influence of Z4 on protease inhibition and beer gushing, Pichia pastoris cells were heterologously transformed with the Z4 coding gene and the protein was recovered from the supernatant of a transformant's liquid culture. Agar diffusion assays showed that the recombinant Z4 protein had an inhibitory effect on proteases present in barley malt. Heat denaturation of the protein impaired protease inhibition and revealed degradation of the structure of Z4. The effect of Z4 on hydrophobin‐induced gushing was analysed by addition of the protein to beers pre‐treated with the class 2 hydrophobin FcHyd5p. Results demonstrated that addition of protein Z4 to gushing beer reduces the overflow volume considerably. Heat treatment again had a negative impact on the gushing reduction capacity of Z4. Copyright © 2014 The Institute of Brewing & Distilling     

Expanding the bovine milk proteome through extensive fractionation

Bovine milk is an agricultural product of tremendous value worldwide. It contains proteins, fat, lactose, vitamins, and minerals. It provides nutrition and immunological protection (e.g., in the gastrointestinal tract) to the newborn and young calf. It also forms an important part of human nutrition. The repertoire of proteins in milk (i.e., its proteome) is vast and complex. The milk proteome can be described in detail by mass spectrometry-based proteomics. However, the high concentration of dominating proteins in milk reduces mass spectrometry detection sensitivity and limits detection of low abundant proteins. Further, the general health and udder health of the dairy cows delivering the milk may influence the composition of the milk proteome. To gain a more exhaustive and true picture of the milk proteome, we performed an extensive preanalysis fractionation of raw composite milk collected from documented healthy cows in early lactation. Four simple and industrially applicable techniques exploring the physical and chemical properties of milk, including acidification, filtration, and centrifugation, were used for separation of the proteins. This resulted in 5 different fractions, whose content of proteins were compared with the proteins of nonfractionated milk using 2-dimensional liquid chromatography tandem mass spectrometry analysis. To validate the proteome analysis, spectral counts and ELISA were performed on 7 proteins using the ELISA for estimation of the detection sensitivity limit of the 2-dimensional liquid chromatography tandem mass spectrometry analysis. Each fractionation technique resulted in identification of a unique subset of proteins. However, high-speed centrifugation of milk to whey was by far the best method to achieve high and repeatable proteome coverage. The total number of milk proteins initially detected in nonfractionated milk and the fractions were 635 in 2 replicates. Removal of dominant proteins and filtering for redundancy across the different fractions reduced the number to 376 unique proteins in 2 replicates. In addition, 366 proteins were detected by this process in 1 replicate. Hence, by applying different fractionation techniques to milk, we expanded the milk proteome. The milk proteome map may serve as a reference for scientists working in the dairy sector.