The Release of Ferulic Acid and Feruloylated Oligosaccharides During Wort and Beer Production

Ferulic acid, a very attractive natural antioxidant is present in beer in free form, but the main form is the bound form as feruloylated oligosaccharides. Previous research showed that feruloylated oligosaccharides more effectively inhibited lipid and Low Density Lipoprotein oxidation than free ferulic acid. The aim of the present study was to evaluate free and bound ferulic acid concentrations throughout the brewing process in experimental mashes (worts, beers during fermentation, maturation and storage), and to conduct a comparison in commercial beers. Another aim of the study was to investigate methods to increase levels of bound ferulic acid in beer due to the potential health benefits. Specifically, the influence of commercial enzyme preparations on both forms of ferulic acid contents was studied. Five commercial enzyme preparations during mashing were examined: Celluclast, Shearzyme, Viscozyme, Cereflo and Ultraflo. In all experimental beers, the concentrations of esterified ferulic acid were 4–6 fold higher than the corresponding free ferulic acid contents, depending on the enzyme preparation used. Ferulic acid contents in the ester form in experimental beers were in the range of 748.4 mg/hL to 1244.3 mg/hL, whereas the contents of free ferulic acid were in the range of 134.6 mg/hL to 275.2 mg/hL. Comparison of free and bound ferulic acid contents in experimental beers, produced using enzyme preparations and commercial beers found in a local market, showed that concentrations of bound ferulic acid in experimental beers were significantly higher than in commercial beers, whereas concentrations of free ferulic acid in experimental and commercial beers were comparable.     

Determination of Prolamins in Beers by ELISA and SDS‐PAGE

Coeliac disease is triggered by exposure to the prolamin protein fraction of wheat, barley, or rye. The prolamin content of five lager beers and one wheat beer were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblotting and seven lager beers and three wheat beers were analyzed by enzyme‐linked immunosorbent assay (ELISA). Most of the lager beers were made from barley and some had varying amounts of rice or corn as adjuncts. One of the beers was “gluten‐free”, having been produced from corn and buckwheat without barley. The lager beer samples were gel‐filtered before ELISA or SDS‐PAGE analysis. Prolamin proteins were found in all but one beer which was made of corn, rice and barley and which was not the “gluten‐free” beer. ELISA analysis was done using a commercially available gluten assay kit. For lager beers, a barley prolamin standard for ELISA was propanol‐extracted from barley malt instead of using the prolamin standard of the gluten assay kit. As expected, the wheat beers contained much higher amounts of prolamins than the lager beers. The samples were studied by SDS‐PAGE to identify different prolamin fractions. Proteins having a relative molecular mass in the range of 8000–17,000 and 38,000 and above were detected in immunoblotting by the prolamin sensitive antibody in the lager beers.     

Content of Individual Phenolic Acids in Worts and Beers and their Possible Contribution to the Antiradical Activity of Beer

Phenolic acids are widely distributed in foods and raw materials. They are easily absorbed by humans due to their simplicity. Once they enter the blood plasma, they act as antioxidants. Beer can be a rich source of phenolic acids in the diet. The aim of this study was to determine the concentrations of phenolic acids in two experimental worts and beers as well as in nine market beers (using HPLC‐UV). An examination of the total antiradical activities of phenolic acids with in vitro model systems (using ABTS and DPPH free radicals), at the concentrations comparable to those detected in beers, was performed. Only low fractions of the main phenolic acids present in barley malt (ferulic, vanillic and p‐coumaric acid) were detected in the experimental worts. Moreover, the concentrations of phenolic acids significantly decreased until the last steps of beer production. The main beer phenolic acids (vanillic and ferulic acid) exerted a lower share of total antiradical activity against both free radicals (calculated as the sum of the individual activities of all acids detected in beer) than the minor phenolic acids (caffeic, chlorogenic, o‐coumaric, sinapic or syringic acid). The synergies, between individual phenolic acids in pairs, were also studied with in vitro model solutions using free radicals. The total antiradical activity of the compounds studied in pairs, was at the most as high as the sum of the antiradical activity of the individual phenolic acids, but in most cases it was considerably lower (i.e. no synergy was detected).     

玉米芯功能性低聚糖分级纯化及抗氧化活性研究

探究玉米芯功能性低聚糖分级纯化前后不同组分的组成、理化性质和抗氧化活性。以玉米芯为原料,采用活性炭柱层析法对玉米芯功能性低聚糖进行分级纯化,对纯化前后组分的聚合度、相对分子质量、阿魏酸基团以及红外光谱特性进行分析,并对酶解低聚糖粗提物(EH)及其纯化组分的自由基清除能力和抑制脂质过氧化能力进行比较分析。结果表明：活性炭水洗脱低聚糖组分(WO)、醇洗脱低聚糖组分(EO)均主要由木糖和阿拉伯糖组成,其中WO含有结合态阿魏酸,为阿魏酰阿拉伯低聚木糖;EO不含结合态阿魏酸,为阿拉伯低聚木糖。EH及其纯化组分WO、EO均具有一定的清除自由基(DPPH·、·OH、ABTS⁺·)能力和抑制脂质过氧化能力,且随浓度的增加而增加,WO抗氧化活性最强,EH次之,EO抗氧化活性较弱。     

CONTROL OF FERULIC ACID AND 4-VINYL GUAIACOL IN BREWING

Phenolic acids in beer are important because they can be decarboxylated to phenols, which usually impart off-flavours. An improved high performance liquid chromatographic system was used to monitor phenolic acids and phenols during the brewing process. Ferulic acid was the most significant phenolic acid found in beers prepared from malted barley. Extraction of ferulic acid from malt involved an enzymatic release mechanism with an optimum temperature about 45°C. Mashing-in at 65°C significantly decreased the release of free ferulic acid into the wort. Wort boiling produced 4-vinyl guaiacol by thermal decarboxylation, in amounts (0.3 mg/L) close to its taste threshold, from worts that contained high contents of free ferulic acid (> 6 mg/L). The capacity of yeasts to decarboxylate phenolic acids (Pof⁺ phenotype) was strong in wild strains of Saccharomyces and absent in all lager brewing yeast and most ale brewing yeasts. Some top-fermenting strains, especially those used in wheat beer production, possessed a weak decarboxylating activity (i.e. Pof^δ). During storage of beers there were appreciable temperature-dependent losses of 4-vinyl guaiacol. These results indicated that the production of 4-vinyl guaiacol is amenable to close technological control.     

AMINO ACID ANALYSIS OF BEER POLYPEPTIDES

The principles of amino acid analysis of proteins and polypeptides are reviewed. Analysis of the amino acid composition of dialysed beer material prepared from a wide variety of commercial and pilot brewery beers showed that the principal amino acids comprised glutamic acid/glutamine, proline, glycine and aspartic acid/asparagine. The results from the analysis of a series of pilot brewery beers brewed under standardised conditions showed that the composition of the grist may influence the amino acid composition of beer polypeptide fractions. Dialysed beer material prepared from beer brewed from grists containing torrified wheat, wheat flour and malted wheat contained greater proportions of glutamic acid/glutamine compared to material prepared from all malt beers. Further fractionation and analysis of dialysed beer material prepared from pilot brewery beers suggested that fractions MW>60000 contained polypeptide material derived from yeast mannan-protein. In addition fractions MW>60000 prepared from beer brewed from grists containing torrified wheat, wheat flour or all malted wheat may contain high molecular weight polypeptide material derived from wheat proteins. The results from the analysis of fraction MW 40,000–60000 prepared from beers brewed from grists containing all malt, 80% malt and 20% torrified wheat and 50% malt and 50% malted wheat are consistent with the presence of polypeptide material derived from cereal albumins and globulins whereas fractions MW 40,000–60000 prepared from beers brewed from 80% malt and 20% wheat flour and 100% malted wheat may contain polypeptide material derived from wheat prolamins and glutelins. The amino acid composition of fraction MW 20,000–40,000 from all pilot brewery beers investigated is consistent with the presence of polypeptide material derived from cereal prolamins and glutelins. The amino acid composition of beer polypeptide fractions may be used to detect the use of wheat adjuncts in beer brewing.     

草酸蒸煮处理和表面活性剂对酶法制备低聚糖阿魏酸酯的影响

低聚糖阿魏酸酯兼具低聚糖的益生菌增殖活性和阿魏酸的抗氧化活性,可通过水解阿拉伯木聚糖获得。本文采用阿魏酸含量最高的玉米皮为原料,研究草酸蒸煮处理和添加表面活性剂对酶解玉米皮制备低聚糖阿魏酸酯的影响。研究结果显示:草酸含量0.6%,料液比1:10(W:V),0.1MPa蒸煮20min后,采用木聚糖酶酶解可获得阿魏酸含量为14.10mg/g的低聚糖阿魏酸酯,并将玉米皮中52.2%的阿魏酸游离出来。酶解过程中,添加表面活性剂Tween-80、Tween-20和Span-80,能显著提高玉米皮的酶解效率。当3者添加量分别为4%、3%和2%时,低聚糖阿魏酸酯中阿魏酸含量分别提高到190%、128%和152%。使用Span-20可降低阿魏酸酯产量。     

Total hordatine content in different types of beers

Hordatines are phenolic secondary metabolites typical of barley. Hordatines withstand at least moderate processing, and thus they are also found in barley malts and beer. So far, no published data on the hordatine content has been available in beers or different styles of beer. The aim of this study was to produce information on the total hordatine content in beers and statistically compare the hordatine content of different beer types. In the current study, hordatines were analysed in 208 beers by high‐performance liquid chromatography equipped with a diode array detector (HPLC‐DAD). The average total hordatine content of all beer samples was 5.6 ± 3.1 mg L^?1 as p‐coumaric acid equivalents (PCAE), with a minimum values 0 to a maximum value 18.7 mg L^?1 PCAE. The total hordatine content correlated positively to the alcohol content in lagers, ales, stouts and porters, but not in wheat beers. There was no statistically significant difference in hordatine content in different types of beer, excluding the non‐alcoholic group of beers. It is noteworthy that non‐alcoholic beers also contained hordatines. More research would be needed to understand how parameters, such as mashing, should be chosen in order to achieve maximum recovery of hordatines in wort and beer.     

对香豆酸、阿魏酸和低聚糖阿魏酸酯对酪氨酸酶的抑制效果

通过测定酶的稳态活力、迟滞时间和动力学参数,研究对香豆酸、阿魏酸及低聚糖阿魏酸酯对酪氨酸酶的抑制效果。结果表明,3种物质对酪氨酸酶单酚酶活性均有抑制作用,其中对香豆酸的抑制作用最强,其次为低聚糖阿魏酸酯和阿魏酸。对香豆酸、低聚糖阿魏酸酯和阿魏酸对单酚酶的IC_(50)值分别为0.75,3.20,9.30 mmol/L。对香豆酸和阿魏酸抑制二酚酶活性,IC_(50)值分别为4.3,12.7mmol/L;但低聚糖阿魏酸酯对二酚酶活力没有影响。对香豆酸能明显延长单酚酶反应的迟滞时间,阿魏酸影响很小,而低聚糖阿魏酸酯则缩短迟滞时间。动力学研究结果显示,阿魏酸和低聚糖阿魏酸酯对单酚酶的抑制作用表现为混合性抑制,而对香豆酸为竞争性抑制。     

The influence of unmalted barley on the oxidative stability of wort and beer

The aim of this study was to investigate the influences of unmalted barley on the brewing process and the quality of the resulting beer‐like beverages, with the main focus on the oxidative stability, using traditional beer analyses, GC‐MS for the determination of aging compounds and electron paramagnetic resonance spectroscopy to determine free radical activity. For the investigation, brews with different barley proportions and 75% barley brews with a colour malt addition, to compensate for a lower colour using barley, were produced. In general, it can be said that beers with a proportion of up to 50% barley achieved a comparable or higher extract yield and final attenuation owing to the combined effectiveness of the malt and microbial enzymes. Although all analytical values were within the normal range according to Methodensammlung der Mitteleuropäischen Brautechnischen Analysenkommission (MEBAK), a slight decrease in total polyphenols and free amino nitrogen content was observed. Also in response to higher barley portions, an increase of higher molecular weight proteins and β‐glucan was detected. Barley is not exposed to heat and oxidative stress in the malting plant, which explains the lower values of the thiobarbituric acid index and colour as an indicator of Maillard reaction products in the resulting wort and beer. Additionally, the results demonstrate a slower increase of aging compounds during beer storage with increasing barley proportions. Furthermore, it was observed that higher barley proportions led to a better oxidative stability indicated by a lower radical generation (T₄₅₀‐value) in wort and an increasing beverage antioxidant index/endogenous antioxidative potential (BAX/EAP value) in the final beverage. The case of ‘barley beers’ showed that the positive effect of barley on the oxidative beer stability was greater than the negative effect of the addition of colour malt, to adjust the colour of a 100% malt beer. In sensory comparison with beer produced with 100% malt, the beers brewed with a barley proportion up to 50% showed a slight flavour preference and up to a 75% equivalent evaluation. Copyright © 2012 The Institute of Brewing & Distilling     

黑小麦功能性低聚糖的分离与组成分析

研究黑小麦麸皮功能性低聚糖的分离制备方法,并对其组成进行分析。采用木聚糖酶酶解黑小麦麸皮不溶性膳食纤维,所得酶解液经活性炭柱层析进行分离,并利用液相色谱和离子色谱对分离组分的单糖组成、低聚糖组成以及阿魏酰基团进行检测。结果表明,活性炭柱层析的水洗组分(WO)、不同浓度醇洗组分(EO)的低聚糖均主要由阿拉伯糖和木糖组成,为阿拉伯低聚木糖。其中,WO含有结合态阿魏酸,为阿魏酰低聚糖。WO的低聚糖聚合度较高,为DP 4~7;醇洗组分EO的低聚糖聚合度较低,为DP 2~4。     

Comparison of beer quality attributes between beers brewed with 100% barley malt and 100% barley raw material

BACKGROUND: Brewing with 100% barley using the Ondea^® Pro exogenous brewing enzyme product was compared to brewing with 100% barley. The use of barley, rather than malt, in the brewing process and the consequences for selected beer quality attributes (foam formation, colloidal stability and filterability, sensory differences, protein content and composition) was considered. RESULTS: The quality attributes of barley, malt, kettle‐full‐wort, cold wort, unfiltered beer and filtered beer were assessed. A particular focus was given to monitoring changes in the barley protein composition during the brewing process and how the exogenous OndeaPro^® enzymes influenced wort protein composition. All analyses were based on standard brewing methods described in ASBC, EBC or MEBAK. To monitor the protein changes two‐dimensional polyacrylamide gel electrophoresis was used. CONCLUSION: It was shown that by brewing beer with 100% barley and an appropriate addition of exogenous Ondea^® Pro enzymes it was possible to efficiently brew beer of a satisfactory quality. The production of beers brewed with 100% barley resulted in good process efficiency (lautering and filtration) and to a final product whose sensory quality was described as light, with little body and mouthfeel, very good foam stability and similar organoleptic qualities compared to conventional malt beer. In spite of the sensory evaluation differences could still be seen in protein content and composition. Copyright © 2011 Society of Chemical Industry     

Variation in gluten protein and peptide concentrations in Belgian barley malt beers

Gluten is the main family of storage proteins found in barley. During malting and brewing, some of the barley malt's proteinaceous material is hydrolysed into peptides or to amino acids. Most of the gluten proteins are removed with the spent grains and with hot‐ and cold‐breaks. However, some gluten proteins and especially gluten‐derived peptides can remain throughout the brewing process and will hamper the gluten‐free (≤20 ppm) status of the beer. In this work, three production batches (a, b and c) of 51 Belgian barley malt beers from 24 breweries were analysed with the sandwich (R7001) and competitive (R7021) Ridascreen gliadin R5‐ELISA to quantify gluten proteins and peptides. Although the majority of the beers contained low‐gluten protein concentrations of ≤20 ppm (a/45, b/47, c/48), only a minority were truly gluten‐free with ≤20 ppm gluten peptides (a/18, b/17, c/15). The grain bill had no influence on the measured gluten concentration, but the use of (combined) clarification techniques and presence of wheat malt in the grist was respectively a positive and negative influence. Ten beers, from four breweries, were gluten free in all analysed samples. These included two wheat beers, reflecting the importance of effective clarification in the management of gluten. These results explore the feasibility of the production of gluten‐free barley malt beers. Copyright © 2018 The Institute of Brewing & Distilling     

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.     

β‐Glucans and Pentosans and their Degradation Products in Commercial Beers

Several commercial beers have been analyzed for their content of β‐glucans, pentosans and their degradation products using high performance liquid chromatography, thin layer chromatography and chemical and enzymic analysis procedures. The beers tested contained high levels of residual high molecular weight pentosan, but much less high molecular weight β‐glucan. The beers also contained sizeable levels of oligosaccharides, especially trisaccharides, reflecting the incomplete degradation of polymeric materials in malting and mashing and the inability of yeast to ferment them. There is substantially more β‐linked glucosyl material in beer than pentosyl substances, although the higher molecular weight of the latter probably makes it more likely to represent soluble fibre. In respect of fibre claims for the beers examined, even for the beer containing the least pentosan, it seems that less than a litre of the product would afford sufficient material.     

Association of non-starch polysaccharides and ferulic acid in grain amaranth (Amaranthus caudatus L.) dietary fiber

The association of ferulic acid, an alkali-extractable phenolic acid in amaranth (Amaranthus caudatus L., Amaranthaceae) insoluble fiber (trans-ferulic acid: 620 microg.g-1, cis-ferulic acid: 203 microg.g-1), and non-starch polysaccharides was investigated. Enzymatic hydrolysis of insoluble amaranth fiber released several feruloylated oligosaccharides that were separated using Sephadex LH-20-chromatography and reversed phase-high performance liquid chromatography (RP-HPLC). Three compounds were unambiguously identified: O-(6-O-trans-feruloyl-beta-D-galactopyranosyl)-(1-->4)-D-galactopyranose, O-(2-O-trans-feruloyl-alpha-L-arabinofuranosyl)-(1-->5)-L-arabinofuranose, and O-alpha-L-arabinofuranosyl-(1-->3)-O-(2-O-trans-feruloyl-alpha-L-arabinofuranosyl)-(1-->5)-L-arabinofuranose. These feruloylated oligosaccharides show that ferulic acid is predominantly bound to pectic arabinans and galactans in amaranth insoluble fiber. 5-O-trans-Feruloyl-L-arabinofuranose was the only compound isolated in pure form from an acid hydrolyzate. This compound may have its origin from pectic arabinans but also from arabinoxylans.     

APPLICATIONS OF FAST PROTEIN LIQUID CHROMATOGRAPHY (FPLC) TO THE ANALYSIS OF THE NITROGENOUS CONSTITUENTS OF BEER

The nitrogenous constituents of beer were investigated by several Fast Protein Liquid Chromatography (FPLC) techniques. Size exclusion chromatography of dialysed beer material using columns of Superose 6 and Superose 12 suggested that beer polypeptide material was distributed across a wide relative molecular mass (Mr) range with discrete fractions of high Mr (Mr 300 000, Mr 500 000), Mr c60 000, Mr c40 000 and relatively low Mr (Mr 5 000–20 000). The composition of fractions Mr >40 000 and Mr 40 000–60 000 was investigated by ion exchange chromatography. Differences were detected in the elution profiles of fractions prepared from beers brewed from grists comprising 100% malt, 80% malt plus 20% torrfied wheat and 100% malted wheat consistent with differences in the polypeptide composition of these fractions. The Superose 12 column, although designed for the fractionation of high molecular weight components, also provided a method of fractionating low molecular weight nitrogenous materials directly from beer (for example fractions containing purine nucleosides). Reverse phase chromatography was employed in the analysis of beer peptides and demonstrated the complex composition of beer peptide fractions.     

PHENOLIC ACIDS IN BEERS AND WORTS

The total contents of phenolic acids measured by high-performance liquid-chromatography were 5–8 mg/litre in beers brewed in Ireland whereas 16–40 mg/litre were present in four other beers. In all beers the predominant phenolic acids were vanillic, p-coumaric and ferulic acids. Free phenolic acids were extracted from Emma barley grains and malt in very small amounts (15–28 mg/kg) but larger quantities (191 mg/kg) were released on mashing the malt. Little change occurred in the contents of phenolic acids on processing a lager wort through to the finished beer. Treatment with excess Polyclar AT removed astringent flavour and phenolic acids from an experimental ale but this flavour loss could not be accounted for by the adsorption of phenolic acids. The flavour threshold for a nine-component phenolic acid mixture in lager was between 50 mg/litre and 100 mg/litre.     

Influence of barley variety, timing of nitrogen fertilisation and sunn pest infestation on malting and brewing

BACKGROUND: This paper presents a multivariate approach to investigate the influence of barley variety, timing of nitrogen fertilisation and sunn pest infestation on malting and brewing. Four spring and two winter barley varieties were grown in one location in southern Europe. Moreover, one of the spring varieties was infested with sunn pest, in order to study the effects of this pest on malting quality, and subjected to different nitrogen fertilisation timing regimes. The samples were micromalted, mashed, brewed and analysed. RESULTS: The data showed that even though the two winter barleys seemed to be the best regarding their physical appearance (sieving fraction I + II > 82%), this superiority was not confirmed in the malt samples, which showed low values of Hartong extract (27.1%) and high values of pH (6.07–6.11) and β‐glucan content (12.5–13.2 g kg^?1), resulting in low‐quality beers. The barley sample subjected to postponed fertilisation had a total nitrogen content (19.5 g kg^?1 dry matter) exceeding the specification for malting barley and gave a beer with a low content of free amino nitrogen (47 mg L^?1) and high values of viscosity (1.99 cP) and β‐glucan content (533 mg L^?1). The beer obtained from the barley sample subjected to pest attack had good quality parameters. CONCLUSION: All spring barleys gave well‐modified malts and consequently beers of higher quality than the winter barleys. Moreover, postponed fertilisation was negatively related to the quality of the final beer, and sunn pest infestation did not induce important economic losses in the beer production chain. Copyright © 2010 Society of Chemical Industry     

Relationship between the chemical composition and the biological activities of food melanoidins

The relationship between the chemical composition and the biological activities of food melanoidin-rich fractions was investigated. Melanoidin-rich fractions were extracted using ultrafiltration (a 10 kDa cut-off) from coffee, barley coffee, dark beer, and traditional balsamic vinegar. All the food melanoidin-rich fractions were formed mainly of carbohydrates, phenolic compounds, and proteins. In dark beer, barley coffee, and traditional balsamic vinegar melanoidins, glucose was the most abundant sugar incorporated into melanoidins. Coffee melanoidins contained the largest amount of phenolic groups, followed by traditional balsamic vinegar melanoidins. The radical scavenging, Fe²⁺-chelating, and heme binding abilities of food melanoidins were investigated under gastric conditions. The melanoidinrich fraction extracted from coffee was the most active, showing the highest radical scavenging, Fe²⁺-chelating, and heme binding activities, compared to barley coffee, dark beer, and traditional balsamic vinegar. The radical scavenging and Fe²⁺-chelating abilities were assigned to the phenolic groups present in food melanoidins.