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     

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.     

Levels of gliadin in commercial beers

The gliadin contents of twenty-eight commercially available beers spanning the range of grist material and alcohol level were determined using a competitive enzyme immunoassay (RIDASCREEN competitive ELISA kit). The gliadin levels ranged from <3 mg/l for gluten-free beers to 145.8 mg/l for filtered American Pale Wheat beers, with: American light lagers (3.9–12.2 mg/l), alcohol free beer (6.5 mg/l), American lagers (7.1–12.3 mg/l), English Pale Ale (7.3 mg/l), English Brown Ale (8 mg/l), European Pale lager (8.3 mg/l), Scottish Ale (12 mg/l), Low carb American lager (12.1 mg/l), American India Pale Ales (11.8–19.3 mg/l), American Pale Ales (14.6–15.3 mg/l), Czech Pilsner (14.9 mg/l), Sweet Stout (17.1 mg/l), Dry Stout (18.9 mg/l), Oktoberfest/Marzen (21.5 mg/l), Russian Imperial Stout (21.3 mg/l), Kolsch (24.9 mg/l), American Porter (28.5 mg/l), and Dunkelweizen (98.2 mg/l) within this range. Ten of the twenty-eight (34%) beers contained less gluten than the guidelines established by Codex Alimentarius Standard (20 ppm gluten). The calculation of gluten is based on the assumption of a 1:1 ratio between gliadin and glutenin.     

An overview of selected specialty beers: developments,challenges and prospects

The brewing industry has devoted much research effort into the development of new technologies and innovations for the expansion of the assortment of specialty beers in response to increased consumer demand. Specialty beers are more or less the catch‐all for beer styles, which do not fit into conventional beer categories and five types of specialty beers are of particular interest – low‐calorie beer, low‐alcohol or nonalcohol beer, novel‐flavoured beer, gluten‐free beer and functional beer. The selected beer types are technologically challenging to produce relative to the traditional approach of ingredient addition, yet offer special appeal to consumers from the perspective of health and flavour. Biological processes that make use of the equipment of a traditional brewery plant should be better exploited in comparison with nonbiological technologies such as thermal and membrane processes. Probiotic beer could be the specialty beer of the future in light of the increasing popularity of probiotics. These beers are reviewed in terms of developments, challenges and prospects.     

Immunochemical determination of gluten in malts and beers.

The gluten content in different varieties of barley and malts, and in different types of beers, was determined by a 'sandwich' enzyme immunoassay (RIDASCREEN Gliadin kit). The gluten levels in barley wheat, rye and spelt malts ranged 18.8-45.0, 44.0-68.0, 41.6 and 21.2 g kg-1, respectively. When various types of beer were compared, the gluten concentration increased as follows: alcohol-free beer (<3.0), lager beers (<3.0-8.7 mg l-1), stouts (9.0-15.2 mg l-1) and wheat beers (10.6-41.2 mg l-1). When 10 Czech lager beers were analysed, using both sandwich and competitive ELISA, the results showed that the latter method provided values several times higher than the former. Gluten balance was carried out during the brewing process, starting from the raw materials and terminating at the final beer. Gluten levels decreased due to precipitation during the mashing process, primary and secondary fermentation and, lastly, as a result of adsorption during beer stabilization. The gluten content in beer is, thus, approximately three orders of magnitude lower than in the raw malt.     

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     

小麦面筋蛋白质酶解产物用作啤酒发泡蛋白的研究

为改善啤酒的泡沫性能,作者分别采用木瓜蛋白酶、胃蛋白酶以及碱性蛋白酶对小麦面筋蛋白进行适度酶解改性,并对其产物用作啤酒发泡蛋白的可行性进行了研究.结果表明,经适度酶解作用后,小麦面筋蛋白在pH 4.5条件下溶解性和泡沫性能得到显著改善(P<0.05),且小麦面筋蛋白酶解产物在啤酒环境中热稳定性较好,经30 min的热处理,含100 mg/L小麦面筋蛋白酶解产物的啤酒浊度与加热前相比增加不显著(p>0.05).小麦面筋蛋白胃蛋白酶酶解产物和碱性蛋白酶酶解产物对啤酒初始泡持性的改善效果都较好,但胃蛋白酶酶解产物对酵母蛋白酶A作用较敏感,对纯生啤酒货架期内泡持性的改善效果不太理想,而碱性蛋白酶酶解产物可明显改善纯生啤酒货架期内的泡沫稳定性.     

Detection of gluten in a pilot-scale barley-based beer produced with and without a prolyl endopeptidase enzyme

ABSTRACT

Immunochemical and mass spectrometric methods were used to examine the gluten composition of a gluten-reduced beer produced by brewing with barley malt in the presence of prolyl endopeptidase (PEP) and a final filtration treatment with diatomaceous earth and perlite. The competitive ELISA is generally considered appropriate for the analysis of hydrolysed gluten, but it is not considered a scientifically valid method for the quantification of gluten in fermented or hydrolysed foods due to the lack of an appropriate reference standard. As no single analytical method can capture the spectrum of gluten-derived products in beer, a comprehensive approach was employed to analyse the intact and hydrolysed fractions of gluten with complementary methods. The combination of PEP addition and diatomaceous earth/perlite filtration was more effective at reducing the concentration of detectable gluten than each of the treatments alone. However, gluten proteins and/or polypeptides were observed in filtered, PEP-treated beers using sandwich ELISA methods, western blot, and bottom-up mass spectrometry. In addition, mass spectrometry results showed that the number of hydrolysed gluten peptides was almost unaffected by the filtration process. Gluten peptides that contained potentially immunopathogenic sequences were identified in the filtered PEP-containing beers by MS. Variability in gluten composition was observed between three replicate pilot-scale productions, suggesting that the gluten profile in beer could differ from batch to batch. As there is uncertainty in the detection and quantification of gluten in hydrolysed and fermented foods, characterisation of hydrolysed gluten by complementary analytical methodologies is recommended.     

Effect of UV‐C Disinfection of Beer — Sensory Analyses and Consumer Ranking

Ultraviolet (UV‐C) light irradiation is gaining rapid acceptance within the food and beverage industry as a non‐thermal disinfection technique. A series of trials, using a pilot scale UV‐C treatment system, were conducted to investigate the effect of UV‐C on beer with specific attention to lightstruck flavour formation. Both commercial and micro‐brewed beers were treated with UV‐C light at 254 nm. Samples were analysed by consumer and trained panels. Sensory analyses revealed that at a low UV‐C level, lightstruck flavour was apparent and this increasingly gave way to a more intense burnt rubber off‐flavour as the UV‐C exposure was increased. A sample enrichment probe technique coupled with a gas chromatography‐mass spectrometry (SEP/GCMS) revealed the presence of lightstruck flavour in all the treated beers.     

Gluten weight in ancient and modern wheat and the reactivity of epitopes towards R5 and G12 monoclonal antibodies

Differences in the level of coeliac‐active gluten epitopes in wheat might have some significance for individuals reporting noncoeliac gluten sensitivity. The aim of this study was to compare the reactivity of epitopes towards ELISA R5 and G12 monoclonal antibodies in ancient (emmer; Khorasan wheat; spelt) and modern wheat (common bread wheat; durum), and to check whether the bread‐making process leads to the degradation of epitopes. Data from ELISA R5 and G12 did not match gluten dry weight in wheat. Bread dough fermentation and extensive baking did not change the reactivity of coeliac‐active epitopes towards monoclonal antibodies. Compared to hexaploid bread‐type wheat (spelt; common bread wheat), ancient and modern pasta‐type tetraploid wheat (emmer; Khorasan; durum) had less epitopes reactive towards ELISA R5 and G12 and might be preferable for wheat‐sensitive individuals looking for food with reduced coeliac‐active epitopes.     

Metabonomics analysis of nonvolatile small molecules of beers during forced ageing

Three kinds of beers with different degrees of ageing were used to examine their nonvolatile small molecules by a metabonomics approach based on ultra performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐Q‐ToF‐MS/MS). Results showed that a total of 2114 compounds were detected in forced‐aged beers with the positive mode of UPLC‐Q‐ToF‐MS/MS. There was a clear separation among three groups of beers with different degrees of ageing in principal component analysis (PCA) model. Sixteen potential metabolite markers related to beer oxidative stability were identified by orthogonal partial least‐squares discriminate analysis (OPLS‐DA). Results from the changes in the spectrum also indicated that some new compounds formed in beer during forced ageing. Thus, the proposed metabonomics approach is a powerful tool to give a fully understanding the ageing process of beer based on the identification of the markers related to oxidative stability.     

Survey of deoxynivalenol and its conjugates deoxynivalenol-3-glucoside and 3-acetyl-deoxynivalenol in 374 beer samples

Beer is one of the most popular beverages worldwide. Malted cereal grains are among the basic ingredients and hence mycotoxin contamination might occur. Previous studies reported the presence of the Fusarium mycotoxins deoxynivalenol (DON) and 3-acetyl-deoxynivalenol (3ADON), as well as of the masked mycotoxin deoxynivalenol-3-glucoside (D3G) in beer. In the present survey, 374?beer samples from 38?countries with a focus on Austrian (156) and German (64) beers were analysed for the presence of D3G, DON and 3ADON. Beers were assigned to the following six categories: pale (217), wheat (46), dark (47), bock (20), nonalcoholic beers (19) and shandies (25). In total, 348 and 289 beers (93 and 77%, respectively) contained D3G and DON at the levels above the limit of detection, whereas 3ADON was not detected in any of the samples. Average concentrations of all beers were 6.9?µg?L^?1 for D3G and 8.4?µg?L^?1 in the case of DON. Nonalcoholic beers and shandies showed the lowest contaminations, 1.5 and 3.2?µg?L^?1 for D3G and 2.7 and 4.4?µg?L^?1 for DON, respectively. In bock beers characterised by a higher gravity, a significant trichothecene load of 14.8?µg?L^?1 D3G and 12.4?µg?L^?1 DON was found. The highest contamination (81?µg?L^?1 D3G, 89?µg?L^?1 DON) was detected in a pale beer from Austria, underlining the importance of this study for food safety. The molar D3G to DON ratio ranged between 0.11 and 1.25 and was 0.56 on average. Concluding, the average contamination of beer is not of toxicological concern for moderate beer drinkers. However, in the case of heavy beer drinkers, beer consumption may considerably contribute to the overall intake of DON, which might even lead to exceeding the maximum tolerable limits established for this Fusarium toxin.     

Breeding of lipoxygenase‐1‐less malting barley variety ‘Satuiku 2 go’

The lipoxygenase‐1‐less (LOX‐less) trait has positive effects on beer quality, in particular, improvement of flavour stability related to the reduction of beer‐deteriorating substances such as trans‐2‐nonenal. ‘Ryohfu’ is the only spring‐sown malting barley variety grown in Hokkaido, located in the northern part of Japan, and has been used in the Japanese brewing industry for over 20 years. ‘Satuiku 2 go’ was developed as the first LOX‐less malting barley variety in Japan by successive back‐crossing with molecular marker‐assisted selection to introduce the LOX‐less trait into the recurrent parent ‘Ryohfu’. The agronomic performance and general malt quality of ‘Satuiku 2 go’ were almost equivalent to those of ‘Ryohfu’. Wort and beer analyses at the pilot‐scale brewing trial indicated that the LOX‐less trait had little effect on the general characteristics. In contrast, the beers made from ‘Satuiku 2 go’ malt exhibited reduced levels of trans‐2‐nonenal and trihydroxyoctadecenoic acid. The sensory evaluation demonstrated the superiority of ‘Satuiku 2 go’ beers stored under differing conditions in terms of staleness. It can be concluded that the LOX‐less trait was effective in different genetic backgrounds of the recurrent parents used for the development of LOX‐less malting barley varieties. Copyright © 2018 The Institute of Brewing & Distilling     

Comparison of Procedures for Resveratrol Analysis in Beer: Assessment of Stilbenoids Stability through Wort Fermentation and Beer Aging

Three resveratrol extraction procedures were compared in beer. Two‐step pre‐cleaning with toluene and cyclohexane allows 76% trans‐resveratrol recovery by solid‐phase extraction (SPE) before RP‐HPLC‐MS/MS analysis. This procedure proved much more efficient than liquid/liquid extraction, which requires solvents that are too hydrophobic for stilbenoids. SPME‐GC‐MS, where the main limiting factor is non‐reproducibility from fibre to fibre, is an interesting alternative for resveratrol quantification in complex mixtures where pre‐cleaning is too difficult. With the optimized SPE procedure, 5 μg.L^?1 trans‐resveratrol was detected in four commercial beers. However, concentrations of stilbenoids in beer should be higher taking into account the presence of cis‐stilbenoids. During fermentation, trans‐resveratrol was partially regenerated from its glucoside, more stable through beer aging. Adding a stilbenoids‐enriched ethanolic hop extract after fermentation significantly increases the beer stilbenoids potential.     

A fundamental study on the relationship between barley cultivar and hordeins in single cultivar beers

The hordein proteins found in beer are not suitable for gluten‐sensitive consumers. Hordeins are storage proteins found in barley and have limited solubility in water. It is not currently known if the nitrogen concentration of barley directly impacts on the hordeins present in beer. In this study a controlled malting on eight barley cultivars was performed and a single cultivar model beer was produced from each. The single cultivar model beers were then examined for differences in content of hordeins. The quality of barley and malt was assessed and the parameters measured were compared with the beer hordeins using a Pearson correlation matrix. The results showed significant differences in the content of beer hordeins, depending on the barley malt used. Correlations between results showed a positive relationship to malt nitrogen and a negative relationship to friability. The results suggest it may be possible to optimize the choice of the barley cultivar and the malting conditions in order to produce a beer low in hordeins. Copyright © 2016 The Institute of Brewing & Distilling     

Assay of gliadin by real‐time immunopolymerase chain reaction

Patients with coeliac disease (gluten‐sensitive enteropathy) are intolerant against gliadins from wheat and the respective proteins from related cereals and have to keep a lifelong gluten‐free diet. For control of gliadin in gluten‐free food sensitive assay techniques are necessary. We developed an immunopolymerase chain reaction (iPCR) assay for gliadin. In this technique immunological detection of gliadin by a monoclonal antibody R5 conjugated with an oligonucleotide is amplified by PCR. For quantification, iPCR was performed as real‐time PCR (real‐time iPCR) in one step. By means of real‐time iPCR, the sensitivity of gliadin analysis was increased more than 30‐fold above the level reached by enzyme immunoassay. Real time‐iPCR using R5 directly conjugated with oligonucleotide was clearly more sensitive than real time‐iPCR applying sequentially biotinylated R5, streptavidin, and biotinylated oligonucleotide. With directly conjugated R5 gliadin was detected at a concentration as low as 0.16 ng/mL corresponding to 16 μg gliadin/100 g food or 0.16 ppm (corresponding to 0.25 g of food extracted in 10 mL of solvent and 25‐fold dilution of the extract prior to analysis). This is the first report applying the highly sensitive technique of iPCR for gliadin analysis. Furthermore, this is the first approach to perform real‐time iPCR in one step without changing the reaction vessels after enzyme immunoassay for subsequent PCR analysis thus minimizing risks of contamination and loss of sensitivity.     

Characterization of Antibodies for Grain‐Specific Gluten Detection

Gluten ingestion causes immunoglobulin E (IgE)‐mediated allergy or celiac disease in sensitive individuals, and a strict gluten‐free diet greatly limits food choices. Immunoassays such as enzyme‐linked immunosorbent assay (ELISA) are used to quantify gluten to ensure labeling compliance of gluten‐free foods. Anti‐gluten antibodies may not exhibit equal affinity to gluten from wheat, rye, and barley. Moreover, because wheat gluten is commonly used as a calibrator in ELISA, accurate gluten quantitation from rye and barley contaminated foods may be compromised. Immunoassays utilizing grain‐specific antibodies and calibrators may help improve gluten quantitation. In this study, polyclonal antibodies raised against gluten‐containing grain‐specific peptides were characterized for their immunoreactivity to gluten from different grain sources. Strong immunoreactivity to multiple gluten polypeptides from wheat, rye, and barley was observed in the range 34 to 43 kDa with anti‐gliadin, 11 to 15 and 72 to 95 kDa with anti‐secalin, and 30 to 43 kDa with anti‐hordein peptide antibodies, respectively. Minimal or no cross‐reactivity with gluten from other grains was observed among these antibodies. The anti‐consensus peptide antibody raised against a repetitive amino acid sequence of proline and glutamine exhibited immunoreactivity to gluten from wheat, rye, barley, and oat. The antibodies exhibited similar immunoreactivity with most of the corresponding grain cultivars by ELISA. The high specificity and minimal cross‐reactivity of grain‐specific antibodies suggest their potential use in immunoassays for accurate gluten quantitation.     

Influence of hop harvest date of the ‘Mandarina Bavaria’ hop variety on the sensory evaluation of dry‐hopped top‐fermented beer

To impart a special hop aroma to beer, dry‐hopping is a technique that is becoming more and more popular with commercial breweries. Nevertheless, until now little was known about the factors that influence the reproducibility (and consistent product quality) of dry‐hopping with flavour varieties. One factor that could influence the sensory impressions and aroma profile compositions of dry‐hopped beers is the hop harvest date. Therefore, to determine the effects of different harvest dates of the flavour variety ‘Mandarina Bavaria’ on the aroma of top‐fermented beer, laboratory‐scale dry‐hopping trials were performed. Besides tasting sessions of brewed beers, relative quantities of selected hop‐derived, as well as beer‐originated aroma compounds, were investigated by headspace–solid‐phase microextraction–gas chromatography–mass spectrometry. Duo–trio tests between the beers hopped with pellets of different harvest dates showed no significant differences (α = 0.05) between them. In addition, these beers had similar profiles in a five‐point profile tasting scheme. On the other hand, relative concentrations of some hop‐derived aroma compounds – especially myrcene, which is known to be able to contribute to beer flavour – increased corresponding to a later harvest date, while beer originated volatiles were not different between the beers. Analytical results combined with the results of sensory evaluations led to the conclusion that the harvest date of Mandarina Bavaria was not a dominant factor in the dry‐hopping aroma of top‐fermented beers. High amounts of fermentation by‐products are likely responsible for masking effects resulting in no sensory distinctness between the samples with different hop aroma compound concentrations. Copyright © 2016 The Institute of Brewing & Distilling     

Gluten‐sensitive enteropathy (celiac disease)

Abstract

Gluten‐sensitive enteropathy (celiac disease—GSE) is induced by dietary wheat gliadin and related proteins in genetically susceptible individuals. Enzyme abnormalities and a lectin‐like gluten toxicity could be implicated in the pathogenesis of the disease, but most evidence suggests that the mucosal lesion of GSE represents an imraunologicaly mediated tissue injury triggered by gluten ingestion within the context of a particular assortment of major histocompatibility complex genes. The process leading to the mucosal damage is as yet still poorly understood, as well as the amino acid sequence(s) of gliadin and related proteins responsible for toxicity. Several in vitro models are available to test toxicity of gliadin amino acid sequences, pathogenesis of mucosal damage, and possible protective mechanisms, but definitive conclusions must rely on in vivo jejunal, and probably rectal, challenge studies in treated celiac patients. Animal models could contribute to the understanding of GSE pathogenesis. Symptomatic GSE affects approximately 1:1000 individuals in Europe, but this figure is likely to underestimate the real prevalence of the disease. In fact, many cases are asymptomatic, and they may be recognized only by means of large screening programs. Furthermore, it is now becoming clear that a condition of latent gluten sensitivity (preceliac disease) can exist in some apparently normal individuals, who present normal (or almost normal) jejunal histology while eating gluten. In some regions of Europe GSE is still presenting more often in the infantile age with classical gastrointestinal symptoms, but in other countries the clinical presentation is changing, coming closer to the adult type of the disease, and the age of onset of symptoms is shifting upward. Liver, joint, hematological, gynecological, and neurological symptoms are increasingly being recognized. A number of diseases have been found to be associated with GSE; among these are: IgA deficiency, IgA nephropathy, sarcoidosis, insulin‐dependent diabetes mellitus, and a range of other autoimmune diseases. The diagnosis of GSE is based on the finding of severe histological lesion of the jejunum while the patient is on a gluten‐containing diet, and on its disappearance once gluten is excluded from the diet. As far as therapy is concerned, a lifelong, strict gluten‐free diet is mandatory for GSE patients. Among other long‐term problems, an increased risk of intestinal lymphoma has been reported in those patients on a normal or even on a gluten‐poor diet.     

A study on kinetics of beer ageing and development of methods for predicting the time to detection of flavour changes in beer

The kinetics of beer ageing were studied based on the development of beer stale flavour with storage time. Results showed that the beer ageing rates at 50 and 60°C were 30.0 and 56 times as fast as those at room temperature, respectively. Based on these findings, two methods (method A and B) for predicting the ‘time to detection of flavour change’ (TDFC) of beer were developed. TDFC is the beer shelf‐life in terms of flavour stability. In method A, beers were stored in a 50°C water bath and the intensity of beer ageing was scored daily. Thus, the range of TDFC of the beer was acquired according to the maximum number of days within which the intensity of beer ageing was ≤2 and the minimum number of days within which the intensity of beer ageing was >2. In method B, the 2‐thiobarbituric acid (TBA) values of a beer were determined before and after 1 day of storage in a 50°C water bath, and the TDFC of the beer was calculated using the equation: where ΔTBA is the increment of TBA value during 1 day of storage at 50°C. Both methods were simple, rapid and accurate. Copyright © 2015 The Institute of Brewing & Distilling