Tracking polysaccharides through the brewing process

Brewing is a highly complex stepwise process that starts with a mashing step during which starch is gelatinized and converted into oligo and/or monosaccharides by enzymes and heat. Previous studies have mostly been restricted to analysing the grain and/or malt prior to entering the brewing process, but here we track the fates of polysaccharides during the entire brewing process.

Carbohydrate Polymers 196 (2018) 465–473 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Tracking polysaccharides through the brewing process a a c d Jonatan U Fangel , Jens Eiken , Aafje Sierksma , Henk A Schols , William G.T Willats ⁎ Jesper Harholta, b,⁎ T , a Carlsberg Research Laboratory, J.C Jacobsens Gade 4, DK-1799, Copenhagen V, Denmark School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK The Dutch Beer Institute, Lawickse Allee 11, 6701 AN, Wageningen, Netherlands d Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, 6708WG, Wageningen, Netherlands b c A R T I C LE I N FO A B S T R A C T Keywords: Polysaccharides Glycan arrays Enzymes Beer Malt Wort Brewing is a highly complex stepwise process that starts with a mashing step during which starch is gelatinized and converted into oligo- and/or monosaccharides by enzymes and heat The starch is mostly degraded and utilised during the fermentation process, but grains and hops both contain additional soluble and insoluble complex polysaccharides within their cell walls that persist and can have beneficial or detrimental effects on the brewing process Previous studies have mostly been restricted to analysing the grain and/or malt prior to entering the brewing process, but here we track the fates of polysaccharides during the entire brewing process To this, we utilised a novel approach based on carbohydrate microarray technology We demonstrate the successful application of this technology to brewing science and show how it can be utilised to obtain an unprecedented level of knowledge about the underlying molecular mechanisms at work Introduction Beer is the most popular alcoholic beverage and the third-most consumed beverage in general after water and tea (Barth-Hass-Group, 2017) Despite of being a 6000–8000 years old practice (Nelson, 2005), the high complexity of beer still proves a significant challenge when identifying and quantifying the most important beer components Consequently, the brewing industry is constantly challenged to optimise practises in relation to product quality and cost effectiveness To produce beer, a stepwise batch process is most commonly used The malted barley is milled, mixed with water (called ‘mash’) and heated up (mashed) to release fermentable sugars and degrade the barley cell wall (Gupta, Abu-Ghannam, & Gallaghar, 2010) The mash is separated into liquid, named ‘wort’ and the spent grains The wort is then boiled and hops are added Afterwards the solids, referred to as the ‘trub’, are removed, and the wort is cooled, transferred to a fermentation tank and yeast is added Following fermentation, the yeast is removed and beer is left to mature before being filtered and bottled Besides starch, many other complex polysaccharides are found in the grain In the case of barley, the cell walls consist of approximately 70% β-(1- > 3)(1- > 4)-glucan (referred to as β-glucan in brewing literature), 25% arabinoxylan, 2% cellulose, 2% mannan and arabinogalactan proteins (AGP) (Fincher, 1975; Lazaridou, Chornick, Biliaderis, & ⁎ Izydorczyk, 2008) Additionally, high molecular pectins and AGPs are present in hops (Oosterveld, 2002) Part of these polysaccharides may end up in the beer as such or as fragment after enzymatic degradation during the brewing process One of the main challenges related to polysaccharides in brewing is filtration, with high molecular weight β-(1- > 3)(1- > 4)-glucans as the main culprit (Kumar, Kumar, Verma, Kharub, & Sharma, 2013) However, studies have also shown a negative correlation between filtration efficiency and high molecular weight water extractable arabinoxylans (Lu, Li, Gu, & Mao, 2005; Stewart et al., 1998) Besides these two polymers, mannans have also been linked to filtration issues within the food industry more widely In coffee, they are known to effect viscosity (Sachslehner, 2000) as well as initiate gel formation and can even create crystalline structures similar to cellulose (Millane & Hendrixson, 1994) Although not yet reported, mannans may also affect the brewing process Colloidal instability is another production issue partly related to polysaccharide content, and one of the most important quality criteria of beer related to long-term stability Most common is chill haze, which usually dissolves at higher temperatures, but may also result in covalently bound irreversible haze complexes (Steiner, Becker, & Gastl, 2010) The role of polysaccharides in haze formation is not well understood, but studies have shown both positive and negative effects of Corresponding authors E-mail addresses: Jonatan.Ulrik.Fangel@carlsberg.com (J.U Fangel), jens.eiken@carlsberg.com (J Eiken), Sierksma@kennisinstituutbier.nl (A Sierksma), Henk.Schols@wur.nl (H.A Schols), William.Willats@newcastle.ac.uk (W.G.T Willats), Jesper.Harholt@Carlsberg.com (J Harholt) https://doi.org/10.1016/j.carbpol.2018.05.053 Received 29 January 2018; Received in revised form 16 May 2018; Accepted 16 May 2018 Available online 22 May 2018 0144-8617/ © 2018 Published by Elsevier Ltd Carbohydrate Polymers 196 (2018) 465–473 J.U Fangel et al Fig A diagram illustrating the brewing process and the steps where samples are collected Modified after Die deutschen Brauer Deutscher Brauer-Bund e.V (Die deutschen Brauer and Deutscher Brauer-Bund, 2018) B shows the results of the carbohydrate analysis The heatmap presents two separate analysis, one for solid (green) and one for liquid (orange) samples The highest value in each heatmap have been set to a 100 and the remaining values adjusted accordingly A cut-off of five have been introduced CDTA: 50 mM diamino-cyclo-hexane-tetra-acetic acid, pH 7.5 NaOH: ml M NaOH with 0.1% v/v NaBH4 Homogalacturonan (HG), Arabinogalactan protein (AGP) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) polysaccharides on haze formation (Siebert, Carrasco, & Lynn, 1996) A second type of beer particles, known as ‘stringy floaters’, can be problematic within specific market sectors These particles consist of protein-carbohydrate complexes characterised by a high content of cysteine residues, with the carbohydrate constituting 30–50% of the complex (Vaag, Riis, & Outtrup, 2003) Polysaccharide-related 466 Carbohydrate Polymers 196 (2018) 465–473 J.U Fangel et al Materials and methods problems can be dealt with to some extent through alteration of the mashing regime, use of enzyme-rich raw materials and/or addition of external enzymes (Steiner, Auer, Becker, & Gastl, 2012) Consequently, small adjustments to the enzyme combinations could lead to major improvements on the aforementioned challenges – but a greater understanding of the polysaccharide content and transformation during brewing is a prerequisite for achieving this One of the newer techniques for plant cell wall polysaccharide analysis is based on plant carbohydrate microarrays, enabling the analysis of hundreds or thousands of molecular interactions simultaneously using very small amounts of analytes (Moller et al., 2007; Pedersen et al., 2012) The method provides information on the occurrence of glyco-epitopes, rather than simply monosaccharide profiles This is made possible through the use of monoclonal antibodies (mAbs) selected to be specific to small (2–12 monosaccharide) motives (‘epitopes’) in polysaccharides The technique is already well established within the plant cell wall community and have seen utilization within wine research, but has yet to be utilised for brewing science (Gao, Fangel, Willats, Vivier, & Moore, 2015; Gao et al., 2016) Although carbohydrates in beer mostly are investigated in relation to sensory or technological properties, a nutritional evaluation could also be valuable for these compounds Dietary fibre is routinely determined in non-liquid plant-based foods, but not in liquids like beer − probably because the official methods for analysis are not readily applicable to beverages Consequently, food composition tables often report zero dietary fibre content in beverages However, drinks are quantitatively important items in any diet and they may contain measurable amounts of soluble dietary fibre (SDF) According to a Spanish study (Diaz-Rubio & Saura-Calixto, 2011), coffee and cocoa contain the highest levels of SDF (around g/L) of all beverages tested Beer contains between and 3.5 g/L SDF (depending on the type of beer), which is comparable to fruit juices from apple, orange and peach The amount of SDF in wine depends on the type of wine making; red wine contains around g/L SDF whereas white wine only 0.2 g/L (Diaz-Rubio & Saura-Calixto, 2011) To put these amounts of fibre in beverages in perspective: according to the European Food Safety Authority a food is regarded as a source of fibre if it contains ≥ g/100 g or ≥1.5 g/100 kcal and is deemed high in fibre if it contains ≥6 g/100 g or ≥3 g/100 kcal On average, fibre intake for adult males in Europe range from 18 to 24 g/d and for females from 16 to 20 g/d, with little variation from country to country Recommendations for fibre intake for adults for most European countries are in the order of 30–35 g/d for men and 25–32 g/d for women (Stephen et al., 2017) Since fibre intake in Europe is below recommended levels, it is important to report that beverages can contribute to the fibre intake For example, in Spain, if beverages are taken into account total fibre intake increases by 10.4% A moderate consumption of beer of 500 ml per day could imply an intake of about g SDF (Diaz-Rubio & Saura-Calixto, 2011) Not only the total amount of fibre is relevant, but also the composition and physiological and nutritional properties Beer is made of barley and therefore contains mainly water soluble arabinoxylans and β-(1- > 3)(1- > 4)-glucans – with β-(1- > 3)(1- > 4)-glucans been shown to lower/reduce blood cholesterol In this study, we demonstrate the implementation of novel high-throughput technology for tracking in detail the fate of polysaccharides throughout the brewing process – from grain to bottled product This allows the brewer to infer the occurrence of polysaccharides and the information indicates to the brewer which enzymes will depolymerize this polysaccharide in the cases where adjustment is required In addition, we compare the microarray-based technique with known quantitative methods for β-(1- > 3)(1- > 4)-glucans to compare its potential for identifying beverages of particular nutritional interest 2.1 Brewing samples Samples were obtained from the Carlsberg Research Laboratory pilot brewery (Carlsberg, Copenhagen, DK) A lager was brewed using 70% malt and 30% barley Mashing was started at 52 °C for 20 min, increased to 65 °C over 13 and held for 60 before increasing to 78 °C over 13 and held for The wort was subsequently boiled for 60 with addition of standard bitter hops Saccharomyces Cerevisiae was used for fermentation over days at 15 °C Samples of 15 ml were collected from 12 steps illustrated in Fig 1A Samples containing both liquid and solids were separated by centrifugation at 4.000g for 10 All solid samples were lyophilised and grinded in a Retsch mixer mill (Retsch GmbH, Hann, Germany) to a fine powder 2.2 Comprehensive microarray polymer profiling (CoMPP) All solid samples were analysed in triplicates of 20 mg material, sequentially extracted with ml 50 mM diamino-cyclo-hexane-tetraacetic acid, pH 7.5 (CDTA) and ml M NaOH with 0.1% v/v NaBH4 (NaOH) Each extraction was carried out in ml eppendorf tubes at 1000 rpm for h followed by 10 centrifugation at 10.000g and the supernatant collected The liquid samples required no pre-treatment and all samples were centrifuged at 10.000g before mixed with arrayjet printing buffer (55.2% glycerol, 44% water, 0.8% Triton X-100) and spotted unto a nitrocellulose membrane, pore size of 0.45 μm (Whatman, Maidstone, UK) using an Arrayjet Sprint (Arrayjey, Roslin, UK) Each sample was printed with four technical replicates and dilutions and probed as described in Pedersen et al (2012) See Table for further information on monoclonal antibodies and please also refer to Rydahl, Hansen, Kračun, and Mravec (2018) The arrays were Table Overview of monoclonal antibodies used throughout this study highlighting epitope specificity and reference code Homogalacturonan (HG), Arabinogalactan protein (AGP) Specificity Code Reference HG partially/de-esterified JIM5 HG partially esterified JIM7 HG partially/de-esterified HG partially/de-esterified Backbone of rhamnogalacturonan I Backbone of rhamnogalacturonan I (1 → 4)-β-D-galactan (1 → 5)-α-L-arabinan Linearised (1 → 5)-α-L-arabinan LM18 LM19 INRA-RU1 Clausen et al (2003), Verhertbruggen et al (2009) Clausen et al (2003), Verhertbruggen et al (2009) Verhertbruggen et al (2009) Verhertbruggen et al (2009) Ralet et al (2010) INRA-RU2 Ralet et al (2010) LM5 LM6 LM13 BS-400-4 LM21 Jones et al (1997) Willats et al (1998) Moller et al (2007), Verhertbruggen et al (2009) Pettolino et al (2001) Marcus et al (2010) LM22 BS-400-2 BS-400-3 LM15 LM25 Marcus et al (2010) Meikle et al (1991) Meikle et al (1994) Marcus et al (2008) Pedersen et al (2012) LM10 LM11 LM27 LM28 JIM20 JIM13 LM2 McCartney et al (2005) McCartney et al (2005) Cornuault et al (2015) Cornuault et al (2015) Smallwood et al (1994) Yates et al (1996) Smalwood, Yates, Willats, Martin, and Knox (1996) (1 → 4)-β-D-(galacto)mannan (1 → 4)-β-D-(galacto)(gluco) mannan (1 → 4)-β-D-(gluco)mannan (1 → 3)-β-D-glucan (1 → 3)(1 → 4)-β-D-glucan Xyloglucan (XXXG motif) Xyloglucan/unsubstituted β-Dglucan (1 → 4)-β-D-xylan (1 → 4)-β-D-xylan/arabinoxylan Grass xylan preperations Glucuronoxylan Extensin AGP AGP, β-linked GlcA 467 Carbohydrate Polymers 196 (2018) 465–473 J.U Fangel et al scanned using a flatbed scanner (CanoScan 9000 Mark II, Canon, Søborg, Denmark) at 2400 dpi and quantified using Array-Pro Analyzer 6,3 (Media Cybernetics, Rockville, MD, USA) For each sample, an average was calculated based on the three sample replicates, the four technical replicates and the four dilutions resulting in 48 measurements per signal value For multivariate data analysis, the raw value was used and for heatmaps, the highest value was set to a 100 and the rest adjusted accordingly For all heatmaps a cut-off five have been introduced presented in Fig 1B It is important to note that the heatmap values not represent absolute levels of polysaccharides, but the relative abundance of each epitope across the sample set Pre-germination, the grain (1) shows a glycan profile with the main polysaccharides being mannan (mAb BS-400-4), β-(1- > 3)(1- > 4)-glucan (mAb BS-400-3) and arabinoxylan (mAb LM11) which is in accordance with previous reports (Lazaridou et al., 2008) After germination, the malt sample (2) showed no change for arabinoxylan (mAb LM11), but reduction for mannan (mAb BS-400-4) and β-(1- > 3)(1- > 4)-glucan (mAb BS-4003), which could indicate a function as storage polymers utilised during germination This reduction in β-(1- > 3)(1- > 4)-glucan content during malting is a direct quality parameter in malting varieties of barley, facilitated by expression of glucanases and under redox control, showing the potential of also using CoMPP in breeding efforts (Betts et al., 2017; Singh et al., 2017) For both grain and malt, the NaOH extract contained the highest amounts with particularly arabinoxylan (mAb LM11) Additionally, a small signal for the presence of rhamnogalacturonan-I was detected in the grain (RG-I) (mAb INRA-RU1) with increased detection in the malt, together with galactan (mAb LM5) and arabinan (mAb LM6) known to form sidechains on the RG-I backbone (Buffetto et al., 2015) With the addition of water to the malt in the mashing tun (3) the sample now constitutes a kind of porridge and a solid and liquid sample were taken at the three temperature steps during mashing For the liquid sample, the prime difference is observed between 52 and 65 °C Here feruloyate groups (mAb LM12) and β-(1> 3)(1- > 4)-glucan (mAb BS-400-3) increased, while arabinoxylan (LM11) and AGP (mAb JIM13) were reduced which is consistent with βglucanases, xylanases and proteases being activated during these processing steps (Vivian, Aoyagui, de Oliveira, & Catharino, 2016) Most notable in the CDTA extraction was the increased detection of homogalacturonan (HG) (mAb JIM5, JIM7, LM18) not observed in malt This suggests the occurrence and activation of pectinases during the mashing and a pectin structure with HG with intermediate methyl esterification as well as egg box domains together with RG-I domains Although traces of uronic acids have been found in barley grains (Englyst, 1989) this is to the authors knowledge, the first description of complex pectin structures in barley grains Following the mash tun the wort was transferred to the lauter tun to separate the husks and non-converted material from the liquid The first liquid to enter the wort kettle is referred to as the first wort Extra water (sparging liquor) was added to wash the remaining sugar out of the milled cereals and is referred to as the weak wort The first wort (4) showed a very similar profile to the mash at 78 °C in the mash tun with the exception of mannan and β-(1> 3)(1- > 4)-glucan being greatly increased This is an effect of the grains used as a natural filter to separate the liquid and an increased extraction takes place The weak wort showed an identical, but diluted profile as would be expected The excess material, spent grains (5), were very similar in glycan profile to that of the solid 78 °C samples from the lauter tun though all signals were increased In the wort kettle (6) the first and weak wort have been mixed and a diluted glycan profile is observed with the same relative amount of polysaccharides as in the lauter tun The addition of hops and subsequent boil did not affect the glycan profile with the exception of reduction in HG (mAb JIM7) and arabinan (mAb LM6) likely due to thermal degradation of corresponding epitopes (Fraeye et al., 2007) The transfer to the whirlpool (7), and separation of the trub (8), yielded no change to the liquid glycomic profile The trub showed small traces of arabinan (mAb LM13), feruloyate groups (mAb LM12) and β-(1- > 3)(1- > 4)-glucan (mAb BS-400-3) probably originating for the liquid trapped in the trub which predominately consists of high molar weight protein (Mathias, Alexandre, Cammarota, de Mello, & Servulo, 2015) Yeast (9) used for fermentation has a cell wall mainly constructed of β-(1- > 3)-glucans and mannoproteins Β-(1- > 3)-glucan was clearly detected (BS-400-2) while mannan in the proteoglycans are α linked and not detectable by plant cell wall antibodies (Lipke & Ovalle, 1998) The addition of yeast showed no alteration of the glycomic profile as seen at the start of 2.3 Multivariate data analysis Patterns within certain microarray datasets were investigated with principal component analysis (PCA) using SIMCA 12 (MKS Data Analytics Solutions, Umeå, Sweden) The data were prior to PCA modelling scaled to unit variance (UV) 2.4 β-(1- > 3)(1- > 4)-glucan samples Samples were prepared in triplicates from five commercially available beers having varying β-(1- > 3)(1- > 4)-glucan amount The samples were analysed using the β-Glucan Assay Kit (Megazyme International, Ireland) following preparation procedure C and E which differs in the isolation of polysaccharides before enzymatic degradation For procedure C, 300 μl of beer is boiled for and 300 μl 95% ethanol is added and vortexed Additional 500 μl of 95% ethanol is added and the solution is centrifuged for 10 at 1800g The supernatant is discarded, and the pellet is suspended in 800 μl 50% ethanol and vortexed before centrifugation for 10 at 1800g and supernatant discarded For procedure E 500 ul of beer was de-gassed by heating to approx 80 °C in a boiling water bath 250 mg of finely milled ammonium sulphate crystals was added and dissolved before incubation for approx 20 h at °C The sample was centrifuged at 1000g for 10 and the supernatant discarded The pellet was washed twice by dissolving the pellet in 100 ul of 50% ethanol followed by additional ml of 50% ethanol and centrifuged for at 1000g A variation of procedure C was also carried out with containing a single 70% precipitation step centrifuged at 10.000g for 10 and the supernatant discarded and will be referred to as C* All three pellets were then dissolved in sodium phosphate buffer (20 mM, pH 6.5) and subjected to lichenase and glucanase treatment as described in the Megazyme protocol For each procedure, a sample was taken prior to the addition βglucosidase for oligosaccharide fragment analysis 2.5 High-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) Glc-β-(1- > 4)-Glc-β-(1- > 3)-Glc (DP3) and Glc-β-(1- > 4)-Glc-β(1- > 4)-Glc-β-(1- > 3)-Glc (DP4) oligomers were quantified with HPAEC-PAD using a Dionex ICS 5000+ DC system equipped with a μm SA-10 column with × 250 mm dimensions and a guard column Run conditions were 0.4 ml/min, column temperature 40 °C, isocratic 100 mM NaOH eluent for 15 Standard for quantification were produced by U/ml lichenase (Megazyme International, Ireland) digestion of known quantities of water soluble medium viscosity β-glucan (Megazyme International, Ireland) in 50 mM MES pH 6.5 assuming an equal molecular PAD response ratio between DP3 and DP4 Results and discussion 3.1 Carbohydrate microarrays To illustrate the capability of the experimental procedure during the entire brewing process it was divided into 12 key steps as seen on Fig 1A Liquid and/or solid material were acquired from each step and analysed with CoMPP The polysaccharide profiles of each step are 468 Carbohydrate Polymers 196 (2018) 465–473 J.U Fangel et al Fig Carbohydrate microarray analysis of 60 commercial beer samples The heatmap shows the relative abundance of cell wall related epitopes with the highest value set to a 100 and the rest adjusted accordingly A cut-off of five have been introduced Arabinogalactan protein (AGP) 469 Carbohydrate Polymers 196 (2018) 465–473 J.U Fangel et al fermentation (10) After end fermentation, subtle changes were observed for most antibodies, mostly attributable the rising alcohol percentage changing the solubility of the polymers in the liquids The following filtration (11) and study of filter content showed yeast to be the dominant remaining component since the analysis yields were identical to that of pure yeast samples The final beer (12) was diluted with water to reach the desired alcohol percentage giving the same profile as observed for the finalised fermentation although reduced The only polysaccharide not following this pattern was β-(1- > 3)(1- > 4)-glucan, which have slightly increased This is most likely due to increased solubility as the alcohol percentage were decreased the other by pectin and glycoprotein Interestingly, the mannan, RG-I, and glycoprotein mAbs are located in the same groups, while the xylan mAbs are divided between them with mAb LM10 + LM11 and mAb LM27 + LM28 separated This indicates a separation based on xylan substructures linked to the specific polysaccharide fingerprint of the beer with mAb LM27 + LM28 characteristic for barley and mAb LM10 + LM11 for wheat Comparing the six categories the alcohol free beers and to some extent the bottom-fermented beers under 6% of alcohol seems to be lower in fibres compared to the other four Additionally, they seem to be characterised by the two mannan antibodies (mAb LM21 and BS-400-4) The bottom-fermented beers over 6% together with top fermented beers show no obvious difference between them and contains a general mixture of all the polysaccharides detected As seen before the wheat beers are separated on the presence of RG-I domains as well as the presence of arabinoxylan In conclusion, our work has shown that carbohydrate microarray technology is a powerful addition to the analytical toolbox for the detailed analysis of polysaccharides during the brewing process, including end products The methods is highly sensitive, high-throughput and requires none to very little sample preparation Microarray are especially valuable for rapidly obtaining semi-quantitative glycan profiles across large numbers of samples and for informing subsequent more quantitative analysis of sub-sets of those samples by slower more conventional biochemical techniques 3.2 Beer analysis In addition to tracking polysaccharides over a single brewing process, we also analysed polysaccharide profiles in a variety of commercial beers and interpreted these data in the context of brewing style and specific conditions Fig shows the analysis of 60 store bought beers, grouped into six categories based on alcohol percentage, top-fermenting or bottom-fermenting yeast, and beers classified as wheat beers Overall, the same polysaccharides observed in the final sample of our brewed beer (Fig 1B) are detected again However it is noteworthy that while most polysaccharides are present in all samples in varying amounts, mannan (mAb LM21, BS-400-4), β-(1- > 3)(1- > 4)-glucan (mAb BS-400-3) and arabinoxylan (mAb LM11) are not detected in several beers While these three polysaccharides showed no obvious pattern across the defined categories, the RG-I signal yielded higher signal strength in the wheat beers compared to the rest of the data set As stated above, we report here the presence of complex pectin structures in barley, and pectic polymers have been reported in wheat grains recently (Chateigner-Boutin et al., 2014) Although both barley and wheat contain pectin, this analysis indicates either a higher amount in the wheat kernels or a different structure leaving the pectin more prone to water extraction compared to that of barley It is possible that the extra RG-I could come from citrus fruit peel, often added to wheat beers, but since the sample set contains wheat beers both with and without this addition this seems unlikely In any event, an increased RG-I signal seem to be a consistent characteristic of the polysaccharide fingerprints of wheat beers It should be noted that sample which is the only sample with high RG-I detection not located in the wheat category, is the alcohol free version of sample 55 and would have been placed next to it if only based on its polysaccharide fingerprint and is indeed a wheat beer The detection of pectin in most samples also highlight the sensitivity of the analysis as it only constitutes minor amount of the total complex polysaccharide amount in grains The substantially reduced relative abundance of mannan and β-(1- > 3)(1> 4)-glucan in some beers most likely reflects the alternation of mashing profiles, use of enzyme rich raw materials or the addition of external enzymes Addition of enzymes during mashing is a well know practice when brewing with un-malted adjuncts and looking at the grain information for samples showing little to no detection of mannan and β-(1- > 3)(1- > 4)-glucan (sample 6, 7, 8, 9, 12, 28, 37, 51, 54 and 55) an overrepresentation of beers with un-malted adjuncts are observed This also explains why the pattern is not related to any beer style but to the brewing method in general In an attempt to reveal underlying patterns in more detail, the microarray raw data was subjected to principal component analysis (PCA) (Fig 3) Looking at the score plot the wheat group is clearly distinguishable from the other samples, but while any difference was very difficult to see in the heatmap between the remaining categories, some separation became apparent The samples are in general spread out based on fibre richness in the liquids indicated by the two arrows with PC1 accounting for 35,5% of the variance and PC2 21,4% As shown by the loading plot the fibres detected seems to cluster together in two groups One dominated by mannan and β-(1- > 3)(1- > 4)-glucan and 3.3 β-(1- > 3)(1- > 4)-glucan quantification β-(1- > 3)(1- > 4)-glucan in beer was quantitatively investigated with several methods and sample preparations in order to assess the effectiveness and precision of the methods and compare them to the carbohydrate microarrays High-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was used as well as the commercially available ‘β-Glucan Assay Kit (Mixed Linkage)’ Five beers with varying amounts of β-(1- > 3)(1- > 4)glucan based on carbohydrate profiling were analysed using three different sample preparations all taken from the β-Glucan Assay Kit (Mixed Linkage) protocol Fig shows the comparison of the two detection methods with three preparations for each of the five beers tested The megazyme kit has a procedure for liquids through ethanol precipitation (C) and a procedure for beer approved by the European brewing convention (E) In addition a simple 70% ethanol precipitation, commonly used to precipitate polysaccharides, were used as well (C*) Since the Megazyme kit utilises a glucose oxidase/peroxidase reagent, HPAEC-PAD was used as well to analyse the fragments This method is able to distinguish oligosaccharides from β-(1- > 3)(1- > 4)-glucan and β-(1> 4)-glucan and can reveal the precision of the three preparation methods C*, C and E Overall, the Megazyme kit, yields a higher or equal signal to that of HPAEC-PAD for all samples The E samples gives the most comparable result between the two analysis methods while C* samples yields the most diverse results However it is the only method able to detect β-(1> 3)(1- > 4)-glucan in samples 37 and containing the lowest β-(1> 3)(1- > 4)-glucan amounts Since the megazyme kit detects glucose of any form, fragments from glucose containing polysaccharides such as starch cellulose and xyloglucan can interfere with the final result if present in the sample Since the HPAEC-PAD can separate the fragments we can measure the presence of β-glucans nor originating from β-(1- > 3)(1- > 4)-glucan of one to five sugars (Table 2) Looking at the maltooligosaccharides present in the samples it is possible to determine how effective the three sample preparations were at removing these Amounts between 157 and 389 mg/L are detected in C* samples where the C samples only contains 12–42 mg/L and Megazyme E less than mg/L This suggests that glucose from sources other than β-(1- > 3)(1- > 4)-glucan may be detected in the Megazyme assay, increasing the final result explaining 470 Carbohydrate Polymers 196 (2018) 465–473 J.U Fangel et al Fig PCA score plot (A) and loading plot (B) of the raw carbohydrate microarray values from 60 commercial beers The colour is according to beer categories in the score plot and polysaccharide categories in the loading plot Principal component (PC), Rhamnogalacturonan I (RG-I), Top or bottom fermented (Top/Bottom) 6% refers to beverage alcohol level Conclusion the increased amount seen in Fig In contrast the Megazyme E procedure appears to be more effective at eliminating maltooligosaccharides, yielding a result close to that of HPAEC-PAD using the same procedure for beer 17 and 60, however little to no detection was observed in beer 23, 37 and suggesting that the increased purification induces a loss of β-(1- > 3)(1- > 4)-glucan Conclusively the C* preparation together with HPAEC-PAD is the fastest preparation protocol and most precise detection method In order to compare the quantitative numbers with the semi-quantitative data from the carbohydrate microarrays, the highest number in both datasets (beer 17) was set to the same amount and the rest adjusted accordingly (Fig 5) Beer 60 is measured to the same level for both assays while 23, 37 and are below the HPAEC-PAD numbers The relative differences between the samples are however correct and the method is thus suitable to rank the beers on β-(1- > 3)(1- > 4)-glucan content To get a precise measurement on β-(1- > 3)(1- > 4)-glucan a second approach has to be utilised In relation to nutritional value this is the first study estimating the amount of β-(1- > 3)(1- > 4)-glucan in beer, measuring values up to 200 mg/L Despite one of the ingredients of beer is a source of β-(1> 3)(1- > 4)-glucan (barley), probably filtration (or possibly the use of enzymes) during the brewing process significantly reduces the β-glucan levels in the beverage (Larsen, Wichmann, Sørensen, & Miller, 2015) Carbohydrate microarrays were established as a novel highthroughput method for polysaccharide fingerprinting during all stages of the brewing process for both the solid and liquid fractions Liquids in particular are rapid to analyse, as no sample preparation is required since brewing is already an extraction procedure of polysaccharides in itself The technique is an attractive addition to the brewing analysis toolbox and offers a great level of detail compared to established methods Besides giving valuable input to understand polysaccharide related issues regarding filtration and colloidal instability, the results can potentially give information regarding enzyme and grain usages in a given beer as this seems to be revealed on the polysaccharide profiles of the 60 shelf beer analysis This information can prove very valuable on how to optimise the addition of enzymes during mashing resulting in a more cost effective brewing with increased long-term stability Furthermore, the analysis can rank the beers according to their levels of dietary fibre matching that of established methods resulting of the selection of samples with potential of higher nutritional value Conflict of interest Aafje Sierksma was employed by the Dutch Beer Institute during the Fig Levels of β-(1- > 3)(1- > 4)-glucan as analysed with HPAEC-PAD and “β-Glucan Assay Kit (Mixed Linkage)” using three different sample preparations C*, C and E High-performance anion exchange chromatography (HPAEC) detect oligomers using pulsed amperometric detection (PAD) detector while the β-Glucan Assay Kit converts all mixed link glucan specifically to glucose which is determined by an enzymatic colour assay using glucose oxidase plus peroxidase and 4aminoantipyrine (GOPOD) 471 Carbohydrate Polymers 196 (2018) 465–473 J.U Fangel et al Table Glucose and maltodextrin levels (mg/L) in five different beers across three different preparation protocols as measured by HPAEC-PAD All numbers are mg/L Glucose Maltose (mg/ml) C* C E Beer Beer Beer Beer Beer Beer Beer Beer Beer Beer Beer Beer Beer Beer Beer 17 60 23 37 17 60 23 37 17 60 23 37 69,3 67,7 74,8 70,7 67,9 1,9 3,0 1,7 1,9 1,4 0,0 0,0 0,0 0,0 0,0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Maltotriose (mg/ml) 3,3 2,9 15,1 11,3 27,3 0,8 2,0 0,7 1,5 0,4 0,0 0,0 0,0 0,0 0,0 24,1 14,6 9,1 13,1 7,4 8,0 4,6 1,6 0,7 0,7 0,0 0,0 0,0 0,0 0,0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Maltotetraose (mg/ml) 1,2 2,0 0,3 2,3 1,6 0,8 0,5 0,5 0,3 0,2 0,0 0,0 0,0 0,0 0,0 28,1 8,9 13,5 8,1 15,5 2,9 4,6 1,7 4,3 3,8 0,0 0,0 0,0 0,0 0,0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Maltopentose (mg/ml) 20,2 10,9 5,9 3,7 3,9 0,8 1,6 0,5 2,3 1,0 0,0 0,0 0,0 0,0 0,0 58,7 20,3 44,0 214,2 30,8 4,2 4,4 2,4 24,9 3,3 0,3 0,3 0,2 0,7 0,2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Total (mg/ml) 10,4 8,4 5,9 18,0 6,8 0,6 1,2 0,3 3,7 0,7 0,1 0,0 0,0 0,3 0,0 87,4 104,6 132,7 83,3 35,2 14,7 12,4 8,4 10,3 2,5 1,3 0,8 0,0 0,4 0,0 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± (mg/ml) 4,2 10,2 6,1 7,5 9,0 1,5 1,7 2,6 0,9 1,2 0,2 0,2 0,0 0,2 0,0 267,5 216,1 274,1 389,3 156,7 31,8 29,1 15,8 42,2 11,6 1,6 1,1 0,2 1,2 0,2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 25,8 28,0 7,5 21,2 39,7 3,9 5,9 3,6 8,7 2,3 0,2 0,2 0,0 0,4 0,0 S0008-6215(03)00272-6 Cornuault, V., Buffetto, F., Rydahl, M G., Marcus, S E., Torode, T A., Xue, J., Knox, J P (2015) Monoclonal antibodies indicate low-abundance links between heteroxylan and other glycans 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novel high-throughput technology for tracking in detail the fate of polysaccharides throughout the brewing process – from grain to bottled product This allows the brewer... adjusted to the same level as the HPAEC-PAD result and the rest adjusted accordingly study and during writing the manuscript She had no role in the design of the study, the analyses, or in the decision... addition to tracking polysaccharides over a single brewing process, we also analysed polysaccharide profiles in a variety of commercial beers and interpreted these data in the context of brewing style