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

β-Glucans and Pentosans and their Degradation Products in Commercial Beers Makoto Kanauchi1, Wakana Ishikura1 and Charles W. Bamforth2,*ABSTRACTJ. Inst. Brew. 117(1), 120–124, 2011Several commercial beers have been analyzed for their content of β-glucans, pentosans and their degradation products using high performance liquid chromatography, thin layer chromatog-raphy 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, espe-cially 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 glu-cosyl 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 suffi-cient material.Key words: beer, beta-glucan, carbohydrate, oligosaccharides, pentosan.INTRODUCTIONBrewers have long been concerned about the mixed linkage β-glucans deriving from the cell walls of the starchy endosperm of barley because of the problems that they can cause in brewing1,3,20. They have paid rather more attention to these viscous polysaccharides than they have to the arabinoxylans, which are also found in the walls but in lesser quantities1,4. However it is now recognized that these too can be problematic as they have similarly high viscosities and, furthermore, are not degraded as exten-sively as the glucans during malting17. The aim, then, has invariably been one of minimizing the quantity of such molecules in wort and beer. However, when approached from a food perspective, complex cereal polysaccharides are championed for their potential benefits in the human diet7,9,11,19,23. On this basis, might incompletely degraded cell wall polysaccharides emerging through the brewing process represent soluble fibre if present in beer12? Fur-thermore, any β-glucan degradation products larger than glucose are not assimilable by the body and may con-stitute prebiotic material6. Such may also be the case for pentosan degradation products10,16.Much attention has been paid to the enzymic mecha-nisms by which the cell wall components are de-graded5,18,21. However there has been a dearth of studies in which comparison has been made of levels of these mole-cules and their digestion products in different types of beer. This is the topic addressed in the present paper.METHODS AND MATERIALSBeersThe beers were purchased locally (Miyagi) and are de-scribed in Table I. These are individual examples of the beers concerned. All were analyzed in duplicate. Carbohydrate analysis by high performanceliquid chromatography (HPLC)Beers were diluted five-fold with deionised water and filtered (Dismic 13HP, Cat. #13HPO45AN, Millipore Co. Millipore Billerica, MA, USA). Carbohydrates were ana-lyzed by HPLC (Shodex GPC – 101, Showa denko K.K.) using an Asahipak GS-220 HQ column (7.5 mm ID × 300 mm) at 50°C. Samples (10 µL) were eluted by water at a flow rate of 0.6 mL per min with detection by refractive index. Quantification was by measuring peak area in com-parison to reference standards.Carbohydrate retention times were determined using standards in the range 25–1,250 mg/L: D(+)-glucose (Cat. #049-00591, Wako, Wako Pure Chemicals Industries Ltd. Osaka, Japan); D(-)-Arabinose (Cat. #013-04572, Wako); D(+)xylose (Cat. #244-00302, Wako); maltose (Cat. #138-00611, Wako); maltotriose (Cat.#137-06541, Wako); D(+)-cellobiose (Cat. #036-07401, Wako); D(+)-cel-lotriose (Cat. #C1167, Sigma-Aldrich Corporation, St. Louis, MO, USA); cellotetraose (Cat. #400402, Seika-gaku Biobusiness Corporation, Tokyo Japan); cellopen-1 Department of Food Management, Miyagi University, 2-2-1 Hata-tate Taihaku-ku Sendai Miyagi, 982-0215 Japan.2Department of Food Science & Technology, University of Califor-nia, Davis, CA 95616-8598, USA.* Corresponding author. E-mail: cwbamforth@ Publication no. G-2011-0316-1112© 2011 The Institute of Brewing & Distilling Table I. Products investigated.Beer DescriptionA American lager, rice adjunct, low bitternessB American lager, corn syrup adjunct, low bitternessC Japanese dry beerD Japaneseall-maltbeerE IrishstoutF German Oktoberfest beerG Japanese high malt beer120JOURNAL OF THE INSTITUTE OF BREWINGVOL. 117, NO. 1, 2011 121taose (Cat. #400404, Seikagaku); barley β-glucan (Cat. #G6513, Sigma-Aldrich); oat spelt xylan (Cat. #X0627, Sigma-Aldrich). The samples were filtered as above. The retention times for standards are shown in Fig. 1. It was observed that the β-glucan was retained on the column to a significant extent, whereas xylan was eluted in the void volume. This could be because the xylan is of a substan-tially higher molecular weight, or because of interactions between the glucan and the column, leading to delayed elution from the column.Carbohydrate analysis by thin layer chromatography (TLC)Samples (2.5 μL) of beer and standards of D(+)-glu-cose, D(-)-arabinose, D(+)-xylose, maltose, maltotriose, D(+)-cellobiose, D(+)-cellotriose, cellotetraose, cellopen-taose, β-glucan or xylan (each solution being 5.0 mg/mL) were spotted onto a TLC plate (silica gel 70 plate, 5 cm × 10 cm, Cat. #193-08381, Wako). The plate was developed with an ethyl acetate, methanol, acetic acid and watermixture (60:20:15:5) before detection of migration usingFig. 1.Calibration trace for HPLC of carbohydrates.Fig. 2. HPLC analysis of beer carbohydrates.an anisaldehyde reagent spray [26 mL ethanol, 1.5 mL anisaldehyde, 1.5 mL sulphuric acid (98%) and 0.5 mL acetic acid] with heating at 100°C for 5 min in an oven. Assay of total β-glucan and total pentosan Total β-glucan was assayed by the method of Bamforth2. Pentosan was assayed as described by Douglas13.RESULTS AND DISCUSSION The carbohydrate spectrum of a range of beer styles was analyzed by HPLC (Fig. 2). All of the beers appeared to contain substantial levels of residual pentosan, as indi-cated by the large peak eluting essentially in the void vol-ume of the column. [Note that very high molecular weight materials other than pentosan will also elute in the void volume. These would include any unconverted starch.] By contrast the peak for high molecular weight β-glucan was much smaller for each of the beers, consistent with previ-ous claims that there is much more extensive degradation of β-glucan than pentosan in malting and brewing17.There were a number of peaks in the various beers that did not coincide with the standard sugars that were run in the study. These eluted before the sugars and are pre-sumed to be oligosaccharides, likely both α- and β-linked, arising from the degradation of β-glucan, pentosan and starch. Table II displays the levels of oligosaccharides and monosaccharides determined from the HPLC analysis. Although the values for the β-glucan degradation products are all cited on the basis of the calibration performed with cellulosic oligosaccharides, it is possible (though not yet verified) that there may be a contribution from oligosac-charides that include a single β1→3 bond: the primary endo-β-glucanases in malt catalyze the hydrolysis of a β1→4 bond on the reducing side of a β1→3 linkage, thereby primarily producing oligosaccharides with a sin-gle β1→3 linkage on the reducing end and varying num-bers of β1→4 linked glucosyl units25. It is presumed thatthese oligosaccharides migrate on HPLC in a similar mode to the purely cellulosic oligosaccharides with an identical degree of polymerization.Perusal of Fig. 1 and of Tables II and III reveals that for all of the beers tested there was much more β-glucan than pentosan, however the molecular mass of the glucan was much lower. A total β-glucan level of the order of 4–6 g/L would at face value mean that 0.2 L of beer or less would satisfy a dietary fibre claim minimum of >0.75 g per serving24 . However, as reviewed by Wood26, it appears only to be high molecular weight β-glucan that has benefit as fibre. Accordingly, it would seem that it is the arabi-noxylan fraction that represents any soluble fibre present in the beer. Assuming a similar minimum level of 0.75 g to allow claims to be made, then at the levels measured here, beer F would deliver a sufficiency when consumed at approximately a third of a litre, whereas for beer E ap-proximately twice this consumption would be needed.Lower molecular weight carbohydrates were also ex-amined by thin layer chromatography (Fig. 3). For each of the beers, the predominant species was a trisaccharide – either α- or β-linked. There were much smaller quantities of disaccharide and arabinose in each of the beers. This is in accord with the data from the HPLC analysis.The absence of xylose from the beers is indicative of an absence of xylan degradation in malting and mashing (c.f. also the abundance of high molecular weight pento-san suggested in Fig. 2). This may relate to the impact of a xylanase inhibitor15. Free arabinose was detected (Table II) showing that some arabinofuranosidase activity was occurring22.The absence of glucose was consistent with the prefer-ential metabolism of this sugar by brewing yeast. With the exception of Beer B, the maltose levels in the beers exam-ined were within the range generally reported4,8 and were vastly less than what is typically found in wort (typically approximately 60 mg/mL for a wort of 10°Plato) (Table II). Similarly, there are measureable quantities of malto-triose in all but one of the beers, such levels again being comparable with those previously reported4,8 and veryTable II. Lower molecular weight carbohydrates in beer. Data represents the mean of duplicate determinations.Carbohydrate (μg/mL)Beer A Beer B Beer C Beer D Beer E Beer F Beer G Cellopentaose 1,401 (±9) 508 (±28) 1,350 (±160) 701 (±1) 585 (±503) 1,462 (±230) 1,804 (±6)Cellotetraose -a1,904 (±7) - 414 (±0) - 342 (±17) -Cellotriose 4,245 (±147) - 3,305 (±53) - 4,589 (±1,923) - 4,391 (±55)Maltotriose 1,902 (±84) 5,414 (±207) 1,349 (±21) 5,387 (±58) - 5,838 (±53) 2,317 (±162)Cellobiose-813(±406)- - - -109(±0) Maltose 1,124 (±68) 6,795 (±99) 1,509 (±20) 1,946 (±196) 928 (±584) 2,953 (±51) 2,559 (±25)Glucose - - - - - - - Arabinose 105 (±43) 493 (±37) 163 (±96) 444 (±57) 102 (±0) 208 (±13) 413 (±69)Xylose - - - - - - -a Not detected.Table III. Total β-glucan and pentosan in beer. Data represents the mean of duplicate determinations.Carbohydrate (μg/mL)Beer A Beer B Beer C Beer D Beer E Beer F Beer Gβ-Glucan 4,109 (±26) 6,042 (±155) 4,099 (±121) 5,369 (±80) 4,182 (±20) 4,011 (±46) 5,081 (±203)Pentosan 882 (±133) 1,115 (±172) 1,004 (±70) 1,805 (±116) 866 (±107) 1,788 (±165) 1,360 (±85)122JOURNAL OF THE INSTITUTE OF BREWINGVOL. 117, NO. 1, 2011 123much less than in conventional worts (approx. 20 mg/mL for wort of 10°Plato).Several factors will impinge on the spectrum of carbo-hydrates found in beer. These will include inter alia grist materials (the malt and its degree of modification, ad-juncts), brewhouse conditions and fermentation condi-tions. Accordingly, it is not possible to simply interpret from the data generated here the mode of production of the beers, which is of course commercially sensitive. It might additionally be noted that the history of the beers, being purchased off the shelf as they were, is unknown and so no consideration of any changes that may have occurred in the beer during storage can be made. However gross changes in the levels of the carbohydrates would be unexpected, especially as there was no visible evidence in the beers of any biological or non-biological breakdown. Furthermore, it must be emphasized that we were not at-tempting to make any definitive comparisons between the beers tested for their absolute content of the various car-bohydrates, recognizing that there may be batch-to-batch variations in the precise levels. Simply, we observed broad similarities in composition from beer to beer in terms of the nature of the carbohydrates that were present.CONCLUSIONSBeer contains high levels of residual high molecular weight pentosan, but much less high molecular weight β-glucan, consistent with the latter being the more exten-sively degraded component from the starchy endosperm cell walls of barley. Beer also contains sizeable levels of oligosaccharides, especially trisaccharides, reflecting theincomplete 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 mo-lecular 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 appears that less than a litre of the product would afford sufficient material.REFERENCES1. 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