PROJECT RESULTS

Identification of Biochemical and Functional Properties of β-Glucans and Arabinoxylans in Barley and Malt that Cause Processing Problems During Malting and Brewing

Drs. Marta S. Izydorczyk and Alexander W. MacGregor

Grain Research Laboratory

Canadian Grain Commission

Winnipeg, Manitoba  R3C 3G8  Canada

Table of Contents:

• Background and Objective
• Procedure and Project Activities
• Results and Discussion
• Conclusions

ARDI Project:

#98-187

Project Status:

Background and Objective:

In terms of production, barley is the second-ranked cereal in Canada with an average annual production of 11.1 million tonnes on the prairies (1.5 million tonnes in Manitoba).  Since the early 1990's the proportion of total barley production used for malting has increased from 10-19 percent; although the domestic use has remained static at about 4%.  Increased exports of both malt and malting barley has accounted for the increased use of barley for malting purposes.  However, the export markets for both malt and malting barley is fiercely competitive and some of our competitors (e.g., Australia) are making strenuous efforts to increase the quality of their malting barley so as to increase their share of the export market.  We must identify, address, and rectify weak quality parameters in Canadian barley if it is to compete successfully in export markets in the future.  β-Glucans and arabinoxylans are high molecular weight components of barley that cause processing and quality problems in brewing if they are not adequately broken down to harmless products during malting.

Malting conditions that can achieve an acceptable breakdown of these components also tend to produce unacceptably high levels of soluble protein in the malt because the hot, dry growing conditions on the prairies tend to produce barley having high levels of protein.  The maltster has a difficult, and sometimes impossible, compromise to make between necessary extensive degradation of β-glucans and arabinoxylans and controlled, limited protein degradation.  One approach to this problem is to identify the structural features of these non-starch polysaccharides that cause processing problems and then devise strategies to ensure that these structures in particular are preferentially degraded during malting. 

The main objective of the proposed research was to increase the malting quality of Canadian barley through increased understanding of the structure and functionality of barley components, more specifically the non-starch polysaccharides.  These polysaccharides in barley - including  β-glucans (BG) and arabinoxylans (AX) - together with the enzymes responsible for their modification, play an important role in barley processing and quality attributes of barley-derived products.  Clear understanding of the molecular basis of functionality of these polysaccharides is necessary to gain control over mechanisms responsible for their behavior during the malting and brewing processes.

Procedure and Project Activities:

Barley (cv. Harrington) and commercial barley malt (cv. Harrington) were used in this study.  Non-starch polysaccharides were sequentially isolated from barley and malt with H₂O (40, 65 and 95^oC), Ba(OH)₂, Ba(OH)₂/H₂O, and NaOH (Cyran et al, 2002, Cereal Chem.79:359).  Monosaccharide composition was determined via gas-liquid chromatography of alditol acetates (Izydorczyk et al, 1998, Carbohydrate Polymers 35:249).  ¹H-NMR spectra  of samples dissolved in D₂O were recorded on a Bruker AM 300 FT spectrometer at 85°C (Bruker Specrtospin Canada, Milton, Ont., Canada).  β- Glucans were digested with lichenase and analyzed according to the method of Izydorczyk et al (1998, Carbohydrate Polymers 35:249).  Weight-average molecular weights (M_w) of polysaccharides were estimated by using the HPSEC-MALLS system comprised 3TSK-gel packed columns (G5000, G3000, G2500; Tosoh Corporation), a DAWN DSP laser-light-scattering detector (Wyatt Technology, USA), refractive index and UV detectors (Waters 410 and Waters 490, respectively).  The rheological properties of arabinoxylans and β-glucans were determined using the Bohlin rheometer (Bohlin Reologi).

Results and Discussion:

The content of β-glucans in barley can be affected by genetic and environmental factors but generally falls between 3 and 6%.  The content of arabinoxylans can be equally high in barley (3-6%).

β-Glucans are unbranched homopolymers of D-Glcp linked via β-1→3 and β-1→4 linkages.  The linkage arrangement is not completely irregular; consecutive blocks of β-1→4 linkages –mostly 2 or 3, but sometimes up to 20 – are separated by single β-1→3 linkages at random (Fig. 1).  The NMR and methylation analyses confirmed the presence of β 1→3 and 1→4 linkages in malting barley.  β-Glucans extracted at higher temperature (65^oC or 95^oC) generally had a higher ratio of β (1→4)/(1→3) linkages than those extracted at 40^oC.  The ratio of cellotriose to cellotetraose DP3/DP4 and the presence of longer cellooligosaccharides (DP>9) was higher in β-glucans extracted at higher temperatures.

Barley β-glucans are high molecular weight (M_w)polymers.  The average M_w of β-glucan fraction extracted at 40^oC was 220,000 whereas those extracted at 65^oC exceeded 1,000,000.  Two alkali-extractable fractions (Ba(OH)₂ and NaOH) showed the average M_w of 1,000,000 and 700,000, respectively (Fig. 2).  β-Glucans are capable of intermolecular aggregations leading to a higher apparent M_w.  When the sample of β-glucans (95^oC extraction) was dissolved in water, the average M_w was about 4,700,000.  When the sample was dissolved in NaOH and subsequently neutralized, the M_w dropped to 2,600,000.

Arabinoxylans consist of a linear chain backbone of β-1→4 linked Xylp residues to which single Araf residues are attached through O-2 and/or O-3 of the xylose units (Fig. 3).

Barley arabinoxylans clearly indicated their potential for gelation via formation of covalent linkages between ferulic acid moieties associated with the arabinoxylan chains.  We have shown that even dilute solution of arabinoxylans can undergo an oxidative gelation (via formation of diferulic acid bridges) resulting in formation of viscous solutions or gels.

We have demonstrated that partially degraded β-glucans are capable of forming a gel-like network structure – such structures are not detected in solutions of intact β-glucans.  This behaviour was attributed to aggregation of the chains along the cellulose like fragments in the chains of β-glucans.

During malting, the majority of β-glucans gets degraded.  We examined the content and composition of non-starch polysaccharides in a commercial malt obtained from cv. Harrington.  We have determined that malt contained only 0.5% of β-glucans - 0.23% was soluble, whereas approximately 0.38% remained insoluble in water but could be extracted with alkali.  Arabinoxylans were hydrolyzed to a lesser extent than β-glucans.  Malt contained 6.7% of arabinoxylans.  The majority of arabinoxylans was, however, still insoluble.  Only 0.8% could be solubilized in water, 3.5% was solubilized in alkali which still leaves about 3% of arabinoxylans resisting solubilization even in alkali.

The molecular weight (M_w) of water-soluble β-glucans was drastically reduced during malting.  The average M_w of the major polymer population in β-glucans preparation was about 36,000 (Fig. 4).

The arabinoxylan fraction was isolated from malt material with water.  The β-glucans present in the water extracts were removed enzymatically.  Arabinoxylan fraction contained about 16 % protein, 0.6% ferulic acid.  This fraction contained mainly xylose (Xyl) and arabinose (Ara) residues –the content of arabinoxylans amounted to 84%.  The ratio of Xyl to Ara (1.29) was similar to arabinoxylans from barley.  In order to obtain a better insight into the structure of malt arabinoxylans – we fractionated this material into smaller and more homogeneous subfractions.

Water-soluble arabinoxylans isolated from malt contained relatively high amounts of unsubstituted Xylp making the ratio of unsubstituted to substituted Xylp residues higher than those reported for water-extractable barley arabinoxylans.  Also, the majority of substituted Xylp carried two Araf residues resulting again in higher ratios of doubly to singly substituted Xylp than those reported for water-extractable barley arabinoxylans.

It is possible to envisage two types of structural domains in arabinoxylans which may have resisted the enzymic degradation during malting.  On the one hand, there are domains densely substituted with arabinose residues and thereby protected from enzymic attack.  On the other hand, there are smooth domains with no arabinose substitution.  Although it is generally accepted that exposed xylan backbones are most susceptible to enzymic degradation by xylanases, it is possible that such domains participate in formation of stable aggregates -- with β-glucans or possibly other polymers -- and, therefore, resist enzymatic degradation.  Both types of structural features may render arabinoxylans resistant to further enzymatic hydrolysis during mashing and eventually be responsible for wort filtration problems.

The average M_w of the major polymeric population of water soluble arabinoxylans in malt was 200,000, however, a small population of arabinoxylans had M_w above 1,000,000 (Fig. 4).  It is possible that these high molecular weight species are a result of polymer aggregation.  It is also possible that a small portion of arabinoxylans is cross-linked via diferulic acid bridges.

Degraded β-glucans might also interact with arabinoxylans.  Enzymic degradation of β-glucans liberates the cellulose-like fragments and because of increased flexibility and diffusion of these fragments in solution a better contact between the liberated cellulose-like fragments and the unsubstituted xylan blocks in arabinoxylans is achieved.  If the interactions are numerous, aggregation and subsequent precipitation might occur (Fig. 5).  The form of the final product, soluble aggregate or precipitate, will probably depend on the concentration of both polymers in solution as well as on the conditions /characteristics of the solvent.

Arabinoxylans and β-glucans can also be found in wort.  Our studies indicated that both polymers are unstable in wort and can easily co-precipitate with proteins and residual α-glucans from the wort solutions (Fig. 6).

Conclusions:

Acknowledgements:

This project was made possible due to funding from the Governments of Manitoba and Canada through the Canada-Manitoba Agri-Food Research and Development Initiative (ARDI).  The Brewing and Malting Barley Research Institute was also a funding partner.

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