Vol. 85, No. 3, 2008 283 Extractability, Structure and Molecular Weight of β-Glucan from Canadian Rye (Secale cereale L.) Whole Meal S. M. Ragaee, 1,2 P. J. Wood, 1 Q. Wang, 1 S. Tosh, 1 and Y. Brummer 1 ABSTRACT Cereal Chem. 85(3):283–288 Oat and barley (1→3)(1→4)-β-D-glucans (β-glucan) are readily ex- tracted by hot water but rye β-glucan is resistant to such extraction. This poor extractability might be due to entrapment within a matrix of arabi- noxylan (AX) cross-linked through phenolic constituents. AX are the major nonstarch polysaccharides of the rye kernel. In this study, several approaches were compared in an effort to determine optimum conditions for extraction of high yields, high molecular weight (MW), and high purity of β-glucan from Canadian rye whole meal. Variables investigated included sodium hydroxide concentrations, extraction time, sample pre- hydration, extraction under low temperature, and prior extraction of AX with barium hydroxide. There was a linear relationship between the strength of NaOH and amount of β-glucan extracted and because MW was essen- tially the same up to 1.0N NaOH, this extraction agent, at room tempera- ture for 90 min, was selected to isolate rye β-glucan. The β-glucan was then purified and structure and molecular weight distribution studied. Rye (Secale cereale L.) is a hardy crop and a good source of dietary fiber (DF). Whole meal rye flour usually contains 15–17% DF, the main components of which are AX (7–12%) (Glitso and Bach-Knudsen 1999; Ragaee et al 2001); β-glucan (1.3–3.11%) (Ragaee et al 2001, unpublished; Demirbas 2005); cellulose (2.5– 2.6); and Klason lignin (1.1–3.0%) (Nilsson et al 1997). Also the storage oligo- or polysaccharide fructan is found in relatively high amounts (3.1%) in rye grain compared with, for example, 1.5% in wheat (Knudsen 1997). Whole meal rye products may have a protective effect against the development of hormone-related can- cers which may be attributed to the content of lignan on hormone metabolism as well as benefits from fiber (Herman et al 1995). Plant lignans such as matairesinol and secoisolariciresinol are present in the plant as glycosides (Herman et al 1995) and rye flour contains 0.33–0.52 mg/kg of matairesinol and 0.27–0.45 mg/kg of secoisolariciresinol (Nilsson et al 1997). Cereal β-glucan has considerable potential as an ingredient for the functional food industry because of its specific physiological effects and attendant health benefits (Wood 2004). In clinical studies, oat β-glucan reduced serum cholesterol levels and attenu- ated postprandial blood glucose and insulin response (Braaten et al 1994; Wood et al 1994b; Tappy et al 1996). Reported levels of β-glucan in dehulled or naked oats and most dehulled or naked barleys range mostly from ≈3 to 8% (Peterson 1991; Wood 1994; Lee et al 1997; Demirbas 2005; Yao et al 2007), in rye ≈2% (Ragaee et al 2001), and in wheat <0.5% (Beresford and Stone 1983). Canadian rye grain contains a potentially func- tional amount (1.8–3.1%) of β-glucan (Ragaee et al 2001, unpub- lished) depending on genotype, location, and year of harvest. Structural features of cereal β-glucan, in particular the ratio of β-(1→3)-linked cellotriosyl to (1→3)-linked cellotetraosyl units, and molecular weight (MW) distribution are primarily responsible for its physical and functional properties, including physiological responses (Tosh et al 2004a; Wood et al 1994a,b). The structure, MW, and physical properties of β-glucan from both oat and barley have been extensively examined. Roubroeks et al (2000a) re- ported on the structure of rye β-glucan but, in general, attention has focused more on AX (Nilsson et al 1996) because β-glucan is a minor component of the dietary fiber compared with AX. How- ever, with an upper range of levels ≈3% and the potential for en- richment in milling fractions (Nilsson et al 1996), the properties of the rye β-glucan merits some attention. Additionally, we are interested in rye β-glucan as part of comparative studies to better understand solution properties and rheological behavior of the cereal β-glucans in general. The rye β-glucan characterized by Roubroeks et al (2000a,b) was of low MW (14K–80K) compared with the value reported in crude extracts by Wood et al (1991b) (1,130K), and was much lower than commonly reported for oat, barley, and wheat (≈400K to 2.5 million) (Beer et al 1997; Cui et al 1999). Although different methods of measurement and MW estimates such as peak MW (M p ) and weight average MW (M w ), have been used, it is clear that these numbers represent major differences in MW distribution but it is not necessarily clear whether such differences are intrinsic to the source or artifacts of extraction. To ensure a representative sample, it is desirable that as much as possible be extracted but without significant degrada- tion. Barium hydroxide is commonly used to extract AX sepa- rately from β-glucan but previous experiments in our laboratory (Brummer et al 2008) showed that the resultant β-glucan was of low molecular weight (Roubroeks et al 2000a,b). Therefore, the main objective of this study was to investigate the extraction of high molecular weight β-glucan from rye whole meal then use the conditions established to isolate a high molecular weight and high purity β-glucan in high yield to study structure and properties. MATERIALS AND METHODS Rye grain (Secale cereale) cultivar AC Rifle grown in western Canada (Lethbridge) in 2004 was kindly provided by J. G. McLeod (Agriculture and Agri-Food Canada, Swift Current, SK). Thermostable-α-amylase (Bacillus lichenformis, E.C. 3.2.1.1, Lot 50901), β-xylanase M6 (from rumen microorganism, E.C. 3.2.1.8, Lot 51206), lichenase (Bacillus subtilis, E.C. 3.2.1.73, Lot 60502), and a standard high purity β-glucan (peak molecular weight, M p = 1,144K) were purchased from Megazyme International Ireland Ltd (Bray, Ireland). All chemicals were of reagent grade unless otherwise specified. Another standard β-glucan (80% β-glucan) (M p = 1,300K), which had been prepared on a large scale at the POS Saskatoon, SK (Wood et al 1989), was used for an assay of β-glucanase in rye whole meal (Roudsari et al, unpublished). Rye kernels were ground to pass through a 0.3-mm screen to obtain rye whole meal. Endogenous enzymes were deactivated by suspending the rye whole meal in aqueous ethanol (70% v/v) and stirring under reflux for 2 hr at a ratio of (1:5 w/v), followed by refluxing for 1 hr with 80% aqueous ethanol (v/v). The super- natant was removed by centrifugation at 5,000 × g for 15 min, and the residue was washed twice with two volumes of ethanol (95%) and dried at 45°C overnight. The enzyme-deactivated dry rye sample was reground using a ball mill to pass through a 0.3-mm screen. 1 Agriculture and Agri-Food Canada, Food Research Program, Guelph, ON N1G 5C9. 2 Corresponding author. Phone: (519)829-2649. E-mail: sragaee@uoguelph.ca doi:10.1094/ CCHEM-85-3-0283 © 2008 AACC International, Inc.