Barley and Oat β‐Glucan Content Measured by Calcofluor Fluorescence in a Microplate Assay

Abstract

Of the various components of barley and oats, one constituent of the cereal cell wall, (1→3,1→4)-β-D-glucan (β-glucan), is important to a wide range of interests. Malting barley breeders strive to develop lines that modify well, yielding malts with low soluble β-glucan levels in Congress worts (less than the American Malting Barley Association guidelines of 100–120 ppm) (http:// www.ambainc.org/ni/Guidelines.pdf) to meet brewers’ needs for low-viscosity/high-filterability wort in the brewhouse. Similarly, barley grain with reduced β-glucan content is more suitable as poultry feed than barley because of a higher β-glucan content (Bergh et al 1999) due to feed digestibility and related issues. In contrast, the U.S. Food and Drug Administration recognizes benefits to human coronary health from including soluble β-glucan in the diet. β-glucan from oat and barley reduced both blood cholesterol levels as well as the glycemic index of foods (Wood 2002, Baik and Ullrich 2008). With the increased interest in barley β-glucan as a food component, there is a need for convenient, cost-effective procedures to analyze the grain for β-glucan content. Probably the most widelyused method for analyzing barley (and oat) β-glucan content in research laboratories is the enzymatic method (McCleary and Codd, 1991) used in Approved Method 32-23 (AACC International 2000). Essentially, the mixed-linkage, linear β-glucan polymers are extracted from grain flour, hydrolyzed to monosaccharides by sequential enzymatic reactions (endo-(1→3,1→4)-βD-glucan 4-glucanohydrolase (lichenase), followed by β-glucosidase), and finally coupled to color generation using glucose oxidase and a chromogenic substrate. Kits providing the principle reagents for this method are commercially available from Megazyme (www.megazyme.com) and require only relatively simple laboratory instruments (spectrophotometer, centrifuge, pipettes, water bath, etc.). However, the enzymatic method does include a number of manipulations, increasing the labor requirement for the method. Also, the costs for the reagent kits can be significant for large sample sets, making use of the kits less feasible for routine high-throughput analysis. A second method for β-glucan determination measures fluorescence increase upon the binding of Calcofluor to high molecular weight β-glucans in grain extracts (Li et al 2008). Commonly, Calcofluor methods for β-glucan analysis involve use of flow injection analysis (FIA). In FIA, an autosampler is used to inject a fixed volume of sample into a stream of buffer or water, which is subsequently mixed with a second stream of Calcofluor reagent. A fluorescence detector measures the increase in fluorescence in the sample bolus in the combined streams due to interaction of the sample β-glucan with the Calcofluor reagent. The sample βglucan is quantified by comparing sample peak area or peak height with a standard curve generated from injections of a dilution series of β-glucan standards. This protocol, while procedurally simpler than the enzymatic method, requires more extensive instrumentation in its most common implementation. Dedicated commercial instrumentation for such flow injection analysis or comparable segmented flow analysis can cost in excess of $100,000. Home-built versions of such FIA instrumentation can be more affordable, although still requiring significant initial expense and maintenance costs. As a result, the FIA instrumentation is commonly found only in high-volume dedicated quality analysis laboratories. Apart from the cost of instrumentation, the Calcofluor methodology requires relatively inexpensive reagents and significantly less labor. A third alternative method, using a dye-binding (colorimetric) procedure analogous to the Calcofluor method that can be read in a microplate spectrophotometer, has been adopted as EBC Method 4.16.3 High Molecular Weight β-Glucan Content of Wort: Colorimetric Method (see Freijee 2005). However, the kit for the colorimetric method (GlucaTest from NovaBiotec Dr. Fechter GmbH, www.novabiotec.de) is not commercially available in North America at the present time. In this note, we show that the Calcofluor method can be adapted to a fluorescent microplate reader, providing a simple means of measuring β-glucan in cereal grains and malt, using inexpensive reagents, and affordable, commonly available instrumentation.