Supplementary material for von Wettstein et al. (2000) Proc. Natl. Acad. Sci. USA 97 (25), 13512-13517.
Methods
Analyses of Experimental Diets.
The diets were ground in a Udy (Udy Analyzer, Boulder, CO) mill with a 0.5-mm screen. Moisture and ash contents were determined according to Association of Official Analytical Chemists methods 930.15 and 942.05, respectively (1). Protein content (n × 5.7) was determined with a Leco FP-428 nitrogen analyzer (Leco, St. Joseph, MI). Neutral and acid detergent soluble fiber, comprising nonstarch polysaccharides and Klason lignin, was determined on an Ankom200 fiber analyzer (Ankom Technology, Fairport, NY). Diet (0.5 g) was placed in filter bags and extracted sequentially in the reaction vessel with neutral and acid detergent solution under positive pressure at 99ºC. Starch was digested with a-amylase in the rinsing solution after draining the neutral detergent. Fiber content was calculated as the difference between dry weights before and after extraction. Activity of endogenous and heat-stable b-glucanase was measured with azo-b-glucan substrate (Megazyme, Wicklow, Ireland). Soluble protein was determined with the detergent-compatible Lowry phosphomolybdic reagent (Dc, Bio-Rad), and enzyme activity was expressed as µg enzyme·g-1 soluble protein. Average b-glucanase activity for malts of Golden Promise and line 5607 were 0.054 mg·g-1 and 4.647 mg·g-1, respectively. Total b-glucan contents of diets and malts were estimated according to McCleary and Mugford (2) by using the Megazyme kit. Water-soluble b-glucans were determined according to Jørgensen (3).Viscosity and b-Glucanase Measurements.
One gram of glandular stomach, small intestine, ceca content, or excreta was weighed out into a centrifuge tube, and 1 ml of water was added. The contents were mixed thoroughly and centrifuged at 25,000 × g for 20 min. The supernatant was collected in 2-ml Eppendorf tubes and recentrifuged (15,000 × g, 5 min). Supernatants free of particles were collected and used for measurements at 30ºC with a Brookfield Viscometer fitted with the CP-40 cone (Brookfield Engineering Laboratories, MA). The samples were then analyzed for heat-stable (1,3-1,4)-b-glucanase activity as follows: the sample (50 ml) was mixed with buffer containing 40 mM sodium acetate and 40 mM sodium phosphate, pH 4.6, and incubated for 30 min at 65ºC. An aliquot of this mixture was used to monitor the hydrolysis of azo-b-glucan (Megazyme) at 65ºC for 30 min. Soluble protein in the extract was measured with the Bio-Rad Dc method. Activity of recombinant enzyme was expressed in mg·g-1 soluble protein. Recombinant heat-stable b-glucanase activity in excrement was also measured as outlined above, but the protein content of the excretal extract was measured with the Coomassie blue dye binding method of Bradford (Bio-Rad).Soluble and Insoluble (1,3-1,4)-b-Glucans in the Gastrointestinal Tract and Excrement.
Contents (50 mg) from the glandular stomach, small intestine, or cecum or of excreta were placed in 2-ml Eppendorf tubes and suspended in 1 ml 80% (vol/vol) ethanol. With the lids tightly secured, the tubes were incubated for 5 min in a boiling water bath. After cooling to room temperature and vigorous vortexing, the insoluble material was collected by centrifugation at 13,000 × g for 10 min. Soluble b-glucans were isolated by three extractions with water as follows: the pellet was suspended in 1 ml of water, vortexed vigorously, and incubated in boiling water for 10 min. Contents were cooled to room temperature, vortexed, and centrifuged (3,000 × g, 10 min). Supernatant was collected into a graduated tube. The pellet was resuspended in another 1 ml of water, vortexed, and centrifuged (3,000 × g, 10 min). Supernatant was collected and added to the graduate tube. The pellet was resuspended in 0.5 ml water, vortexed, and centrifuged (3,000 × g, 10 min); the supernatant pooled to the graduate tube; and the pellet was saved. The volumes of supernatants in the graduate tubes were adjusted to 2.5 ml with water, and 20 ml of a 2 M sodium phosphate buffer (pH 6.5) was added. Insoluble b-glucans were retrieved from the pellet remaining after extraction of the soluble b-glucans. The pellet was suspended in 1 ml of 50 mM HCl; the lid was secured; and tubes were incubated for 10 min in a boiling water bath. After cooling and vortexing, the suspension was centrifuged at 1,500 × g for 10 min, and the supernatant collected in a graduated tube. The extraction was repeated; the combined supernatants were adjusted to 2 ml; and 0.5 ml of 2 M Na-PO4-buffer (pH 6.5) was added. Digestion of soluble and insoluble b-glucans with lichenase (Megazyme) was performed by adding 30 ml (»1 unit) of lichenase to the graduated tubes, mixing the contents well, and incubating the tubes in a water bath with shaking at 50°C for 1 h. After lichenase digestion, aliquots (0.1 ml) were accurately dispensed on the bottom of three Eppendorf tubes. Fifty ml of b-glucosidase (»1 unit) was added to two of these tubes, and to the third, the blank, 50 ml of sodium acetate buffer (50 mM, pH 4.0) was added. Tubes were incubated at 50°C for 30 min, and 1 ml of glucose oxidase/peroxidase reagent (Megazyme) was added to all tubes and incubated for a further 30 min. Blank (sodium acetate buffer + water) and glucose standards (15 ml and 30 ml) were included in each set of samples analyzed. The amount of b-glucans was estimated from absorbance of glucose at 510 nm. Calculations of the amount of b-glucans were done according to McCleary and Glennie-Holmes (4).Western Blot Analyses with Heat-Stable (1,3-1,4)-b-Glucanase-Specific Antibody.
Acetone powder was prepared from digesta of the glandular stomach, the intestine, the ceca, and the excrements of chickens fed the barley diet with added transgenic malt. Acetone powders from cecum digesta of chickens fed corn diet, barley diet, and barley diet with added normal Golden Promise malt were used as controls. Material (200 mg) was weighed into 2-ml Eppendorf tubes, suspended in 2 ml of acetone with a glass rod, and centrifuged for 5 min at 15,000 × g. The acetone wash was repeated, and the pellet was dried in air. Urea was removed from the excrement samples by extraction with 2-ml aliquots of ethanol before acetone extraction. For isolation of heat-stable (1,3-1,4)-b-glucanase, the air-dried pellets were suspended in 300 µl of 40 mM sodium acetate, 40 mM sodium dihydrogen phosphate (pH 4.6). (For excrements, 600 µl was required.) After incubation at 65ºC for 30 min and centrifugation at 15,000 × g (10 min), protein content and heat-stable b-glucanase activity were determined in the supernatant. For total protein extraction, the acetone powders were suspended in 300 µl of a solution containing 0.1 M Tris·HCl (pH 8.8), 1% SDS, 0.1% b-mercaptoethanol, and the capped tubes were incubated for 5 min in a boiling water bath. After cooling and centrifugation at 15,000 × g (10 min), the supernatants were collected. The proteins were separated by electrophoresis in a 15% polyacrylamide gel containing 1% SDS and transferred onto a nitrocellulose membrane by using a Bio-Rad semidry blotter and a solution containing 2.93 g·liter-1 glycine, 5.81 g·liter-1 Tris, and 200 ml of methanol. Electroblotting was performed for 45 min at 15 V. The membrane was incubated for 1 h in a solution of 20 mM Tris·HCl (pH 7.5), 0.5 M NaCl, 0.05% Tween-20, and 5% (vol/vol) nonfat milk to block reacting groups. The membrane was then incubated in the above solution overnight with an antibody raised against the heat-stable (1,3-1,4)-b-glucanase expressed in Escherichia coli (dilution 1:2,000). Excess antibody was removed by three successive washes with 20 mM Tris·HCl (pH 7.5), containing 0.5 M NaCl, 0.05% Tween 20, and 5% (vol/vol) nonfat milk. The blots were incubated with the secondary antibody (peroxidase-linked goat anti-rabbit monoclonal IgG; Sigma) at 1:20,000 dilution in the nonfat milk solution, and excess antibody was removed and stained for peroxidase activity in a solution of 0.01% H2O2, 0.5 mg·ml-1 4-chloro-a-naphtol in Tris·HCl (pH 7.5).References:
1. Association of Official Analytical Chemists (1990) Official Methods of Analysis (Association of Official Analytical Chemists, Gaithersburg, MD), 15th Ed.
2. McCleary, V. B. & Mugford, D. C. (1997) J. AOAC Int. 80, 580-583.
3. Jørgensen, K. G. (1988) Carlsberg Res. Commun. 53, 277-285.
4. McCleary, V. B. & Glennie-Holmes, M. (1985) J. Inst. Brew. 91, 285-295.