The total amount of β-glucan in the grain was measured using the enzymes in Mixed-linkage beta-glucan kit (Megazyme, K-BGLU). A grain was ground in 1 mL 50% (v/v) ethanol using Multi-beads shocker MB2000. The sample was incubated at 100 °C for 5 min. After the centrifugation at 1800 × g for 10 min, the pellet was resuspended in 1 mL 50% (v/v) ethanol and centrifuged at 2800 × g for 10 min. The pellet was resuspended in 1 mL of 20 mM sodium phosphate buffer (pH 6.5) and mixed with 2.5 units of lichenase. The sample was incubated at 50 °C for 1 h. Then, the sample was adjusted to 4 mL with 200 mM sodium acetate buffer (pH 4.5). The aliquot (50 μL) was mixed with β-glucosidase at 50 °C for 10 min. The glucose amount in the sample was measured using GOPOD reagent in the kit.
Mixed linkage beta glucan kit
Manufactured by Megazyme
Sourced in Ireland
The Mixed-Linkage Beta-Glucan Kit is a laboratory tool designed for the quantitative determination of mixed-linkage beta-glucan content in cereal grains, food, and feed samples. The kit provides a reliable and standardized method for measuring this important dietary fiber.
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5 protocols using mixed linkage beta glucan kit
1
Quantifying β-Glucan Content in Grains
2
Mixed-Linkage Beta-Glucan Quantification
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Mixed linkage (1, 3;1, 4)-β-glucan in plant biomass was determined according to the manufacturer’s description of the mixed-linkage beta-glucan kit (Megazyme). An additional 5 ml H2O was added to the samples (750 mg milled leaf biomass) in the initial incubation step in a water bath (100°C). Calculation of the (1, 3;1, 4)-β-glucan content followed the instructions of the manufacturer’s manual.
3
BARLEYmax Granola Nutrient Composition
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The study food included 40 g of BARLEYmax granola (TEIJIN LIMITED, Tokyo, Japan) containing 20.4 g of BARLEYmax. It consisted of brown sugar syrup, oats, puffed brown rice, coconut, rice oil, prune puree, dried mango, dried papaya, dried pineapple, and citric acid. Regarding the nutritional facts, 40 g of the study food contained 169 kilocalories, 3.32 g of protein, 5.76 g of fat, 23.08 g of sugar, 5.72 g of dietary fiber, and 0.48 g of resistant starch. Dietary fiber constituted 1.2 g of β-glucan and 2.32 g of fructan. The nutrient components were analyzed by the Japan Food Research Laboratories (Tokyo, Japan). The amount of total resistant starch, β-glucan, and fructan were measured using the resistant starch kit, mixed-linkage beta-glucan kit, and fructan kit, respectively (Megazyme, Sydney, Australia).
4
Barley Flour β-Glucan Interaction Assay
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The LAB carrying the gtf gene were further assayed in duplicate for their interaction with the β-glucans contained in a sterilized barley flour (BF) medium, prepared by adding 5% barley flour (w v-1) to deionized water. Each isolate was inoculated in tubes containing 10 mL of BF medium to reach a final cell concentration of approximately 2 x 108 cells mL-1. The β-glucan content of the liquid medium was determined immediately before inoculation and after 24 h of fermentation at 30°C using a commercial mixed-linkage beta-glucan kit (Megazyme, Bray, Ireland) according to the assay procedure suggested by the kit manufacturer for liquid samples (including alcohol precipitation). The results were expressed as the mean variance (%) (positive or negative) of the β-glucan content before and after fermentation ± standard deviation.
5
Determination of Beta-Glucan in Bread
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A Mixed-Linkage Beta-Glucan kit (McCleary method) from Megazyme International Ireland (Wicklow, Ireland) was used for the determination of beta glucan. Sodium phosphate buffer (4.0 ml, 20 mM, pH 6.5) was added to 1 g bread samples and stirred on a vortex mixer.
Lichenase (EC 3.2.1.73) was added and incubated for 1 h at 50°C. Sodium acetate buffer (5.0 ml, 200 mM, pH 4.0) was added and mixed on a vortex mixer. The tubes were centrifuged at 3,500 g for 10 min. Aliquots (0.1 ml) were incubated with β-glucosidase (EC 3.2.1.21) in 50 mM sodium acetate buffer (pH 4.0) at 50°C for 10 min. Finally, 3.0 ml of GOPOD reagent was added, incubated at 50°C for 20 min and absorbance measured at 510 nm against a reagent blank. The glucose content was calculated using Megazyme-Calc, a calculation sheet provided by the manufacturer. All analyses were done in triplicate and nutrient contents are expressed as g/100 g fresh weight.
Lichenase (EC 3.2.1.73) was added and incubated for 1 h at 50°C. Sodium acetate buffer (5.0 ml, 200 mM, pH 4.0) was added and mixed on a vortex mixer. The tubes were centrifuged at 3,500 g for 10 min. Aliquots (0.1 ml) were incubated with β-glucosidase (EC 3.2.1.21) in 50 mM sodium acetate buffer (pH 4.0) at 50°C for 10 min. Finally, 3.0 ml of GOPOD reagent was added, incubated at 50°C for 20 min and absorbance measured at 510 nm against a reagent blank. The glucose content was calculated using Megazyme-Calc, a calculation sheet provided by the manufacturer. All analyses were done in triplicate and nutrient contents are expressed as g/100 g fresh weight.
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