Figures & data
Fig. 1. Size transition of sword beans and weight transition of absorbed water during soaking.
Notes: (A) Comparison of sword beans (upper) and soybeans (lower). The white bar is 1 cm. Sword beans (open circles) and soybeans (closed circles) were soaked in 10 volumes (v/w) of distilled water (B-E). The long axis (B), minor axis (C), and thickness (D) of beans were measured with micrometer calipers. Data represent the average ± standard deviation of 10 beans. The intersection represents the direction of the size measurement. The double-headed arrows show the long axis (B), minor axis (C), and thickness (D). The weight of the absorbed water was estimated by subtracting the initial weight of the dried beans from that of the soaked beans (E). The ratio was calculated by dividing the weight of the absorbed water by the initial weight of the dried beans. Data represent the average ± standard deviation of 5 beans.
Table 1. Comparison of the size and weight between dried beans.
Fig. 2. Protein extraction from sword beans.
Notes: (A) Extract A was prepared by heating followed by squeezing (a). Extract B was prepared by squeezing without heating (b). Extract B was heated (c) and then squeezed (d) for the preparation of Extract C. (B) The protein concentrations of Extract A (1), Extract B (2), and Extract C (3) were determined using the Bradford method with bovine serum albumin as a standard. Data represent the average ± standard deviation of three independent experiments. (C) The precipitate was collected by heating Extract B in the preparation of Extract C. The white bar represents 2 cm. (D) Sword bean proteins were separated on an SDS-polyacrylamide gel (12.5% polyacrylamide). Proteins corresponding to 10 μg of soybean protein (lane 1), 10 μg of sword bean protein from Extract B (lane 2), and 1 μg of sword bean protein from Extract B (lane 3) were electrophoresed on an SDS-polyacrylamide gel (12.5% polyacrylamide) and stained with Coomassie Brilliant Blue R-250.
Fig. 3. Temperature-dependent precipitate formation of sword bean proteins.
Notes: The effects of heating at various temperatures on the precipitation of sword bean proteins were analyzed. After heating, samples were separated into supernatants and precipitates. (A) The protein concentration of the supernatants (open circles) was determined using the Bradford method with bovine serum albumin as a standard. The ratio was calculated by dividing the concentration of the supernatant by that of the initial sample without heating and centrifugation. The weight of the wet precipitate was measured using a balancer. The precipitation efficiency was calculated by dividing the weight of the precipitate by that of the initial sample (closed circles). Data represent the average ± standard deviation of three independent experiments. (B) Sword bean proteins in Extract B (lane 1) were centrifuged (lane 2). The samples were heated at 25 °C (lane 3), 50 °C (lane 4), 55 °C (lane 5), 75 °C (lane 6), or 100 °C (lane 7) and then centrifuged. The proteins in 12-μL reaction mixtures were subjected to SDS-PAGE (12.5% polyacrylamide) and stained with Coomassie Brilliant Blue R-250.
Fig. 4. Magnesium chloride-dependent precipitation of sword bean proteins.
Notes: Samples were prepared by centrifugation of Extract B. Distilled water (white bars) or 200 mM magnesium chloride (black bars) was added to 9 volumes of sample, and samples were heated at 25, 50, 75, or 100 °C. After heating at each temperature for 15 min, the mixtures were incubated on ice for 15 min and then separated into supernatants and precipitates by centrifugation. (A) The protein concentration of the supernatant was determined in the same way. The ratio was calculated by dividing the concentration of the supernatant by that of the initial sample plus distilled water without heating and centrifugation. Data represent the average ± standard deviation of three independent experiments. (B) The precipitation efficiency was calculated by dividing the weight of the precipitates by that of the initial sample. Data represent the average ± standard deviation of three independent experiments. (C) Distilled water (lanes 1, 3, 5, and 7) or 200 mM magnesium chloride (lanes 2, 4, 6, and 8) was added to 9 volumes of sample after heating at 25 °C (lanes 1 and 2), 50 °C (lanes 3 and 4), 75 °C (lanes 5 and 6), or 100 °C (lanes 7 and 8). After heating at each temperature for 15 min, the mixtures were incubated on ice for 15 min and then separated into supernatants and precipitates by centrifugation. Supernatant proteins in 12-μL reaction mixtures were subjected to SDS-PAGE (12.5% polyacrylamide) and stained with Coomassie Brilliant Blue R-250.
Fig. 5. Schematic model for the precipitation of sword bean proteins under different conditions.
Notes: Circles show proteins precipitated by heating at a high temperature (>90 °C). Triangles show proteins soluble at the high temperature. Open symbols show proteins that were not precipitated by adding magnesium chloride. Closed symbols show proteins that were precipitated by adding magnesium chloride. Arrows 1 and 2 indicate heating at more than 90 °C and the addition of 20 mM magnesium chloride at temperatures ranging from 25 to 75 °C, respectively.
Supplemental material
