Reversible changes of canavalin solubility controlled by divalent cation concentration in crude sword bean extract

Abstract

Canavalin is a vicilin-class (7S) storage protein found in sword bean (Canavalia gladiata). Our previous report indicated that canavalin is precipitated by the addition of 20 mM MgCl₂ to crude sword bean extract. Here, we examined the solubility changes induced by the addition of Mg²⁺ and Ca²⁺ at various concentrations. Canavalin tended to be insolubilized at relatively low concentrations of MgCl₂ (< 20 mM) and solubilized at relatively high concentrations (> 20 mM). In addition, canavalin was slightly insolubilized in the presence of NaCl. Overall, the results revealed that solubility changes are reversible and depend on the concentration of divalent cations. Therefore, we suggested a reaction scheme that describes the effects of divalent cations on the solubility of canavalin, which would facilitate the study of its physiological function and the application of canavalin in the food processing industry.

Graphical abstract

The solubility changes of canavalin are reversible and depend on the concentration of divalent cations.

Key words:

• 7S globulin
• divalent cation
• canavalin
• reversible solubility change
• sword bean

White sword bean (Canavalia gladiata) is a part of the traditional diet in the Asian tropics and subtropics^1,2) and is relatively resistant to pests and most diseases.³⁾ Since the dried beans contain approximately 25% crude proteins,^1,4) they are good potential sources of protein. We have previously reported a method for the extraction of some proteins from sword bean using only distilled water.⁵⁾ In addition, the predominant protein in the crude extract was determined to be canavalin.⁵⁾

Canavalin is the major storage protein of sword bean and jack bean (Canavalia ensiformis) with a molecular mass of 47.6 kDa, classified as 7S seed globulin or legume vicilin.⁶⁾ It was first isolated from jack bean,⁷⁾ and later its primary structure was determined in both bean species using cDNA sequence.^8–11) These two primary structures differ in only two out of 419 amino acids of canavalin^12,13) and have high homology with other legume 7S globulins,^12–15) which probably shows that they may also similar tertiary structure, physiological function, and physicochemical properties. Although the tertiary structure of sword bean canavalin has not been yet determined, several X-ray crystal structure analyses showed that jack bean canavalin is a homotrimer.^16–20) The tertiary structure highly resembles that of soybean β-conglycinin, a soybean 7S globulin.¹²⁾ Soybean 7S globulin is precipitated by the addition of divalent cations.²¹⁾ This characteristic of soybean 7S globulin is utilized for the processing of tofu. If canavalin were also to possess similar characteristics, this protein could be useful for the manufacture of processed foods.

In our previous study, we described a method for the extraction of proteins from sword bean in distilled water and their precipitation by the addition of 20 mM MgCl₂ in the crude extract.⁵⁾ This was the first study that reported the precipitation of canavalin in the presence of MgCl₂ and showed that a detailed investigation of canavalin precipitation might be important for the study of its physiological function and physicochemical properties.

In this study, we examined the effects of the addition of MgCl₂, CaCl₂, and NaCl to the crude sword bean extract at various concentrations on the solubility of canavalin. Our findings might provide useful information for the study of the physiological functions and physicochemical properties of canavalin and its application in food industry.

Materials and methods

Materials

White sword beans were purchased from Morika Beiten (Nara, Japan) and general chemical reagents from Wako Pure Chemical Industries (Osaka, Japan).

Preparation of sword bean extract

A sword bean extract was prepared as described previously.⁵⁾ Briefly, soaked sword beans were ground for 5 min in eight volumes (v/w) of distilled water using a hand blender (CSB-77JBSTRW; Cuisinart, Stamford, CT) on ice, and the suspension was sieved through a cotton cloth. The extract was centrifuged at 9,100 × g for 10 min at 4 °C, and the supernatant was used for analysis.

SDS–PAGE

Samples were mixed with 0.33 volumes of SDS (0.25 M Tris-HCl at pH 7.0, containing 4% SDS, 5% 2-mercaptoethanol, and 40% glycerol) and incubated in a water bath at 100 °C for 5 min. SDS–PAGE was conducted using sample volumes corresponding to 0.25 μL of the sword bean extract; this volume contained approximately 4.0 μg proteins in the absence of divalent cations.⁵⁾ SDS–PAGE was carried out on 10% polyacrylamide gels at a constant current of 12.5 mA for 2.5 h as described previously.²²⁾ Proteins were stained with 0.25% Coomassie Brilliant Blue R-250. The molecular weight standard was purchased from Life Technologies (Tokyo, Japan).

Analysis of supernatant proteins

The sword bean extract was incubated at 25 °C for 5 min. Next, 0.11 volumes of MgCl₂, CaCl₂, NaCl, or distilled water (control) were added to the incubated sample at various concentrations, mixed, and incubated at 25 °C for 15 min and on ice for 5 min. The mixtures were centrifuged at 9,100 × g for 20 min at 4 °C. The supernatant was diluted 15-fold with distilled water. The supernatant proteins were analyzed by 10% SDS–PAGE.

Determination of residual canavalin ratio

The intensity of canavalin bands on SDS-polyacrylamide gel was quantified by ImageJ (National Institutes of Health, Bethesda, MD).²³ The residual canavalin ratio was expressed as the percentage of band intensity of the sample with added metal chloride to that of the sample with distilled water. Data were expressed as mean ± standard deviation of three independent experiments.

Analysis of insoluble canavalin solubilization

Insoluble canavalin was prepared by the addition of 15 mM MgCl₂ or 10 mM CaCl₂ to sword bean extract. Canavalin precipitated in the former was suspended in 60 mM MgCl₂ or distilled water, whereas that precipitated in the latter was suspended in 200 mM CaCl₂ or distilled water. The mixtures were centrifuged at 9,100 × g for 20 min at 4 °C. The supernatant was diluted 15-fold with distilled water to adjust the concentration of divalent cations to less than 15 mM. The precipitates were dissolved in a volume of 8 M urea equal to that of the initial extract. Proteins were analyzed using 10% SDS–PAGE.

Analysis of soluble canavalin insolubilization

Soluble canavalin was prepared by the addition of 60 mM MgCl₂ or 200 mM CaCl₂ to sword bean extract. The solutions were diluted 4- or 20-fold, respectively, with distilled water. The diluted mixtures were centrifuged at 9,100 × g for 20 min at 4 °C. The MgCl₂ supernatant was diluted threefold with distilled water such that the MgCl₂ concentration was 5 mM. The CaCl₂ concentration of the CaCl₂ supernatant is adjusted to 10 mM. The precipitates were prepared as described for the analysis of insoluble canavalin solubilization. Proteins were analyzed using 10% SDS–PAGE.

Curve fitting analysis

The residual canavalin ratio was plotted against each metal chloride concentration. The plots were fitted to the following equation using the KaleidaGraph 4.5 software (Synergy Software, PA, USA):

where r is the residual canavalin ratio at a metal chloride concentration C, r_max is the maximum ratio, r_min is the minimum ratio, C_m is the midpoint metal chloride concentration, and d is the slope coefficient at the midpoint.^24,25) Data represent the average ± standard deviation of five independent experiments.

Results

Effects of MgCl₂ and NaCl on canavalin solubility

We first investigated the MgCl₂ and NaCl concentration-dependency of canavalin precipitation (Fig. 1). The results showed that the minimum ratio of residual canavalin was 7.1 ± 3.5% at 15 mM MgCl₂, whereas the maximum was 78.4 ± 5.6% at 50 mM MgCl₂. Therefore, canavalin was insolubilized at relatively low concentrations of MgCl₂ (< 20 mM) and solubilized at relatively high concentrations (> 20 mM). When NaCl was added into the crude extract, canavalin solubilization was slightly decreased, and the minimum ratio of residual canavalin was 84.9 ± 3.3% at 25 mM NaCl. To investigate whether canavalin solubilization was highly decreased at NaCl concentrations of more than 50 mM, we also studied it at the range of 100–400 mM (Fig. 2). The results showed that at 100–350 mM, approximately 75% of canavalin remained in the supernatant (Fig. 2(B)). The minimum ratio of residual canavalin was 75.1 ± 1.2% at 200 mM NaCl, whereas the maximum was 92.2 ± 5.3% at 400 mM NaCl. Therefore, although canavalin was soluble in the presence of Na⁺, Mg²⁺ had a more significant effect on canavalin solubility.

Fig. 1. Effects of magnesium chloride (MgCl₂) and sodium chloride (NaCl) concentrations on canavalin solubility.

Notes: MgCl₂ (A) and NaCl (B) were added to sword bean extract at concentrations of 0–50 mM. MgCl₂ and NaCl were added to the sword bean extract at a concentration of 5 mM (lane 2), 10 mM (lane 3), 15 mM (lane 4), 20 mM (lane 5), 25 mM (lane 6), 30 mM (lane 7), 35 mM (lane 8), 40 mM (lane 9), 45 mM (lane 10), and 50 mM (lane11). Distilled water was used instead of MgCl₂ as a control (lane 1). The mixture was centrifuged, and the supernatant was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The arrows show canavalin bands. The proportion of residual canavalin in the supernatant was estimated from band intensity using ImageJ (C). Open and closed circles indicate MgCl₂ and NaCl, respectively. Data were expressed as mean ± standard deviation of three independent experiments.

Changes in canavalin solubility induced by MgCl₂

Canavalin reaction to MgCl₂ was divided into the insolubilization phase in the range of 0–20 mM MgCl₂ and the solubilization phase in the range of 20–50 mM MgCl₂ (Fig. 1(C)). Therefore, the effect of MgCl₂ on canavalin solubility was investigated separately in each phase (Fig. 3). In the range of 0–20 mM MgCl₂, soluble canavalin was highly decreased with the increasing concentration of MgCl₂ (Fig. 3(A)). The reduction of canavalin in the supernatant was more than 90% (Fig. 3(B)) at concentrations higher than 12 mM MgCl₂, reaching a maximum reduction at 16 mM MgCl₂ (5.9 ± 2.3%). In the range of 20–60 mM, supernatant canavalin was sigmoidally increased with the increasing concentration of MgCl₂, reaching a plateau at 50 mM MgCl₂ (Fig. 3(C) and (D)). The highest amount of supernatant canavalin was 91.2 ± 7.0% at 55 mM MgCl₂. These results showed that canavalin tends to be insolubilized at relatively low concentrations of MgCl₂ (< 20 mM) and solubilized at relatively high concentrations (> 20 mM).

Fig. 2. Effects of high sodium chloride (NaCl) concentrations on canavalin solubility.

Notes: NaCl was added to the sword bean extract at a concentration of 0–400 mM. NaCl was added to the sword bean extract at a concentration of 50 mM (lane 2), 100 mM (lane 3), 150 mM (lane 4), 200 mM (lane 5), 250 mM (lane 6), 300 mM (lane 7), 350 mM (lane 8), and 400 mM (lane 9). Distilled water was used instead of MgCl₂ as a control (lane 1). Proteins were analyzed (A) and quantified (B) as described in Fig. 1. Data were expressed as mean ± standard deviation of three independent experiments.

Effect of CaCl₂ on canavalin solubility

To investigate the specificity of divalent cations, we also examined the effect of CaCl₂ on the solubility of canavalin (Fig. 4). Canavalin reaction to CaCl₂ was divided into the insolubilization phase in the range of 0–10 mM CaCl₂ (Fig. 4(A) and (C)) and the solubilization phase in the range of 20–60 mM CaCl₂ (Fig. 4(B) and (D)). Canavalin completely disappeared in the range of 9–10 mM (Fig. 4(C)); it was sigmoidally increased by approximately 75% in the range of 10–140 mM (Fig. 4(D)); whereas 75.1 ± 1.2% was solubilized at 200 mM (Data not shown). These results indicated that CaCl₂ played a similar role with that of MgCl₂ and that the concentration of divalent cations significantly affects the solubility of canavalin.

Fig. 3. Analysis of canavalin solubility induced by magnesium chloride (MgCl₂).

Notes: The decreasing (A and B) and increasing (C and D) intensity of canavalin band was investigated as described in Fig. 1. (A) MgCl₂ was added to the sword bean extract at a concentration of 2 mM (lane 2), 4 mM (lane 3), 6 mM (lane 4), 8 mM (lane 5), 10 mM (lane 6), 12 mM (lane 7), 14 mM (lane 8), 16 mM (lane 9), 18 mM (lane 10), and 20 mM (lane11). Distilled water was used instead of MgCl₂ as a control (lane 1). (B) MgCl₂ was added to the sword bean extract at a concentration of 20 mM (lane 2), 25 mM (lane 3), 30 mM (lane 4), 35 mM (lane 5), 40 mM (lane 6), 45 mM (lane 7), 50 mM (lane 8), 55 mM (lane 9), and 60 mM (lane 10). Distilled water was used instead of MgCl₂ as a control (lane 1). Proteins were quantified as described in Fig. 1(B and D). Data were expressed as mean ± standard deviation of three independent experiments.

Comparison between effects of Mg²⁺ and Ca²⁺ on canavalin solubility

To compare the effect of Mg²⁺ and Ca²⁺ on canavalin solubility, the midpoint concentrations (C_m) in the insolubilization and solubilization phases were determined by curve fitting analysis (Figs. 3(B), (D) and 4(C), (D)). As summarized in Table 1, the C_m value of MgCl₂ in the insolubilization phase was similar to that of CaCl₂. However, MgCl₂ induced the insolubilization of canavalin at 2 mM (Fig. 3(B)), whereas CaCl₂ at the same concentration did not (Fig. 4(C)). The C_m value of MgCl₂ in the solubilization phase was lower than that of CaCl₂ (Table 1). In addition, canavalin was almost completely recovered by the addition of MgCl₂ (Fig. 3(D)), but only incompletely by the addition of CaCl₂ (Fig. 4(D)). Overall, the results suggested that the effect of Mg²⁺ on the solubilization or insolubilization of canavalin was stronger than that of Ca²⁺.

Table 1. Midpoint concentrations for the insolbulization and solubilization of canavalin.

	Insolubilization		Solubilization
	Cm^a (mM)	R^2^b	Cm^a (mM)	R^2^b
MgCl2	3.9	0.998	40.0	0.992
CaCl2	3.9	0.998	50.0	0.996

^a C_m indicates the midpoint concentration. The values were determined by the curve fitting analysis using the command for the sigmoidal equation of a graphic soft, KaleidaGraph 4.6.

^b R² indicates the coefficient of determination for the curve fitting analysis and was estimated using the software.

Reversibility of canavalin solubility

We examined whether insolubilized canavalin was solubilized in distilled water or in the presence of MgCl₂ and CaCl₂ (Fig. 5). Canavalin was first insolubilized by the addition of 15 mM MgCl₂ (Fig. 5(A), lanes 2 and 3), and most canavalin was observed in the precipitate fraction (Fig. 5(A), lane 3). The precipitated proteins were suspended in distilled water (Fig. 5(A), lanes 4 and 5), and only a small amount was dissolved. In contrast, the highest amount of precipitated proteins was dissolved in 60 mM MgCl₂ (Fig. 5(A), lanes 6 and 7), and then, the insolubilization of resolubilized proteins was increased with decreasing concentrations of MgCl₂ from 60 to 15 mM (Fig. 5(A), lanes 8 and 9).

Fig. 4. Effects of calcium chloride (CaCl₂) concentration on canavalin solubility.

Notes: CaCl₂ was added to the sword bean extract at a concentration of 0–10 mM (A and C) and 10–140 mM (B and D). Proteins were analyzed (A and B) and quantified (C and D) as described in Fig. 1. (A) CaCl₂ was added to the sword bean extract at a concentration of 1 mM (lane 2), 2 mM (lane 3), 3 mM (lane 4), 4 mM (lane 5), 5 mM (lane 6), 6 mM (lane 7), 7 mM (lane 8), 8 mM (lane 9), 9 mM (lane 10), and 10 mM (lane11). Distilled water was used instead of CaCl₂ as a control (lane 1). (B) CaCl₂ was added to the sword bean extract at a concentration of 10 mM (lane 2), 20 mM (lane 3), 30 mM (lane 4), 40 mM (lane 5), 50 mM (lane 6), 60 mM (lane 7), 70 mM (lane 8), 80 mM (lane 9), 90 mM (lane 10) 100 mM (lane11), 120 mM (lane 12), and 140 mM (lane 13). Distilled water was used instead of CaCl₂ as a control (lane 1). Data were expressed as mean ± standard deviation of three independent experiments.

Canavalin was first insolubilized by the addition of 10 mM CaCl₂ (Fig. 5(B), lanes 2 and 3). The precipitated proteins were suspended in distilled water (Fig. 5(B), lanes 4 and 5), and only a small amount was dissolved. The precipitated proteins were also suspended in 200 mM CaCl₂ (Fig. 5(B), lanes 6 and 7), in which the highest amount was dissolved. The insolubilization of resolubilized proteins was increased with the decreasing concentration of CaCl₂ from 200 to 10 mM (Fig. 5(B), lanes 8 and 9). These results indicated that CaCl₂ also induces reversible solubility changes in canavalin.

It was also examined whether canavalin solubilized in the presence of Mg²⁺ and Ca²⁺ at relatively high concentrations was precipitated by decreasing the concentration of MgCl₂ or CaCl₂, respectively (Fig. 6). Canavalin was solubilized by the addition of 60 mM MgCl₂ (Fig. 6(A)) or 200 mM CaCl₂ (Fig. 6(B)), and the soluble proteins were precipitated with the decreasing concentration of MgCl₂ (Fig. 6(A), lane 5) or CaCl₂ (Fig. 6(B), lane 5), respectively. The results indicated that canavalin was reversibly solubilized and insolubilized by the changing concentrations of Mg²⁺ and Ca²⁺.

Fig. 5. Solubility changes to insolubilized canavalin.

Notes: Magnesium chloride (MgCl₂; A) and calcium chloride (CaCl₂; B) were added to the sword bean extract at a concentration of 15 mM and 10 mM, respectively. Distilled water was used instead of salt as a control (lane 1). The mixture was separated into the supernatant (lane 2) and precipitate (lane 3). The precipitate was suspended in the same volume of distilled water, and then separated into the supernatant (lane 4) and precipitate (lane 5). The precipitate was also suspended in 60 mM MgCl₂ or 200 mM CaCl₂. The suspension was separated into the supernatant (lane 6) and precipitate (lane 7). The supernatant was diluted 4-fold or 20-fold, respectively. The diluted solution was separated into the supernatant (lane 8) and precipitate (lane 9). Proteins in the precipitate were dissolved in 8 M urea and subjected to 10% SDS–PAGE.

Discussion

In our previous study, we indicated that canavalin is precipitated by the addition of 20 mM MgCl₂,⁵⁾ whereas in the present study, we showed that canavalin was insolubilized in the presence of divalent cations at relatively low concentrations and solubilized in the presence of the same cations at relatively high concentrations. In addition, we formulated a schematic representation of the reversible induction of canavalin insolubilization and solubilization by changing concentrations of Mg²⁺ and Ca²⁺ (Fig. 7). Soluble canavalin was prepared as described previously⁵⁾ and precipitated by the addition of MgCl₂ or CaCl₂ at relatively low concentrations, but not by the addition of NaCl (arrow 1). The insolubilized canavalin was not resolubilized in distilled water (arrow 2), but only in the presence of Mg²⁺ and Ca²⁺ at relatively high concentrations (arrow 3). In addition, the soluble canavalin was not precipitated by the addition of 60 mM Mg²⁺ or 200 mM Ca²⁺ (arrow 5), but it was with the decreasing concentration of Mg²⁺ and Ca²⁺ (arrow 4). Based on these results, we assumed that the soluble canavalin prepared by the addition of relatively high concentration of divalent cations to the crude extract was the same with that prepared by the addition of relatively high concentration of divalent cations to insoluble canavalin.

Fig. 6. Solubility changes to solubilized canavalin.

Notes: Magnesium chloride (MgCl₂; A) and calcium chloride (CaCl₂; B) were added to the sword bean extract at a concentration of 60 and 200 mM, respectively. Distilled water was used instead of salt as a control (lane 1). The mixture was separated into the supernatant (lane 2) and precipitate (lane 3). The supernatant was diluted 4- or 20-fold, respectively. The diluted solution was separated into the supernatant (lane 4) and precipitate (lane 5). Proteins in the precipitate were dissolved in 8 M urea and subjected to 10% SDS–PAGE.

Fig. 7. Schematic representation of canavalin solubility.

Notes: Arrows show changes in the concentration of divalent cations. Cross mark indicates no progress. “Low” indicates 15 mM Mg²⁺ or 10 mM Ca²⁺. “High” indicates 60 mM Mg²⁺ or 200 mM Ca²⁺.

In the solubilization phase, the C_m value was determined at 40 mM for Mg²⁺ and at 50 mM for Ca²⁺. The results clearly indicated that the insolubilized canavalin was more easily solubilized in a solution containing Mg²⁺ than in a solution containing Ca²⁺. In the insolubilization phase, the C_m value was determined at 3.86 mM for Mg²⁺ and 3.93 mM for Ca²⁺. The similar C_m values indicated that the effects of Mg²⁺ and Ca²⁺ on canavalin insolubilization do not differ significantly. However, the sigmoidal graphs of canavalin insolubilization (Figs. 3(B) and 4(C)) suggested that the protein shows a higher sensitivity to Mg²⁺ than to Ca²⁺ from the different initial concentrations for the insolubilization. Our previous study indicated that different metal ion species influence the C_m value of protein association during tofu formation.²⁴⁾ The C_m value is significantly negatively correlated with the stability constant of EDTA for metal ions, and this strong correlation influences the interaction between metal ions and the carboxyl groups of soymilk proteins. Studies on the primary structure of canavalin^10,11) indicate that the 419 amino acid residues of the matured form of canavalin include 36 glutamic acid residues and 23 aspartic acid residues. Therefore, changes in canavalin solubility might be also induced by the interaction between metal ions and the carboxyl groups of the protein. However, it was difficult to clarify the mechanism underlying the modulation of solubility change, because the sword bean extract contained many non-proteinaceous substances, such as polysaccharides, that might also interact with canavalin. Thus, further studies are needed to reveal the underlying molecular mechanism.

Sword bean contains abundant calcium and magnesium ions.²⁶⁾ Based on studies by Eknkaya et al.,²⁶⁾ we estimated the Ca²⁺ and Mg²⁺ concentrations in our sword bean extract to be approximately 4.6 and 8.8 mM, respectively. The divalent cations inherently present in sword bean affect the primary solubility change of canavalin. Thus, our results must be interpreted with consideration of the fact that the C_m values in this study indicate the midpoint concentration for the insolubilization and solubilization in the sword bean extract prepared by the method we describe, but might not represent the essential features of canavalin. At present, since highly pure canavalin is insoluble in distilled water (Fig. 5), it is experimentally difficult to determine the C_m value of purified canavalin for the divalent cations. However, it would be important to determine the C_m value of the unrefined canavalin in the extract to facilitate the use of canavalin in food processing. For example, tofu is also processed by the addition of divalent cations to extracts of soybean, which also contains abundant divalent cations. As we have previously reported,⁵⁾ the methodologies used in tofu production could be applied to the development of processed foods using canavalin. In our previous study, we determined C_m values for tofu formation, and a comparison of C_m values between various metal ions clarified the role of metal ions in tofu formation.²⁴⁾ The C_m value for canavalin precipitation would thus be useful information for the development of processed foods using canavalin.

Some proteins are precipitated by the addition of salts, whereas others are solubilized. For example, during tofu formation, soybean proteins are precipitated by an increase in divalent cations,^24,27) while chymosin B, β-lactoglobulin B and pumpkin seed globulin demonstrate salting-in behavior—that is, they are solubilized in the presence of high salt concentrations.²⁸⁾ In the present study, canavalin was insolubilized by the addition of divalent cations at relatively low concentrations, but was solubilized by the addition of the same cations at relatively high concentrations. Briefly, canavalin solubility can be modulated by increasing divalent cation concentration. The modulation of canavalin solubility is interesting from the viewpoint of protein chemistry. With increase in the divalent cation concentration, soluble canavalin decreased in the insolubilization phase, but increased in the solubilization phase. Therefore, canavalin insolubilization was altered to solubilization in a salt concentration-dependent manner. The phase transition is a unique property of canavalin. We assumed that the transition might be induced by the interaction between canavalin and non-proteinaceous substances or/and by some structural changes. However, the underlying molecular mechanism remains unclear, and further studies are needed.

The reversible change in solubility would be useful for the establishment of an inexpensive and simple method for the purification of canavalin. Studies on the primary structure of canavalin^10,11) indicated that the 419 amino acid residues of the matured form of canavalin include 52 leucine residues. A previous study describes that the consumption of leucine in combination with exercise is effective in the prevention and improvement of sarcopenia.²⁹⁾ It is also reported that, since whey proteins have relatively high leucine content (approximately 9.5 g of leucine per 100 g of proteins), the intake of whey proteins is effective to the prevention and improvement of sarcopenia.^30,31) We estimated the leucine content in canavalin to be 14.3 g of leucine per 100 g of canavalin, which is approximately 1.5 times higher than that of whey proteins. The high leucine content of canavalin indicates that the intake of canavalin might be more effective than that of whey proteins. In addition, another study reports that the intake of animal proteins such as whey proteins increases carcinogenic risk.³²⁾ This risk would be reduced if leucine intake was via canavalin, a plant protein, rather than via whey proteins. These findings indicate the desirability of using canavalin for the development of new processed foods.

In conclusion, we provided a schematic model of canavalin solubility changes induced by concentration changes of divalent cations in the crude extract. Canavalin solubility was affected by the addition of MgCl₂ or CaCl₂, but not of NaCl. Therefore, divalent cations play an important role in canavalin solubility. We also determined the C_m values for the insolubilization and solubilization phase of canavalin. Thus, our data provide important information regarding the physicochemical properties of canavalin in crude extracts of white sword bean that would be useful for the study of the physiological functions of canavalin and its application in food industry.

Author contributions

KN and YA conceived and designed the experiments. KN performed the experiments. KN and YA analyzed the data and wrote the paper. YA reviewed and edited the manuscript. KN and YA read and approved the manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by the Tojuro Iijima Foundation for Food Science and Technology [grant number 11 (individual research, 2014)].

Acknowledgments

We would like to thank Editage (www.editage.jp) for English language editing.