Chemical structure and gelation characteristics of a purified gel derived from sword beans (Canavaliagladiata).

Herein, a new method was developed to obtain a crude extract from sword beans at a higher extraction efficiency. The crude extract formed a gel at 8 °C, which melted at 70 °C, and lyophilization of the purified gel produced a powder that could be dissolved in distilled water at a concentration of 7 % (w/w) or less. A 3 % powder solution gelled at 12 °C and melted at 60 °C. The infrared spectrum of the gel powder was consistent with that of starch. Furthermore, a 4-aminobenzoic acid ethyl ester-labeling analysis revealed that glucose was the constituent sugar in the powder, and the powder solution reacted strongly in a starch-iodine test. These observations confirmed that the gelling substance was starch. However, the melting and gelling temperatures were dissimilar to those of other starches frequently used in the food industry. Thus, our results provide valuable information for using sword bean starch as a novel food material.

Development of Tofun: A new sweet confection made from soymilk and honey.

Tofu, a common part of the Asian diet, is soft, making it a good candidate for developing foods for people with dysphagia. Honey can coagulate soymilk proteins due to high levels of gluconic acid. In the present study, we aimed to investigate the soymilk-coagulating ability of 19 honey varieties to develop a soft sweet for people with dysphagia. We found that honey gathered from flora on Awaji Island had a higher coagulating ability than those of 18 other tested honey varieties. Based on the volume-to-weight ratio of honey, trial products were processed at honey concentrations of 20%, 25%, and 30% (w/w). The hardness, adhesiveness, and cohesiveness of products were examined according to the Consumer Affairs Agency's permissible criteria for foods for people with dysphagia. The hardness and adhesiveness of products at 25% and 30% honey concentrations were significantly higher than those at 20% honey concentration. Overall, the product evaluation was consistent with Permission Criteria II (can be swallowed after slight maceration). In terms of physical properties and product cost, the added honey concentration was determined to be 25% (w/w) for commercialization. The sweet was named "Tofun" and commercialized via collaboration with a local company as part of our community development efforts. The introduction of Tofun will encourage further innovation in the sugar confectionery industry.

Direct comparison of the tofu-like precipitate formation by adding different coagulants: magnesium chloride and glucono-δ-lactone.

Tofu is produced by adding a coagulant such as MgCl2 and glucono-δ-lactone (GDL) in soymilk. However, the molecular mechanism of tofu formation by adding these coagulants has been compared between the results obtained under different conditions. In this study, the formation of a tofu-like precipitate (TLP) by adding GDL was directly compared with that formed by adding MgCl2 under the same conditions except for the coagulants. The effects of both the coagulants were almost the same on the changes in the precipitate weight, supernatant protein concentration, and urea-soluble protein concentration, indicating that a common contributing factor induces TLP formation. However, the effects of the coagulants on pH were largely different, suggesting that pH reduction is not an absolute requirement in TLP formation induced by adding MgCl2. Moreover, the findings of this study revealed that the decrease in the surface charge of soymilk proteins is a common initiation factor for TLP formation, whereas the intermolecular hydrophobic interaction is an important factor for the formation of urea-insoluble precipitates. Overall, these findings will be useful in discovering new coagulants to enhance the quality characteristics of tofu.

Honey-mediated aggregation of soymilk proteins.

Gluconic acid, the major organic acid in honey, is a partial hydrolysate of glucono-δ-lactone, typically used as a coagulant in preparing tofu. The present study aimed to examine the coagulation potential of five different types of honey at different concentrations, upon addition to soymilk. In some samples, aggregates formed in the upper layer at a higher honey concentration, while in others, aggregates precipitated at an intermediate honey concentration. Both phenomena were reproduced by adding different mixtures of glucono-δ-lactone and glucose, indicating that gluconic acid concentration and total sugar content of honey can trigger soymilk coagulation. Interestingly, honeys with a high concentration of gluconic acid showed a low total sugar content. Furthermore, in a trial product, the mixture of blended honey with soymilk was determined to be pasty. Our results indicate that honey can coagulate soymilk, which may provide a new and convenient method to prepare soymilk-based industrial products.

Structural transitions of sword bean canavalin in response to different salt concentrations.

Canavalin is the major storage protein in sword beans (Canavalia gladiata) and belongs to the 7S seed globulin class. Canavalin solubility can be reversibly altered via the addition of MgCl2 and CaCl2 at different concentrations; specifically, it is insoluble at lower concentrations and soluble at higher concentrations. However, the addition of NaCl does not induce the insolubilization of canavalin. In this study, it was determined that the addition of NaCl causes the nearly complete solubilization of MgCl2-precipitated canavalin in the presence of high concentrations. Moreover, using gel filtration we examined the quaternary structures of soluble canavalin in the bean extract and in the presence of high-concentration salts. Results indicated that canavalin was present in the monomer form within crude extracts with distilled water. Alternatively, we identified trimeric soluble canavalin in the presence of high concentrations of NaCl or MgCl2. Our study revealed that the quaternary structures of sword bean soluble proteins differ in crude extract compared to those in high-concentration salt solutions. The three-dimensional structure of ß-conglycinin, which is a typical 7S-seed globulin in soybean, has a trimer form in the presence of high concentrations of NaCl. However, it remains unclear whether ß-conglycinin is present as trimers in soybean seed. Our findings serve as an important reference to analyze 7S globulin characteristics.

Divalent magnesium cation concentrations determine the formation of tofu-like precipitates with differing urea solubilities.

Different coagulant concentrations can induce urea-soluble precipitates (USPs) and urea-insoluble precipitates (UIPs) during tofu-like precipitate formation. In this study, MgCl2 concentration-dependent changes in USPs were quantified using a method based on urea solubility differences in order to investigate the factors affecting USP versus UIP formation. The addition of various Mg salts revealed that anions influence the solubility of proteins in both USPs and UIPs. Moreover, addition of MgCl2/NaCl mixtures, in which the Cl- concentration was held constant, demonstrated that Mg2+ was essential for UIP formation but not for USP formation and that Cl- was inconsequential for both USP and UIP formation. NaCl addition showed that the driving force for USP formation was salting-out due to the presence of cations. Overall, our data indicated that the Mg2+ concentration determined the separation of USPs and UIPs. These results will help elucidate the molecular mechanisms mediating the separation of silken tofu and regular tofu.

Sword bean variants and different pretreatments influence protein extraction and protein properties.

Two variants of the sword bean (Canavalia gladiata), namely the white sword bean (WSB) and the red sword bean (RSB), are known. The MgCl2 concentration-dependent canavalin solubility showed different behavior among the extracts from distinct beans prepared by distinct pretreatments. Pretreatment and bean selection are important factors for use in food chemical and biochemical experiments.

A crude sword bean (Canavalia gladiata) extract is gelated by cooling.

White sword bean (Canavalia gladiata) seeds have the potential to be utilized in the manufacturing of processed foods owing to their high protein and carbohydrate content. Our previous reports explored the use of the sword bean as a source of food materials by preparing extracts in distilled water. In the present study, we found that one such extract can be gelated by cooling. The gelling substances were extracted by boiling and simultaneously stirring a suspension containing ground beans. Few proteins were present in the gelated extract. We also examined the conditions under which gelation occurred and the gel melting temperature. The extract gelated at temperatures below 10 °C, and the resulting gel melted at those above 65 °C. This is the first report that gelling substances can be extracted from sword beans in large quantities. We expect that this gelling agent can be used for the production of processed foods.

Espresso coffee foam delays cooling of the liquid phase.

Espresso coffee foam, called crema, is known to be a marker of the quality of espresso coffee extraction. However, the role of foam in coffee temperature has not been quantitatively clarified. In this study, we used an automatic machine for espresso coffee extraction. We evaluated whether the foam prepared using the machine was suitable for foam analysis. After extraction, the percentage and consistency of the foam were measured using various techniques, and changes in the foam volume were tracked over time. Our extraction method, therefore, allowed consistent preparation of high-quality foam. We also quantitatively determined that the foam phase slowed cooling of the liquid phase after extraction. High-quality foam plays an important role in delaying the cooling of espresso coffee.

Trans 18-carbon monoenoic fatty acid has distinct effects from its isomeric cis fatty acid on lipotoxicity and gene expression in Saccharomyces cerevisiae.

Epidemiological studies have suggested that an excess intake of trans-unsaturated fatty acids increases the risk of coronary heart disease. However, the mechanisms of action of trans-unsaturated fatty acids in eukaryotic cells remain unclear. Since the budding yeast Saccharomyces cerevisiae can grow using fatty acids as the sole carbon source, it is a simple and suitable model organism for understanding the effects of trans-unsaturated fatty acids at the molecular and cellular levels. In this study, we compared the physiological effects of Δ9 cis and trans 18-carbon monoenoic fatty acids (oleic acid and elaidic acid) in yeast cells. The results obtained revealed that the two types have distinct effects on the expression of OLE1, which encodes Δ9 desaturase, and lipotoxicity in are1Δare2Δdga1Δlro1Δ and gat1Δ cells. Our results suggest that cis and trans 18-carbon monoenoic fatty acids exert different physiological effects in the regulation of gene expression and processing of excess fatty acids in yeast.

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

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.

Precipitation of sword bean proteins by heating and addition of magnesium chloride in a crude extract.

Sword bean (Canavalia gladiata) seeds are a traditional food in Asian countries. In this study, we aimed to determine the optimal methods for the precipitation of sword bean proteins useful for the food development. The soaking time for sword beans was determined by comparing it with that for soybeans. Sword bean proteins were extracted from dried seeds in distilled water using novel methods. We found that most proteins could be precipitated by heating the extract at more than 90 °C. Interestingly, adding magnesium chloride to the extract at lower temperatures induced specific precipitation of a single protein with a molecular weight of approximately 48 kDa. The molecular weight and N-terminal sequence of the precipitated protein was identical to that of canavalin. These data suggested that canavalin was precipitated by the addition of magnesium chloride to the extract. Our results provide important insights into the production of processed foods from sword bean.

Detection of an interaction between prion protein and neuregulin I-ß1 by fluorescence resonance energy transfer analysis.

Cellular prion protein (PrP) copurifies with neuregulin type I-ß1 (NRG I-ß1), but no interaction has been detected by a general immunoprecipitation study. We speculate that PrP interacts with NRG I-ß1. Here, the interaction of PrP with NRG I-ß1 was detected by measuring fluorescence resonance energy transfer (FRET) between enhanced blue (EBFP) and enhanced green (EGFP) fluorescent protein-fusion proteins. Full-length PrP interacted with EGFP in addition to NRG I-ß1. From this result, we deduced that PrP interacts with EGFP through its unstructured N-terminal domain. We therefore detected FRET between PrP deleting the N-terminal domain and NRG I-ß1. In contrast, the C-terminal domain of PrP interacted with NRG I-ß1 and the proteins dissociated completely in the presence of sodium chloride. This interaction occurs at the nanomolar level, which is important for the reaction to be functional in organisms. We concluded that PrP interacted with NRG I-ß1 through its C-terminal domain.

Role of calcium-binding sites in calcium-dependent membrane association of annexin A4.

Annexin A4 (Anx4) is a cytosolic calcium-binding protein with four repeat domains, each containing one calcium-binding site (CBS). The protein interacts with the phospholipid membrane through the CBS-coordinated calcium ion, although the role of each CBS in the calcium-dependent association is unclear. To determine the role of each CBS, 15 CBS-abolished variants were produced in various combinations by substitution of a calcium-liganding residue on each CBS by Ala. Various mutant combinations produced different influences on calcium-dependent membrane-binding behavior and on the sodium-dependent dissociation of membrane-bound Anx4. Our data suggest the interaction of Anx4 with the lipid membrane consists of strong and weak interactions. CBSs I and IV mediate formation of strong interactions, while CBSs II and III are important for weak interactions. We also suggest Anx4 binds the lipid membrane through CBSs I and IV in the cytoplasmic fluids.

Initiation of protein association in tofu formation by metal ions.

Magnesium and calcium ions are important factors in making tofu. However, the molecular role of these ions remains unclear in tofu formation. We have previously shown that magnesium chloride concentration-dependent produced silken tofu-like (SP) and regular tofu-like (RP) precipitates, but was an inconsequential factor for the retention of tofu. We investigated in this present study, the effect of various metal chlorides on the metal chloride concentration-dependent changes in tofu formation. These changes occurred in a similar manner to that of the magnesium ion, in which SP formation was followed by RP formation. It is interesting that the midpoint concentration for the formation of SP and RP represented a good correlation with the stability constant of EDTA. This correlation demonstrated the possibility that metal ions would interact with the carboxyl groups of soy proteins. We consider from these results that metal ions were the initiators of protein association in tofu formation.

Magnesium chloride concentration-dependent formation of tofu-like precipitates with different physicochemical properties.

A wet precipitate is generated in the process of making tofu by adding a coagulant to the basic soymilk ingredient. We investigated the magnesium chloride concentration-dependent change in the precipitate weight. The wet precipitate weight dramatically increased following a short plateau phase at a low concentration of magnesium chloride. It is interesting that this weight slightly decreased following a long plateau phase at a high concentration. These low and high concentrations respectively induced precipitates with a smooth surface and rough surface. The precipitate with a smooth surface had a higher water content than that with a rough surface. These precipitates also had obviously different solubility in various chemical reagents. The different properties indicate that these precipitates were formed by different intermolecular interactions. These results can be utilized to more clearly understand the mechanisms involved in tofu formation.

High hatching rates after cryopreservation of hydrated cysts of the brine shrimp A. franciscana.

Cysts of Artemia franciscana are known to be extremely tolerant to UV and ionizing radiation, hypoxia, dryness, osmotic pressure, and temperatures. However, when cysts are hydrated, their resistance to extreme environmental conditions is markedly reduced, and they subsequently enter a developmental sequence. The hatching rate of hydrated cysts declined when they were rapidly frozen after a short period of hydration but slow freezing improved hatching rates after 6-h hydration (1.4 g H2O per g dry wt). We observed that trehalose content in hydrated cysts was greatly reduced up to 6-h time. DSC analysis showed different thermal profiles at two cooling rates, suggesting the formation of a minuscule ice crystal inside the cells. High hatching rates can be obtained from highly hydrated cysts at slow cooling rate.

Network structure and forces involved in perilla globulin gelation: comparison with sesame globulin.

Scanning electron micrographs show that perilla globulin gel had a finer network structure than sesame α-globulin gel. The effects of various reagents on the gel formation and solubility of perilla and sesame gels were compared. The contribution of disulfide bonds to the formation and stability of perilla gel was greater than to sesame gel, despite having the same subunit structure.

Production of a recombinant full-length prion protein in a soluble form without refolding or detergents.

Recombinant prion protein has been produced in insoluble form and refolded following solubilization with denaturants. It is, however, preferable to use a soluble recombinant protein prepared without artificial solubilization. In this study, a soluble recombinant prion protein was produced in Escherichia coli cells by coexpression of neuregulin I-ß1 and purified to high purity.

Subunit structure and functional properties of the predominant globulin of perilla (Perilla frutescens var. frutescens) seeds.

Approximately 40% of defatted perilla seeds consists of proteins which are primarily composed of globulin (84%). The amino acid profile of perilla proteins demonstrated balanced amounts of all essential amino acids, except for lysine. The molecular mass of the predominant globulin was estimated to be 340 kDa by gel filtration. This globulin was separated into three intermediary subunits (54, 57 and 59 kDa) by SDS-PAGE. It is suggested from these results that the globulin exists as a hexamer. A treatment with 50 mM dithiothreitol enabled the intermediary subunits to be separated into three acidic subunits (31-34 kDa) and four basic subunits (23-25 kDa). It is interesting that this subunit structure is the same as that of sesame α-globulin, despite them coming from different families. Compared to sesame α-globulin, the heat-induced gel of perilla globulin had better water-holding ability, despite it displaying the same degree of gel hardness.