Naofumi Kitabatake

Promotion of germination and shoot elongation of some plants by alginate oligomers prepared with bacterial alginate lyase

Abstract Depolymerization of sodium alginate (average molecular weight: 25,700) from an edible seaweed Eisenia bicyclis with bacterial alginate lyase yielded oligosaccharide(s) with an average molecular weight of 1,800 together with other components. The proliferation and/or differentiation of plants was markedly enhanced in the presence of the oligosaccharide(s), although that of mammalian (HeLa) cells and an algae (Chlamidomonas sp.) was not. The results indicate that the depolymerization products of alginate containing oligosaccharine-like compounds specifically affect the proliferation and/or differentiation of higher plants.

Irreversible thermal denaturation and formation of linear aggregates of ovalbumin

Abstract Changes in several physicochemical properties related to the thermal denaturation of ovalbumin have been investigated at neutral pH and low ionic strength. The far-UV circular dichroism (CD) spectrum at 80°C indicated small secondary structural changes compared with those induced by addition of guanidine hydrochloride (GuHCl). The near-UV CD spectrum and difference absorption spectrum (250–320 nm) showed completely irreversible micro-environmental changes around the aromatic amino acid residues upon heat treatment (at ≥67°C). In the sedimentation measurements of heated ovalbumin solutions a sharp, single peak, corresponding to soluble aggregates of low polydispersity, appeared, and these aggregates were observed as linear polymers by transmission electron microscopy. After 2 h of heating at 75°C at pH 7.0, the intrinsic viscosity was ~20 times higher than the native one. We conclude that, under these conditions, although the globule form of ovalbumin molecule did not alter drastically upon thermal denaturation, partially denatured molecules which would expose the hydrophobic area(s) aggregate immediately and linear polymers (high-molecular-weight soluble aggregates) were formed.

Recognition of Native and/or Thermally Induced Denatured Forms of the Major Food Allergen, Ovomucoid, by Human IgE and Mouse Monoclonal IgG Antibodies

Human sera obtained from children with egg allergy reacted well with both native and heated ovomucoid (OM). Ovalbumin is present in egg white in a 5 times greater quantity than OM; however, it easily aggregates and becomes difficult to extract by heating. For accurate food allergen labeling of processed food, therefore, OM should be evaluated with the determination of egg white protein in consideration of heat denaturation. Three kinds of monoclonal antibodies and sandwich ELISA tests were established which are able to recognize the native and/or heat-denatured forms of OM. The usefulness of these characteristic mAbs and ELISA tests are discussed in relation to allergen labeling, monitoring food processing, and movement or change of dietary protein in vivo.

Structure of glycinin and ovalbumin gels

Abstract Research on the structures of heat-induced gels of glycinin and ovalbumin is reviewed. Both glycinin and ovalbumin gels are thought to have a so-called ‘string of beads’ structure. In glycinin gels one building unit of the string (a bead) has been thought to be a whole molecule. This means that heat-denatured glycinin molecules do not dissociate into their constituent subunits. An alternative explanation which includes dissociation and reassociation of glycinin has been presented. Heated ovalbumin solutions make transparent gels, turbid gels, clear solutions or turbid suspensions containing aggregates, depending on the pH, ionic strength and protein concentration of the medium. The transparent solutions contain linear polymers of high molecular weight. Ovalbumin gels are thought to be formed by a network of linear polymers. The ‘string of beads’ model has been accepted as the structure of some globular protein gels including glycinin, ovalbumin, serum albumin, ribonuclease and lysozyme. However, how the heat-denatured asymmetric protein molecules combine to make an ordered string has not been explained.

Heat-induced Transparent Gel Formation of Bovine Serum Albumin

The formation of transparent gels by 6% bovine serum albumin (BSA), pH 7.5, was examined by one- and two-step heating methods. Heating of the BSA solutions at various NaCl concentrations produced transparent gels at 25-50mM NaCl and transparent sols at 0-20 mM NaCl (one-step heating method). The transparent sol obtained by heating without NaCl was reheated after mixing with various amounts of NaCl (two-step heating method I). The result was almost identical to that obtained by the one-step heating method. However, when the first heating was done with 10 mM NaCl, transparent gels were obtained over a wide range of NaCl concentrations with a second heating (two-step heating method II). Analyses of sols obtained at various NaCl concentrations by gel permeation chromatography and transmission electron microscopy showed the presence of linear polymers in the heated BSA sol (10 mM NaCl) and gel networks formed by the linear polymers (20 mM NaCl). The mechanism of transparent gel formation in BSA may be similar to that in ovalbumin.

Critical molecular regions for elicitation of the sweetness of the sweet-tasting protein, thaumatin I

Thaumatin is an intensely sweet‐tasting protein. To identify the critical amino acid residue(s) responsible for elicitation of the sweetness of thaumatin, we prepared mutant thaumatin proteins, using Pichia pastoris, in which alanine residues were substituted for lysine or arginine residues, and the sweetness of each mutant protein was evaluated by sensory analysis in humans. Four lysine residues (K49, K67, K106 and K163) and three arginine residues (R76, R79 and R82) played significant roles in thaumatin sweetness. Of these residues, K67 and R82 were particularly important for eliciting the sweetness. We also prepared two further mutant thaumatin I proteins: one in which an arginine residue was substituted for a lysine residue, R82K, and one in which a lysine residue was substituted for an arginine residue, K67R. The threshold value for sweetness was higher for R82K than for thaumatin I, indicating that not only the positive charge but also the structure of the side chain of the arginine residue at position 82 influences the sweetness of thaumatin, whereas only the positive charge of the K67 side chain affects sweetness.

The cysteine-rich domain of human T1R3 is necessary for the interaction between human T1R2-T1R3 sweet receptors and a sweet-tasting protein, thaumatin.

Thaumatin is an intensely sweet-tasting protein perceived by humans but not rodents. Its threshold value of sweetness in humans is 50nM, the lowest of any sweet-tasting protein. In the present study, the sites where sweet receptors interact with thaumatin were investigated using human embryonic kidney 293 (HEK293) cells expressing the sweet receptors T1R2-T1R3. Chimeric human- mouse sweet receptors were constructed and their responses to sweeteners were investigated. The human (h) T1R2- mouse (m) T1R3 combination responded to sucralose but not to thaumatin, clearly indicating that a T1R3 subunit from humans is necessary for the interaction with thaumatin. Furthermore, results obtained from using chimeric T1R3s showed that the cysteine-rich domain (CRD) of human T1R3 is important for the interaction with thaumatin. The CRD of T1R3 would be a prominent target for designing new sweeteners.

High-resolution structure of the recombinant sweet-tasting protein thaumatin I

Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at a concentration of 50 nM. The crystal structure of a recombinant form of thaumatin I produced in the yeast Pichia pastoris has been determined to a resolution of 1.1 Å. The model was refined with anisotropic B parameters and riding H atoms. A comparison of the diffraction data and refinement statistics for recombinant thaumatin I with those for plant thaumatin I revealed no significant differences in the diffraction data. The R values for recombinant thaumatin I and plant thaumatin I (F(o) > 4σ) were 9.11% and 9.91%, respectively, indicating the final model to be of good quality. Notably, the electron-density maps around Asn46 and Ser63, which differ between thaumatin variants, were significantly improved. Furthermore, a number of H atoms became visible in an OMIT map and could be assigned. The high-quality structure of recombinant thaumatin with H atoms should provide details about sweetness determinants in thaumatin and provide valuable insights into the mechanism of its interaction with taste receptors.

Traditional non-alcoholic beverage, Togwa, in East Africa, produced from maize flour and germinated finger millet.

The traditional non-alcoholic beverage in East Africa, togwa, produced from the flour of maize and germinated finger millet (finger millet malt), was investigated. The preparation techniques of togwa observed in the rural villages of East Africa are described, and the temperature and pH profile of togwa during its manufacture are also shown. Maize and finger millet malt should be the source of starch and amylase, respectively. Maize flour slurry was heated once with stirring up to around 80°C and cooled to about 50°C, and then finger millet malt flour was added to the warm porridge paste and kept at about 50°C for 20 min. The consistency of the paste was suddenly reduced by addition of finger millet malt flour and the gel paste changed to viscous liquid, which was kept in a container and incubated at ambient temperature for 15 h. After incubation it became sweet and was ready to drink without removal of any insoluble materials. Changes in the concentration of glucose and lactic acid of togwa during its maturation period were measured using portable devices until 70 h incubation at the rural village of Tanzania in a dry season. Glucose level increased with incubation and reached the threshold value of sweetness; 24 h incubation later, the lactate level increased and pH decreased. The preparation techniques of a traditional alcoholic beverage, pombe, were also investigated in the same rural locality, and the differences and characteristics of both traditional beverages are discussed.

Melting of heat-induced ovalbumin gel by pressure

The effects of pressure on heat-induced gels of ovalbumin were examined. A heat-induced gel containing 7% ovalbumin and 10 mmol/dm3 Na-phosphate buffer, pH 7.0, melted completely with pressure at 600 MPa for 20 min at 20°C. The melted sample gelled again after the pressure ceased. Most of the heat-induced gels of ovalbumin prepared under various conditions (different protein concentrations, ionic strengths, pHs and heating methods) melted partly with pressure at 600 MPa for 20 min at 20°C, and gelled again at atmospheric pressure. However, a heat-induced gel of glycinin and cold-set gels of gelatin or agarose did not melt with pressure at 400–700 MPa for 20 min at 20°C. The pressure denaturation of ovalbumin and the renaturation of heat-denatured ovalbumin caused by pressure were also examined. The pressure denaturation of ovalbumin was partly reversible and heat-denatured ovalbumin renatured somewhat with treatment at 600 MPa for 20 min at pH 7.0 and low ionic strength.