Effect of Proteolytic Modification on Texture and Mastication of Heat-Treated Egg White Gels.

Raw egg white undergoes sol-gel transition by heat treatment, which changes it to an elastic gel. Here, protease treatment to render a new texture to heated egg white gel was applied. Protease-treated gels exhibited ductile flow without obvious rupture points. Transmission electron microscopy analysis showed that in protease-treated gels, protein aggregates were distributed more homogeneously compared with that observed in the untreated control, probably because ovalbumin was digested into small peptides as revealed by SDS-PAGE. The properties of the gel were evaluated by sensory tests and by measuring the movement of the masseter muscle, using surface electromyography. Results showed that maximum bite force and mastication duration were decreased for the protease-treated gels, which were evaluated as being softer, smoother, less elastic and better textured. Overall, our results indicate that protease-treated egg white gel has superior qualities and is easier to swallow than the untreated gel. PRACTICAL APPLICATIONS: In the food industry, the use of egg white is limited compared with that of egg yolk and whole eggs. In this study, we performed protease treatment to generate a new food material with smoother and softer texture compared with heat treated egg white. Our findings may expand the consumption of egg white, which can be consumed by people with mastication and swallowing disorders, and reduce the waste of egg white as a surplus product.

Identification of bitterness-masking compounds from cheese.

Bitterness-masking compounds were identified in a natural white mold cheese. The oily fraction of the cheese was extracted and further fractionated by using silica gel column chromatography. The four fractions obtained were characterized by thin-layer chromatography and nuclear magnetic resonance spectroscopy. The fatty acid-containing fraction was found to have the highest bitterness-masking activity against quinine hydrochloride. Bitterness-masking activity was quantitated using a method based on subjective equivalents. At 0.5 mM, the fatty acid mixture, which had a composition similar to that of cheese, suppressed the bitterness of 0.008% quinine hydrochloride to be equivalent to that of 0.0049-0.0060% and 0.5 mM oleic acid to that of 0.0032-0.0038% solution. The binding potential between oleic acid and the bitter compounds was estimated by isothermal titration calorimetry. These results suggest that oleic acid masked bitterness by forming a complex with the bitter compounds.

Wheat cysteine proteases triticain alpha, beta and gamma exhibit mutually distinct responses to gibberellin in germinating seeds.

We cloned three novel papain-type cysteine proteases (CPs), triticain alpha, beta and gamma, from 1-d-germinating wheat seeds. Triticain alpha, beta and gamma were constituted with 461, 472 and 365 amino acid residues, respectively, and had Cys-His-Asn catalytic triads as well as signal and propeptide sequences. Triticain gamma contained a putative vacuole-sorting sequence. Phylogenetic analysis showed that these CPs were divided into mutually different clusters. Triticain alpha and gamma mRNAs were expressed in seeds at an early stage of maturation and at the stage of germination 2d after imbibition, while triticain beta mRNA appeared shortly after imbibition. The expression of mRNAs for triticain alpha and gamma was suppressed by uniconazol, a gibberellin synthesis inhibitor. All the three CP mRNAs were strongly expressed in both embryo and aleurone layers. These results suggest that triticain alpha, beta and gamma play differential roles in seed maturation as well as in digestion of storage proteins during germination.

Application of an Aspergillus saitoi protease preparation to soybean curd to modify its functional and rheological properties.

An Aspergillus saitoi protease preparation, Molsin, was found to contain beta-glucosidase as well as protease activities. Application of Molsin to soybean curd improved its functionality by converting the contained isoflavone glycosides to their aglycones through beta-glucosidase, and also modified the rheological property into a creamy consistency through protease. The enzymatically modified soybean curd was characterized by a ductility flow having no particular rupture point.

Gliadain, a gibberellin-inducible cysteine proteinase occurring in germinating seeds of wheat, Triticum aestivum L., specifically digests gliadin and is regulated by intrinsic cystatins.

We cloned a new cysteine proteinase of wheat seed origin, which hydrolyzed the storage protein gliadin almost specifically, and was named gliadain. Gliadain mRNA was expressed 1 day after the start of seed imbibition, and showed a gradual increase thereafter. Gliadain expression was suppressed when uniconazol, a gibberellin synthesis inhibitor, was added to germinating seeds. Histochemical detection with anti-gliadain serum indicated that gliadain was present in the aleurone layer and also that its expression intensity increased in sites nearer the embryo. The enzymological characteristics of gliadain were investigated using recombinant glutathione S-transferase (GST)-progliadain fusion protein produced in Escherichia coli. The GST-progliadain almost specifically digested gliadin into low molecular mass peptides. These results indicate that gliadain is produced via gibberellin-mediated gene activation in aleurone cells and secreted into the endosperm to digest its storage proteins. Enzymologically, the GST-progliadain hydrolyzed benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin (Z-Phe-Arg-NH(2)-Mec) at K(m) = 9.5 microm, which is equivalent to the K(m) value for hydrolysis of this substrate by cathepsin L. Hydrolysis was inhibited by two wheat cystatins, WC1 and WC4, with IC(50) values of 1.7 x 10(-8) and 5.0 x 10(-8) m, respectively. These values are comparable with those found for GST-progliadain inhibition by E-64 and egg-white cystatin, and are consistent with the possibility that, in germinating wheat seeds, gliadain is under the control of intrinsic cystatins.

Differential expression of wheat aspartic proteinases, WAP1 and WAP2, in germinating and maturing seeds.

Two aspartic proteinase (AP) cDNA clones, WAP1 and WAP2, were obtained from wheat seeds. Proteins encoded by these clones shared 61% amino acid sequence identity. RNA blotting analysis showed that WAP1 and WAP2 were expressed in both germinating and maturing seeds. The level of WAP2 mRNA expression was clearly weaker than that of WAP1 in all tissues of seeds during germination and maturation. APs purified from germinating seeds were enzymatically active and digested the wheat storage protein, gluten. To elucidate the physiological functions of WAP1 and WAP2 in seeds, we investigated the localisation of WAP1 and WAP2 by in situ hybridisation. In germinating seeds investigated 24h after imbibition, both WAP1 and WAP2 were expressed in embryos, especially in radicles and shoots, scutellum, and the aleurone layer. In maturing seeds, WAP1 was expressed in the whole embryo, with slightly stronger expression in radicles and shoots. WAP1 was also expressed in the aleurone layer 3 weeks after flowering. Strong signals of WAP1 mRNA were detected in the whole embryo and aleurone layer 6 weeks after flowering. On the other hand, WAP2 was scarcely detected in seeds 3 weeks after flowering, and thereafter weak signals began to appear in the whole embryo. WAP1 and WAP2 were expressed widely in germinating and maturing seeds. Such diversity in site- and stage-specific expression of the two enzymes suggests their differential functions in wheat seeds.