Junko Funaki

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‐NH2‐Mec) at Km = 9.5 µm, which is equivalent to the Km value for hydrolysis of this substrate by cathepsin L. Hydrolysis was inhibited by two wheat cystatins, WC1 and WC4, with IC50 values of 1.7 × 10−8 and 5.0 × 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.

Wheat cysteine proteases triticain α, β and γ 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.

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.

Utilization of Oryzacystatin for Regulating the Ripening of Squid Shiokara, a Traditional Japanese Salted and Fermented Seafood

Shiokara is a fermented seafood composed of sliced squid mantle muscle ripened with fresh squid liver. Preliminary sensory evaluation by using the ranking test revealed that the hardness of squid muscle in shiokara was reduced within 7 d of ripening. During the process of ripening, muscle proteins were digested by proteinases present in squid liver. The degradation of paramyosin and myosin heavy chain was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hardness of squid mantle muscle in shiokara was reduced with the degradation of paramyosin and myosin heavy chain. This degradation was mainly caused by E-64-sensitive cysteine proteinases. To control the hardness of shiokara, we used rice seed oryzacystatin, which suppresses proteolysis by papain-like cysteine proteinases. When oryzacystatin was added 4 d after the start of shiokara ripening, the muscle protein degradation stopped, without further muscle softening. These results show that oryzacystatin is useful to control the ripening of shiokara by regulating its hardness.

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 β-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 β-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.

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

Abstract 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.

A Predictive Model Based on Surface Electromyography to Assess the Easinessof Deglutition of Dysphagia Diets

Dysphagia diet is used for the people who have disorder of swallowing caused by aging or cerebral arterial diseases. The current standards for dysphagic diets are based on their physical characteristics. However, parameters that reflect the easiness of swallowing are also critical. Here, we developed a method to objectively evaluate the easiness of deglutition. First, we collected 68 terms that describe food textures related to easiness of deglutition, and selected 54 commercial dysphagia diets as samples. Using these terms and samples, we conducted a texture-perception questionnaire survey, and the results were subjected to a correspondence analysis. Referring to the results of this analysis, 10 textures that represent the easiness of deglutition were selected and dysphagia diets corresponding to each texture were selected as well. Then, sensory evaluation and surface electromyography (sEMG) of the anterior triangle of the neck (submental triangle) were recorded using these samples. We developed a predictive model for the easiness of deglutition by applying a partial least squares (PLS) regression technique to the sensory evaluation and sEMG data. Parameter fitting of the cross-validation model was significant (R2, 0.87; RMSE, 0.34). The model accuracy was further investigated by fitting the model to test data, and results were again significant (R2, 0.89; RMSE, 0.10). This indicates that our predictive model using sEMG measurements was highly accurate. Evaluating the easiness of deglutition with this predictive model will help identify and develop new foods that make swallowing easier for patients with dysphagia.