Effect of fruit maturation on N-glycosylation of plant-derived native and recombinant miraculin.

Miracle fruit, Synsepalum dulcificum, produces a unique taste-modifying protein, miraculin (MIR), which has an attractive potential for commercial application as a novel low-calorie sweetener. To establish a stable supply system for MIR, a previous study established a platform for recombinant MIR (rMIR) production in tomato plants and demonstrated that native miraculin from miracle fruit (nMIR) and rMIR were almost identical in their protein modifications with N-glycan. However, neither N-glycosylation nor the influence of fruit maturation on the structural changes of N-glycan have been fully characterized in detail. Here, with a focus on N-glycosylation and the contribution of fruit maturation to N-glycan, we reanalyzed the N-glycosylation of the natural maturation stages of nMIR and rMIR, and then compared the N-glycan structures on MIRs prepared from the fruit at two different maturation stages. The detailed peptide mapping and N-glycosylation analysis of MIRs provided evidence that MIRs have variants, which were derived mainly from the differences in the N-glycan structure in nMIR and the N-glycosylation in rMIR and not from the cleavage of the peptide backbone. N-Glycan analysis of MIRs from the maturation stage of fruits demonstrated that N-glycan structures were similar among nMIRs and rMIRs at every maturation stage. These results indicated that the heterogeneously expressed rMIRs had the same characteristics in post-translational modifications, especially N-glycosylation and N-glycan structures, throughout the maturation stages. This study demonstrated the potential of recombinant protein expressed in tomato plants and paves the way for the commercial use of rMIR.

Effects of different miracle fruit products on the sensory characteristics of different types of sour foods by descriptive analysis.

Miracle fruit's potential benefit is encouraging as a powerful antioxidant and sweetness enhancer due to its novel ability to modify sour taste to sweet. However, further studies are needed to evaluate the practicality of different miracle fruit products. Thus, this study aimed to characterize the taste modification effects of different miracle fruit products on different sour foods. For this, 10 trained subjects (80% female, 50% white, Mean ± SE = 27.3 ± 3.9 years old) performed a Quantitative Descriptive Analysis using a Latin Square design with three replications. One session was composed of pre- and posttest for miracle fruit administration. Five food samples (green apple, goat cheese, lemonade, plain fat-free yogurt, and cucumber pickle) and four miracle fruit products (three different brands of miracle fruit pill-Y, G, M and one powder product-P) were used and data were analyzed using analysis of variance and principal component analysis. The typical effects of miracle fruit, sweetness increase and sourness suppression were observed for all food samples albeit to different degrees. Y and M pills were most impactful while powder product was least, and goat cheese and yogurt had the most pronounced impact at increasing sweetness. Prior Miracle Fruit administration significantly decreased bitterness and increased creaminess in yogurt. However, it increased off-flavor in lemonade and pickle. Results suggest that miracle fruit usage for increasing sweetness is effective but the degree of taste-modifying effect differs according to the types of miracle fruit product and food samples applied. PRACTICAL APPLICATION: This research confirms miracle fruit's unique taste-modifying abilities and demonstrates a high potential as a sweetness enhancer to benefit human health. The results can be used to optimize miracle fruit's application and can be applied by food industry and health care provider to develop clinical remedies or disease prevention strategies.

Miraculin, a taste-modifying protein is secreted into intercellular spaces in plant cells.

A taste-modifying protein, miraculin, is highly accumulated in ripe fruit of miracle fruit (Richadella dulcifica) and the content can reach up to 10% of the total soluble protein in these fruits. Although speculated for decades that miraculin is secreted into intercellular spaces in miracle fruit, no evidence exists of its cellular localization. To study the cellular localization of miraculin in plant cells, using miracle fruit and transgenic tomato that constitutively express miraculin, immunoelectron microscopy, imaging GFP fusion proteins, and immunological detection of secreted proteins in culture medium of transgenic tomato were carried out. Immunoelectron microscopy showed the specific accumulation of miraculin in the intercellular layers of both miracle fruit and transgenic tomato. Imaging GFP fusion protein demonstrated that the miraculin-GFP fusion protein was accumulated in the intercellular spaces of tomato epidermal cells. Immunological detection of secreted proteins in culture medium of transgenic tomato indicated that miraculin was secreted from the roots of transgenic tomato expressing miraculin. This study firstly showed the evidences of the intercellular localization of miraculin, and provided a new insight of biological roles of miraculin in plants.