Hiroshi Oyama

We developed new fermented foods —Moringa tempe Rm and Rs—from Moringa seeds fermented under aerobic conditions with Rhizopus oligosporus and Rhizopus stolonifer, respectively. In this study, Moringa tempe Rm and Rs were treated under additional anaerobic conditions to enhance their functional ingredients. The amounts of essential amino acids, branched-chain amino acids, and gamma-aminobutyric acid in the free amino acids of anaerobically treated Moringa tempe Rm were approximately five, seven, and eight times higher than those in samples maintained under aerobic conditions, respectively. Similarly, the anaerobically treated Moringa tempe Rs exhibited approximately six, eight, and six times higher levels of these compounds, respectively. Additionally, the total polyphenol contents of both anaerobically treated Moringa tempe Rm and Rs increased significantly. However, the antioxidant activities of both tempes did not differ significantly between aerobic or anaerobic conditions. Altogether, our results demonstrate that anaerobic fermentation effectively improves the nutritional quality.

Coagulation is an important process in the context of water purification; and the seed protein of the moringa tree (Moringa oleifera) is a remarkably effective coagulant. The laboratory course described here is designed to provide high-school students with a stepwise, hands-on experience in investigating the protein-rich coagulant found in Moringa seeds. First, the seed powder was applied to model polluted water containing fine clay, food dyes, copper sulfate, and bacteria. This treatment changed the polluted water into clear water via coagulation; all students were convinced that the coagulation-inducing agent was a thermostable cationic protein. Finally, basic biochemical techniques (e.g., chromatographic separation and electrophoresis) were used to show that the target coagulant is a dimeric protein composed of 6.5 and 4.5 kDa subunits. Overall, this made it possible for the students to gain a deeper understanding (more comprehensive than the information taught in formal classes) of protein structure and its real-world implications. This stepwise exercise can be applied to research-based learning programs in high school, as it is an effective learning tool.

We previously reported that Lys175 in the region of the active site of chymotrypsin (Csin) could be site-selectively modified by using an N-hydroxy succinimide (NHS) ester of the peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester [NHS-Suc-Ala-Ala-Phe^P(OPh)₂]. In this study, the Lys175-selective modification method was expanded to incorporate functional groups into Lys 175 in Csin. Two types of peptidyl phosphonate derivatives with the dansyl group (Dan) as a functional molecule, Dan-β-Ala-[Asp(NHS) or Glu(NHS)]-Ala-Ala-(R)-Phe^P(OPh)₂ (DanD and DanE, respectively), were synthesized, and their action was evaluated when modifying Lys175 in Csin. Ion-exchange chromatography (IEC), fluorescence spectroscopy, and LC-MS/MS were used to analyze the products from the reaction of Csin with DanD or DanE. By IEC and LC-MS/MS, the results showed that DanE reacted with Csin more effectively than DanD to produce the modified Csin (DanMCsin) bearing Dan at Lys175. DanMCsin exhibited an enzymatic activity corresponding to 1/120 of Csin against Suc-Ala-Ala-Phe-pNA. In addition, an effect of Lys175 modification on the access of the proteinaceous Bowman–Birk inhibitor to the active site of DanMCsin was investigated. In conclusion, by using a peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester, we demonstrated that a functional group could be incorporated into Lys175 in Csin.

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